Post on 22-Oct-2021
The Pennsylvania State University
The Graduate School
College of Medicine
AN INVESTIGATION OF MU-OPIOID RECEPTOR INTERACTING PROTEINS
WITHIN THE MU-OPIOID RECEPTOR SIGNALING COMPLEX
A Dissertation in
Genetics
by
Stephanie Justice-Bitner
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Doctor of Philosophy
December 2012
The dissertation of Stephanie Justice-Bitner was reviewed and approved* by the following:
Robert Levenson
Distinguished Professor of Pharmacology
Co-Director of MD/PhD Program
Dissertation Advisor
Chair of Committee
Laura Carrel
Associate Professor of Biochemistry and Molecular Biology
Vincent Chau
Professor of Cellular and Molecular Physiology
Victor Ruiz-Velasco
Associate Professor of Anesthesiology
David Spector
Professor and Distinguished Educator of Microbiology and Immunology
Co-chair, Intercollege Graduate Program in Genetics
*Signatures are on file in the Graduate School
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ABSTRACT
Opioids are powerful analgesic drugs that lead to the development of tolerance, physical
dependence, and addiction with prolonged use or abuse. Chronic exposure appears to cause
alterations in the neuronal morphology of brain regions implicated in opioid dependence and
addiction. Many of these changes reflect the inhibitory effects of these drugs and other opioid
receptor agonists on neuron size, neurite outgrowth, dendritic arborization, and neurogenesis that
occur in brain regions important for reward processing, learning, and memory. The mu-opioid
receptor (MOR) is the G-protein coupled receptor (GPCR) primarily responsible for mediating
the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and
heroin. However, the precise role of the MOR in the development of these neuronal alterations
remains elusive. A growing body of evidence suggests that G-protein-coupled receptor (GPCR)
signaling is modulated by proteins that bind GPCRs and form multiprotein signaling complexes.
A number of proteins that interact with the MOR have recently been identified and have been
shown to affect MOR biogenesis, trafficking, and signaling. Therefore, identifying and
characterizing MOR interacting proteins (MORIPs) may help to elucidate the underlying
mechanisms of opioid addiction.
Using a combination of conventional and modified membrane yeast two-hybrid (MYTH)
screening methods, a cohort of novel MOR interacting proteins (MORIPs) was identified. The
interaction between the MOR and a subset of MORIPs was validated in pull-down, co-
immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR
as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with
the delta (DOR) and kappa (KOR) opioid receptors. Finally, three of these proteins showed
altered expression in the brains of morphine treated mice.
The ubiquitin pathway has been shown to be altered in the addiction process, therefore
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two novel MORIPs, seven in absentia 1(SIAH1) and 2 (SIAH2), were of particular interest due to
their known function as E3 ligases in the ubiquitin pathway. The effects of altering SIAH1
and/or SIAH2 protein expression on opioid receptor ubiquitination and protein levels was
investigated using human embryonic kidney (HEK) stably transfected with MOR, delta opioid
receptor (DOR), or kappa opioid receptor (KOR). These experiments suggest that SIAH2 is an
E3 ligase for the MOR, while both SIAH1 and SIAH2 may conjugate ubiquitin to DOR and
KOR. The ubiquitination by SIAH2 appears to signal for degradation of the MOR, whereas
SIAH1 seems to act as a shuttle protein or chaperone assisting in degradation. In contrast, the
ubiquitination of DOR and KOR may be a signal for degradation or trafficking of receptors.
Additionally, opioid agonist treatment of neuroblastoma cells promoted the formation of
MOR/SIAH1 complexes. Based on the known function of these newly identified MORIPs, the
interactions forming the MOR signalplex are hypothesized to be important for MOR signaling
and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP
interactions provides a conceptual framework for defining the cellular mechanisms of MOR
signaling in brain and may be critical for determining the physiological basis of opioid tolerance
and addiction.
v
TABLE OF CONTENTS
LIST OF FIGURES ................................................................................................................. viii
LIST OF TABLES ................................................................................................................... x
LIST OF COMMON ABBREVIATIONS .............................................................................. xi
ACKNOWLEDGEMENTS ..................................................................................................... xiii
Chapter 1 Literature Review .................................................................................................... 1
1.1 Opioid Drugs .............................................................................................................. 1 1.1.1 Analgesia and Addiction ................................................................................. 1 1.1.2 Opioid Pharmacology ...................................................................................... 3
1.2 The Opioid System ..................................................................................................... 5 1.2.1 The Opioid Receptors ...................................................................................... 6 1.2.3 Mu-Opioid Receptor Genetic Variations ........................................................ 8 1.2.4 Dimerization of Opioid Receptors .................................................................. 17 1.2.5 Endogenous Opioid Peptides .......................................................................... 19
1.3 Neurobiology of Opioid Exposure ............................................................................. 21 1.3.1 Mechanisms of Opioid Addiction ................................................................... 21 1.3.2 Animal Models of Addiction-Like Behavior .................................................. 22 1.3.3 Opioid Receptor Knockout Studies ................................................................. 23 1.3.4 Physiological and Psychological Adaptations to Opioid Exposure ................ 26 1.3.5 Regulation of MOR ......................................................................................... 29
1.4 GPCR Interacting Proteins ......................................................................................... 31 1.4.1 MOR Interacting Proteins ............................................................................... 32 1.4.2 Identifying Candidate MORIPs ....................................................................... 35
1.5 Rationale and Hypothesis ........................................................................................... 36
Chapter 2 Identification of MOR Interacting Proteins ............................................................. 38
2.1 Introduction ................................................................................................................ 38 2.2 Experimental Procedures ........................................................................................... 40
2.2.1 Membrane Yeast Two-Hybrid Screen ............................................................. 40 2.2.2 Conventional Yeast Two-Hybrid Screen ........................................................ 41 2.2.3 Mapping of Binding Regions .......................................................................... 42 2.2.4 Glutathione S-transferase Pulldown Assay ..................................................... 42 2.2.5 Cell Culture ..................................................................................................... 44 2.2.6 Co-immunoprecipitation and Western blot Analysis ...................................... 45 2.2.7 Immunofluorescence Microscopy ................................................................... 46
2.3 Results ........................................................................................................................ 46 2.3.1 Identification of Novel MOR Interacting Proteins .......................................... 46 2.3.2 Mapping of Binding Regions .......................................................................... 51 2.3.3 Validation of MOR/MORIP interaction by GST Pulldown ............................ 53
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2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured
Mammalian Cells ............................................................................................. 55 2.3.5 Immunofluorescence Microscopy ................................................................... 57
2.4 Discussion .................................................................................................................. 59
Chapter 3 Functional Analysis of the MOR/SIAH Interaction ................................................ 64
3.1 Introduction ................................................................................................................ 64 3.2 Experimental Procedures ........................................................................................... 68
3.2.1 Mapping of binding sites ................................................................................. 68 3.2.2 Cell culture and transfections .......................................................................... 69 3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome ............... 70 3.2.4 Detection of ubiquitinated MOR using TUBEs .............................................. 70 3.2.5 siRNA knockdown of SIAH proteins .............................................................. 71 3.2.6 Co-immunoprecipitation and western blot analysis ........................................ 71 3.2.7 Quantitation of Western blots ......................................................................... 73
3.3 Results ........................................................................................................................ 73 3.3.1 Mapping of the MOR binding site on SIAH1 ................................................. 73 3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin ...................... 75 3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid
receptors ........................................................................................................... 78 3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but
increased ubiquitination of DOR and KOR. ..................................................... 81 3.3.5 The MOR undergoes degradation via the proteasome and lysosome ............. 83 3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels.............. 85 3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but
decreases DOR and KOR protein ..................................................................... 86 3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but
increases DOR and KOR protein ..................................................................... 89 3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells.............................................. 91 3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells ...................................... 92 3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the
ubiquitination of the MOR and an increase in MOR protein levels ................. 94 3.4 Discussion .................................................................................................................. 96
Chapter 4 Effect of Opioid Exposure on MORIPs .................................................................. 104
4.1 Introduction ................................................................................................................ 104 4.2 Experimental Procedures ........................................................................................... 107
4.2.1 Cell culture and drug treatment ....................................................................... 107 4.2.2 Co-immunoprecipitation and Western blot analysis ....................................... 108 4.2.3 Animal Care and Drug Treatment ................................................................... 109 4.2.4 Tissue Preparation and Protein Analysis ......................................................... 110
4.3 Results ........................................................................................................................ 111 4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid
receptors ........................................................................................................... 111 4.3.2 Interaction of MOR and MORIPs in Rodent Brain ......................................... 114 4.3.3 The Effect of Morphine on MORIP Expression ............................................. 117
4.4 Discussion .................................................................................................................. 119
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Chapter 5 Overall Discussion .................................................................................................. 123
REFERENCES ........................................................................................................................ 140
viii
LIST OF FIGURES
Figure 1.1: Non-selective agonists and antagonists at opioid receptors .................................. 5
Figure 1.2: Structural similarity between opioid receptors ...................................................... 7
Figure 1.3: Mouse strains show varied responses to morphine and other opioid drugs .......... 10
Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants .................... 13
Figure 2.1: Mapping of MORIP binding regions ..................................................................... 52
Figure 2.2: GST pull down of MORIPs and MOR .................................................................. 54
Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR ........................ 56
Figure 2.4: Co-localization of MOR and MORIPs .................................................................. 59
Figure 3.1: Mapping of SIAH1 binding site ............................................................................ 74
Figure 3.2: SIAH1 decreases the ubiquitination of the MOR .................................................. 77
Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and KOR .......... 80
Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases
ubiquitination of DOR and KOR ..................................................................................... 82
Figure 3.5: Inhibitor treatment of HEK-MOR cells ................................................................. 85
Figure 3.6: SIAH1 induces MOR degradation......................................................................... 86
Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels ................. 88
Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels ................. 90
Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2................................................... 92
Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels ......................... 93
Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels................................ 94
Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation and
increases MOR protein levels .......................................................................................... 96
Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex
formation .......................................................................................................................... 112
Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation .......................... 114
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Figure 4.3: MORIP expression and interaction with MOR in mouse brain ............................. 116
Figure 4.4: MORIP expression in brain regions of morphine treated mice ............................. 118
Figure 5.1: MOR interaction map ............................................................................................ 124
Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR cells ...... 130
Figure 5.3: Simplified model of Siah protein-mediated MOR degradation ............................. 132
Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of the
MOR ................................................................................................................................ 137
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LIST OF TABLES
Table 1.1: Legal Definitions of Schedule I and Schedule II Controlled Substances ............... 2
Table 1.2: Association studies investigating A118G SNP in opioid dependence in
humans ............................................................................................................................. 16
Table 1.3: Opioid receptor dimers with related G protein-coupled receptors .......................... 18
Table 1.4: Endogenous opioid peptides ................................................................................... 20
Table 1.5: Summary of opioid receptor knockout mice studies............................................... 26
Table 1.6: Previously identified MORIPs ................................................................................ 33
Table 2.1: PCR primers used for cloning MORIPs ................................................................. 44
Table 2.2: Putative MORIPs identified in MYTH screen ........................................................ 48
Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens ................. 50
Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants ..................................... 69
Table 3.2: Summary of SIAH overexpression and knockdown experiments .......................... 99
xi
LIST OF COMMON ABBREVIATIONS
AUP1 ancient ubiquitous protein 1
bp base pairs
coIP co-immunoprecipitation
CSN5 COP9 signalosome subunit 5
DOK4 docking protein 4
DOK5 docking protein 5
DOR δ-opioid receptor
ECL enhanced chemiluminescence
FBS fetal bovine serum
GPCR G protein-coupled receptor
GST glutathione S-transferase
HEK human embryonic kidney
HRP horseradish peroxidase
IL intracellular loop
IP immunoprecipitation
kDa kilodalton
KO knockout
KOR κ-opioid receptor
MOR μ-opioid receptor
MORIP μ-opioid receptor interacting protein
MYTH membrane yeast two-hybrid
PAGE polyacrylamide gel electrophoresis
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PBS phosphate buffered saline
PVDF polyvinylidine difluoride
SIAH1 seven in absentia homolog 1
SIAH2 seven in absentia homolog 2
SDS sodium dodecyl sulfate
SNP single nucleotide polymorphism
VAPA vesicle-associated membrane protein-A
VTA ventral tegmental area
WLS Wntless
Y2H yeast two-hybrid
xiii
ACKNOWLEDGEMENTS
I am forever grateful to all of those who have contributed to the completion of my
graduate work. First, I would like to thank Dr. Robert Levenson, my thesis advisor, for his
support, encouragement, and patience. I am grateful to the members of my thesis committee, Drs.
Vincent Chau, Victor Ruiz-Velasco, and Laura Carrel for their advice and support throughout my
graduate school career. I especially thank Dr. Vincent Chau for his technical advice and support.
I would also like to thank the past and present members of the Levenson Lab, who have made the
lab an enjoyable place to work, especially Dr. Jessica Petko for her unending encouragement,
advice, and friendship.
I extend gratitude to my collaborators, including Drs. Igor Staljar, Tom Ferraro, and
Jessica Petko for their assistance and advice. I am also indebted to the faculty and staff of the
Pharmacology Department and the Genetics Program.
Finally, I want to acknowledge my family members. I thank my parents for their
continued love and support throughout my schooling. I thank my husband’s aunt for providing
me a place to live, while working on my research. Lastly, I thank my husband for having the
patience to care for our dogs and home miles away while I worked on my graduate work.
Chapter 1
Literature Review
1.1 Opioid Drugs
1.1.1 Analgesia and Addiction
Opioid analgesics are invaluable in the treatment of severe, chronic, and/or
unremitting pain symptoms. The effectiveness of these drugs in relieving pain has been
known for hundreds of years (Dhawan et al., 1996). Unfortunately, these pain-relieving
benefits come with a number of negative side effects, which limits their use for only the
most severe pain conditions. The major side effects of opioid analgesics include
respiratory depression, alterations in heart rate and blood pressure, severe constipation,
and mood changes. The most significant of these effects are respiratory depression and
mood changes. The most common cause of death in cases of opioid agonist overdose is
respiratory depression. The reported feelings of euphoria and relaxation that typically
occur shortly after administration of opioid agonist drugs have been implicated in the
abuse liability of these drugs. Psychologically, this euphoric state is considered to be the
motivating or rewarding factor that drives an individual to use these drugs inappropriately
(Bailey and Connor, 2005; Corbett et al., 2006).
Due to their potential for abuse, opioid agonist drugs fall under the auspices of the
Controlled Substances Act, and are considered either Schedule I or Schedule II controlled
substances depending on whether or not the drug is clinically useful (Table 1.1). For
example, heroin (diacetylmorphine) has been deemed to have no medical use in the
United States, and is therefore listed as a Schedule I controlled substance. On the other
hand, morphine is considered to have an important medical use and is therefore listed as a
Schedule II controlled substance. The existence of the Controlled Substances Act affirms
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the larger problem that these drugs pose from a biological, societal, and legislative
perspective.
Table 1.1: Legal definitions of Schedule I and Schedule II controlled substances
These definitions were reproduced verbatim from Section 812 of the Controlled
Substances Act that was enacted into law by the Congress of the United States as Title II
of the Comprehensive Drug Abuse and Prevention and Control Act of 1970. It should be
noted that three more schedules exist, but only one opioid mixed agonist/antagonist,
nalorphrine, is schedule III. Schedule III drugs have less potential for abuse than
schedule I and II, but can still cause substance dependence.
Schedule I
(A) The drug or other substance has a high potential for abuse.
(B) The drug or other substance has no currently accepted
medical use in treatment in the United States.
(C) There is a lack of accepted safety for use of the drug or
other substance under medical supervision.
Schedule II
(A) The drug or other substance has a high potential for abuse.
(B) The drug or other substance has a currently accepted
medical use in treatment in the United States or a currently
accepted medical use with severe restrictions.
(C) Abuse of the drug or other substances may lead to severe
psychological of physical dependence.
In the 2010 National Survey on Drug Use and Health, it was estimated that 22.6
million Americans 12 years or older had used an illicit drug or used a prescription drug
inappropriately in the past year. Of these 22.6 million, an estimated 9.0 million people
3
were current users of drugs other than marijuana and 5.3 million of these current drug
users were using prescription pain killers (opioid drugs) or heroin. Opioid drugs are the
third most abused drug behind alcohol and marijuana. The use of prescription opioid
drugs among first-time initiators of drug abuse was found to be almost equal to
marijuana. However, the ability of many users to obtain opioid drugs from a friend or
relative for free could lead to an increase of opioid drug users.
The simplest way to resolve these issues would be the development of an
analgesic drug that is as efficacious as known opioid analgesics, but does not have
potential for abuse. Some researchers have called this endeavor “the exciting but vain
quest for the Holy Grail” (Corbett et al., 2006). When considering the efforts made in the
diverse field of opioid-related research, the sum total of work can all be traced back to
this goal of finding or creating a drug that can alleviate severe pain without being
addictive.
1.1.2 Opioid Pharmacology
Opioid compounds have been used since antiquity; long before the opioid
receptors and their peptide ligands were discovered. It wasn’t until 1805 that the first and
prototypical opioid agonist, morphine, was isolated from opium. Morphine is invaluable
as an analgesic agent, but it can induce a sense of euphoria, which contributes to non-
medical use and abuse (Dhawan et al., 1996). Receptor knockout studies in mice have
shown that both the analgesic and rewarding properties of morphine are mediated by the
mu-opioid receptor (MOR) (Matthes et al., 1996). Since these drugs are such powerful
analgesics, much effort has gone into synthesizing new drugs that retain the pain-
relieving qualities of morphine, but are less rewarding.
4
The quest to find a better opioid drug has led to the development of a large library
of compounds. This library consists of substances that act as agonists, antagonists, and
mixed agonist-antagonists. Morphine is the archetypal opioid agonist that mediates its
effects by binding to and activating opioid receptors. Antagonists, such as naloxone and
naltrexone, block the effects of agonists by competing for receptor binding sites and
preventing activation of receptors by agonists. Antagonists are useful in clinical settings
to rapidly reverse the effects of opioid agonist overdoses. Mixed agonist-antagonists are
compounds, such as nalorphine (n-allylnormorphine), which can act as an agonist at one
opioid receptor subtype, but also act as an antagonist at another opioid receptor subtype.
The development of these mixed action drugs initially held the promise of promoting one
effect (analgesia) while controlling another (addiction), but these drugs have still had
enough abuse liability to remain problematic (Snyder, 2004).
Most of these opioid drugs were created by making small chemical modifications
to morphine. Morphine is a naturally occurring plant alkaloid isolated from opium,
which is harvested from the unripe seed capsule of the poppy plant, Papaver somniferum
(Dhawan et al., 1996). When the structure of morphine and naloxone is compared, all
that is required to convert a potent agonist into an antagonist is an ethylene side-chain
(Figure 1.1). Although this effect is not fully understood, the fact that small differences
in chemical structure can have large impacts on pharmacological activity is clear from
studies that show that the opioid binding sites in the brain are stereospecific (Simon et al.,
1973; Terenius, 1973).
5
Figure 1.1: Non-selective agonists and antagonists at opioid receptors
Each of the compounds above contains the pentacyclic structure of morphine. This basic
structure contains a benzene ring with a phenolic hydroxyl group at position 3 and an
alcohol hydroxyl group at position 6 and at the nitrogen atom. The addition of relatively
simple side chains to this basic structure results in the altered activity of the resulting
compound. Reproduced from (Dhawan et al., 1996).
1.2 The Opioid System
Given the centuries-old knowledge and use of opioid drugs, it is surprising that
the existence of an endogenous opioid receptor was not postulated until the 1950s
(Lasagna, 1954). Since that time, pharmacological studies (Martin et al., 1976) and the
cloning of specific genes have led to the identification of the components of the
endogenous opioid system (Kieffer and Gaveriaux-Ruff, 2002).
6
The endogenous opioid system is composed of a subfamily of G-protein coupled
receptors (GPCRs) and their peptide ligands, most of which are derived from precursor
proteins. Many important physiological functions are regulated by this system, including
respiration, gastrointestinal motility, hormone secretion, thermoregulation, immune
function, mood, and pain modulation (Dhawan et al., 1996). The anatomical expression
profile of opioid receptors and ligands throughout the central and peripheral nervous
system and in other parts of the body, such as the gastrointestinal tract, explains why this
system is involved in so many physiological functions (Snyder, 2004; Snyder and
Childers, 1979). Our knowledge of the endogenous opioid system is largely derived from
the major effects and side effects that are seen when exogenous opioid drugs act on the
endogenous receptors (Dhawan et al., 1996; Snyder, 2004).
1.2.1 The Opioid Receptors
There are four main subtypes of endogenous opioid receptors, which are
designated delta (DORkappa (KOR), mu (MOR), and nociceptin (NOR). All four
receptors belong to the Class A (rhodopsin-like) GPCRs, and share similar structural
features, including an extracellular amino-terminus, seven transmembrane spanning
domains linked by three extracellular and intracellular loops, and an intracellular
carboxy-terminus. The opioid receptors are often posttranslationally modified through
glycosylation and palmitoylation (Minami and Satoh, 1995), and exert their major
downstream effects through pertussis toxin-sensitive heterotrimeric G-proteins (Gαo)
(Johnson et al., 1994; Ueda et al., 1988; Wong et al., 1988). Each receptor is encoded by
a single gene in humans and presumably forms a single gene product (Knapp et al., 1994;
Wang et al., 1994; Zhu et al., 1995), although a number of splice variants have been
reported for the MOR (Pasternak, 2004). Amino acid sequence alignments show that the
receptors are homologous to each other (MORand KOR 68% identical, MOR and DOR
66% identical, KOR and DOR 58% identical; NOR shares 50-60% identity with DOR,
, and MOR) with most of the variation between receptors occurring in the
7
extracellular loops and the carboxy-terminus (Dhawan et al., 1996). Figure 1.2
summarizes the structural similarities and differences between the opioid receptor types.
Recombinant chimeric receptor studies have found that the extracellular regions are
responsible for the specificity of ligand binding (Kong et al., 1994; Onogi et al., 1995;
Xue et al., 1994). Additionally, single nucleotide polymorphisms can alter ligand
binding. For example, an amino acid substitution at residue 40 (Asn to Asp) of the MOR
has been shown to increase -endorphin binding affinity (Bond et al., 1998) and alters the
functional properties of the receptor (Kroslak et al., 2007).
Figure 1.2: Structural similarity between opioid receptors
Model for the mu-opioid receptor in the rat. Amino acid residues that are conserved
between the mu-opioid receptor and both delta- and kappa- opioid receptors are shown in
black. Gray indicates an amino acid conserved with two opioid receptors and white
indicates non-conserved residues. Branched structures indicate potential N-linked
glycosylation sites. Reproduced from Minami and Satoh, 1995.
8
The distribution of opioid receptors has been reviewed by Le Merrer et al (2009)
and will not be addressed in detail in this chapter. Opioid receptors are primarily
expressed in the cortex, limbic system, and the brain stem. Ligand binding studies have
shown that the three opioid receptors exhibit an overlapping expression pattern in most
brain structures; however, some structures exhibit a higher expression of one receptor
over the others. For example, the MOR is more highly expressed in the thalamus when
compared to either the DOR or the KOR. Interestingly, MORs and KORs are often
found together in most brain regions, whereas the distribution of DORs is more restricted.
Importantly, all three receptors have been shown to have expression in areas of the brain
believed to be involved with addiction, such as the hippocampus, ventral tegmental area,
and the nucleus accumbens (Le Merrer et al., 2009). The distribution of opioid receptors
also correlates with the reported functions of the opioid system. For example, activation
of MORs located in the periaqueductal grey area (PAG) leads to analgesia, while
activation of NORs in the same area reduces the analgesic effect of MOR agonists. This
difference is due to the location of these receptors within the PAG. MORs are located on
presynaptic inhibitory interneurons, which leads to disinhibition of the neural output from
the PAG, whereas NORs are located on postsynaptic neurons, which leads to inhibition
of the neural output from the PAG. This anatomical difference explains the opposing
effects of these two opioid receptors, despite the fact that both receptors are coupled to
the same downstream effectors (Corbett et al., 2006). Knockout studies in mice have
further confirmed the role of these receptors in pain modulation, reward, and behavior
(Kieffer and Gaveriaux-Ruff, 2002).
1.2.3 Mu-Opioid Receptor Genetic Variations
Opioid agonists used in the treatment of pain have a narrow therapeutic index and
large interpatient variability in response. Patients may respond better to one opioid than
another with respect to both analgesia and side effects (Pasternak, 2004). Even in
relatively homogeneous patient cohorts, dosage requirements can vary substantially. For
example, in a study of postoperative hip replacement therapy encompassing over 3,000
9
patients, morphine dosage requirements varied almost 40-fold (Aubrun et al., 2003).
There also is great variability in the responses to these drugs, which may lead to the
development of addiction (Trigo et al., 2010; Volkow and Li, 2005). Similar
observations have also been seen in animal models (Figure 1.3). The analgesic
sensitivity of various strains of mice to a fixed morphine dose was observed to vary
substantially, with BALB/c mice the most sensitive and CXBK mice being the least
sensitive (Picker et al., 1991). When similar doses of opioids were given to CD-1 and
CXBK mice, there were more varied differences in analgesia depending on the opioid
drug administered (Figure 1.3) (Chang et al., 1998; Rossi et al., 1996). In addition to
inbred mouse strains, inbred rat strains have also shown variation in response to opioid
drugs. Among these inbred strains, Lewis and Fischer rats are the most often compared
because of their behavioral and neuronal molecular differences in responding to drugs.
Lewis rats tend to be more responsive in behavioral tests because they more easily self-
administer morphine and other drugs, such as cocaine (Ambrosio et al., 1995). In a more
recent study, Lewis rats were found to escalate both the dose and total amount of heroin
administered, whereas Fischer rats did not escalate either (Picetti et al., 2012). These
observed varied responses in animal models and in human subjects are believed to be
mediated by splice variants and polymorphisms in the MOR (Kasai and Ikeda, 2011).
10
Figure 1.3: Mouse strains show varied responses to morphine and other opioid
drugs
These two graphs represent some of the mouse strain studies that have been reported in
the literature. (A) The strains of mice received a fixed dose of morphine and were
assessed for analgesia using the radiant heat tailflck assay (Pick et al., 1991). (B) Doses
of opioids were chosen to give similar responses between CD-1 mice. These same doses
were then administered to CXBK mice (Rossi et al., 1996; Chang et al., 1998). Images
reproduced from Paskernak, 2004.
There are at least three different categories of factors that contribute to the
vulnerability of developing addiction: environmental factors, such as cues and
conditioning; drug-induced factors, which lead to molecular and neurobiological changes;
and genetic factors, which represent 40-60% of the risk for developing addiction (Kreek
et al., 2005). Included in these genetic factors are opioid receptor splice variants and
A
B
11
single nucleotide polymorphisms (SNPs), both of which have been studied in animal
models and in humans.
Data from animal models has provided important information regarding the role
of genetic factors in addiction. In mu-opioid receptor knockout mice, homozygous mice
have lower nociceptive thresholds than heterozygous knockout mice that have 50% less
wild-type receptor density. In turn, these heterozygous mice have lower nociceptive
thresholds than wild-type mice with normal mu-opioid receptor densities (Sora et al.,
1997). Mouse-strain comparison studies in recombinant inbred mouse lines have
provided additional evidence to implicate genetic factors in addiction. The CXBK mouse
strain exhibited reduced responses to some opioid agonists. Like the heterozygous MOR
knockout mice, CXBK mice express approximately 50% less MOR compared with
progenitor strains (BALB/c and C57BL/6) and have a similar phenotype (Ikeda et al.,
1999; Ikeda et al., 2001).
The human MOR gene (OPRM1) spans over 200 kilobases and consists of 11
exons that combine to yield splice variants (Xu et al., 2009). The most abundant
transcript, MOR-1, which consists of exons 1, 2, 3, and 4, is approximately 15 kb in
length, and encodes 400 amino acids (Ide et al., 2005). Mouse MOR-1 shares 94%
amino acid identity to human MOR-1. The first splice variant to be discovered was
human MOR-1A. This variant was shown to encode for only 392 amino acids and differs
from MOR-1 only at its 3’end (Bare et al., 1994). Since this initial discovery, over 28
splice variants from the mouse OPRM1 gene (Doyle et al., 2007a; Doyle et al., 2007b;
Pan, 2005) have been isolated with similar splicing patterns observed in rats (Pasternak et
al., 2004) and humans (Pan, 2005; Pan et al., 2003). The majority of these splice variants
yield receptors that differ from each other at the C-terminus. Splicing typically occurs
between exon 3 and more than 10 downstream exons, yielding at least 16 carboxyl
terminal variants (Figure 1.3). Although these full-length variants share a common
binding pocket (as seven transmembrane domains are conserved), they display significant
differences in mu opioid receptor agonist-induced G protein activation (Bolan et al.,
2004; Oldfield et al., 2008; Pan et al., 2005; Pasternak et al., 2004). These studies used
[35
S] GTPγS binding assays (measures G-protein activation) to show that the potency and
12
efficacy of opioid agonists, such as DAMGO and morphine, differed among splice
variants. Additionally, these variants have different distributions in the types of cells and
regions of the central nervous system (Abbadie et al., 2000a; Abbadie et al., 2000b;
Abbadie et al., 2000c; Abbadie et al., 2001; Schnell and Wessendorf, 2004). For
example, in the dorsal horn of the spinal cord, cells express either MOR-1 or MOR-1C,
but not both together (Pasternak, 2005).
In addition to the 3’end splice variants described above, there are also 5’ end
splice variants that contain exon 11. This exon is located 28-30 kb upstream from exon
1(species dependent). To date, nine exon 11-associated splice variants have been
identified (Xu et al., 2009). Interestingly, three of the transcripts generate the same
protein as MOR-1, while others predict a protein with six transmembrane domains,
and/or a protein with one transmembrane domain (Pan et al., 2001). These variants also
show differing expression patterns in the brain (Abbadie et al., 2004). Recently, a
naltrexone derivative, iodobenzoylnaltrexamide (IBNtxA), was synthesized in the
Pasternak laboratory (Majumdar et al., 2011). This new opioid drug is a potent analgesic,
but lacks the common opioid side effects, such as respiratory depression, constipation, or
physical dependence. In studying this new drug, Pasternak and colleagues found that
IBNtxA analgesia persisted in triple knockout mice (no MOR-1, DOR, or KOR). In
contrast, there was a loss of both IBNtxA analgesia and receptor binding in exon 11
knockout mice (Majumdar et al., 2011). These results demonstrate that exon splice
variants may be of interest in designing opioid drugs with analgesic power and little
abuse liability.
13
Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants
Exons are represented by boxes, while introns are represented by horizontal lines.
Translational start sites are indicated by downward lines on exon boxes. Translation stop
sites are indicated by upward lines on exon boxes. Exons are numbered by the order of
their isolation and/or similarity to exons identified in the mouse. C-terminal splice
variants would include MOR-1A, MOR-1B-1B5, MOR-1O, MOR-1S, MOR-1X, MOR-
1Y, Mu3, SV1, and SV2. Exon 11 splice variants would include MOR-1G1, MOR-1G2,
MOR-1H, and MOR-1i. Reproduced from Xu et al., 2009.
14
According to the dbSNP database and the NCBI database of genetic variation, the
OPRM1 gene contains over 700 single nucleotide polymorphisms (SNPs). This genetic
variation differs greatly between different races and ethnicities. For example, the minor
allele frequency (MAF) of A118G, which is a well-studied nonsynonymous SNP in exon
1 leading to an Asn40Asp substitution, are 0.047 in the African population, 0.154 in the
European population, 0.485 in the Japanese population, 0.14 in the Hispanic population,
and 0.08 in the Bedouin population (Gelernter et al., 1999). Many SNPs have been
studied to determine their association with pain sensitivity. Four SNPs (G-172T, A118G,
IVS1-C2994T, and IVS2 +G31A) were analyzed in association studies with pain.
Among these SNPs, two (IVS1-C2994T and IVS2 +G31A) were found to be significantly
associated with pain sensitivity scores and pressure pain thresholds, respectively
(Fillingim et al., 2005; Huang et al., 2008; Shabalina et al., 2009). However, there have
been no other reports showing an association between these two SNPs and pain-related
traits. Significant associations have also been observed in studies of the A118G SNP.
The G-allele carriers of the A118G SNP showed higher pressure pain thresholds and pain
tolerance thresholds (Fillingim et al., 2008).
Other than the highly studied A118G SNP, few other polymorphisms have been
investigated for functional significance. Among those that have been studied are two
promoter SNPs, G-554A and A-1320G. G-554A decreases transcription of MOR, but is
rare with a MAF of less than 0.001. In contrast, the A-1320G variant increases
transcription of MOR (Bayerer et al., 2007). Three others, G779A, G794A, and T802C,
are located in exon 3 and cause a decrease in receptor coupling and signaling (Befort et
al., 2001; Koch et al., 2000; Wang et al., 2001). Other OPRM1 polymorphisms have
been identified and associated with either pain or opioid dependence, but have no in vitro
evidence for function. These include C17T (A6V), which is found primarily in African
Americans (Crystal et al., 2012; Hoehe et al., 2000) and C440G (S147C), which is rare
with an MAF of less than 0.006 (Glatt et al., 2007).
The MOR mediates analgesia and side effects, such as nausea and respiratory
depression, of opioid drugs. The analgesic and side effects of opioids were abolished or
reduced in homozygous or heterozygous MOR-deficient mice, indicating that MOR gene
15
dosage is related to the clinical efficacy of these drugs. Although many SNPs in the
OPRM1 gene have been investigated in regard to opioid sensitivity, the one most often
studied is A118G. The A118G SNP has been shown to be associated with the analgesic
and side effects of opioids. In these studies, opioid dose (Ginosar et al., 2009; Reyes-
Gibby et al., 2007) and consumption (Lotsch et al., 2002; Sia et al., 2008) were greater in
G-allele carriers when compared to AA individuals. G-allele carriers exhibited a lower
analgesic efficacy and a lower incidence of side effects than AA individuals (Kasai and
Ikeda, 2011; Mague and Blendy, 2010; Somogyi et al., 2007). However, other studies
have found no such associations (Kasai and Ikeda, 2011; Mague and Blendy, 2010). The
A118G SNP has been studied in a variety of substance abuse studies, including those for
alcohol, nicotine, heroin, and alcohol. Findings differ greatly from study to study (Kasai
and Ikeda, 2011; Somogyi et al., 2007). As opioid dependence is the focus of this work,
a summary of the studies on the A118G allele and opioid dependence are shown in Table
1.2.
Many OPRM1 gene variations have been identified and analyzed for their
associations with pain sensitivity, opioid sensitivity, and susceptibility to drug
dependence. These studies have revealed significant associations between genetic
variations with regard to opioid sensitivity and vulnerability to substance dependence.
However, not every analysis finds a significant association; therefore, the significance of
variations in the OPRM1 gene is still debatable. More research into the mechanisms
underlying MOR expression and function are required to settle this debate.
16
Table 1.2: Association studies investigating A118G SNP in opioid dependence in
humans
This table summarizes association studies done with the A118G SNP and heroin
dependence. Risk or protection is associated with having at least one copy of the SNP.
These studies indicate that A118G SNP can have varied outcomes depending on the race
or ethnicity studied and the sample size. Adapted from (Mague and Blendy, 2010).
Effect Finding Population
(number of subjects)
Reference
Risk G118 associated with
heroin dependence
Swedish (139 heroin-
dependent, 149 control)
(Bart et al.,
2004)
Risk 90% of G118 were
heroin users
European Caucasian (118) (Drakenberg et
al., 2006)
Risk Higher frequency of
G118 in opioid
dependent subjects
Indian (126 opioid-
dependent and 156 control)
(Kapur et al.,
2007)
Risk G118 associated with
heroin dependence
Chinese men (200 heroin
dependent and 97 control)
(Szeto et al.,
2001)
Protective A118 associated with
heroin in Indian, but
not East Asian
populations
Indian (20 dependent, 117
control), Malaysian (25
dependent, 131 control),
Chinese (52 dependent,
156 control)
(Tan et al.,
2003)
Protective
A118 associated with
heroin dependence in
Hispanics
African American (46
dependent, 16 controls),
Caucasian (60 dependent,
44 controls), Hispanic (116
dependent, 78 controls)
(Bond et al.,
1998)
None No association found Chinese (48 heroin
dependent, 48 controls)
(Shi et al.,
2002)
17
1.2.4 Dimerization of Opioid Receptors
Opioid receptors belong to the G protein-coupled receptor (GPCR) Class A
superfamily. GPCRs can form homo- and heterodimers. Importantly, these dimers can
have distinct pharmacological properties. Therefore, dimerization can be seen as a way
for an organism to increase the functional variety of GPCRs. A large body of evidence
for the existence of GPCRs as dimers and oligomers has accumulated in recent years.
GPCR dimerization has been shown to be a physiological process that modifies receptor
pharmacology and regulates function. Heterodimerization can generate receptors with
characteristics distinct from either single receptor, leading to altered pharmacological
properties (George et al., 2002).
Within the opioid receptor family, DORs have been shown to interact with both
KORs and MORs to form heterodimers (Gomes et al., 2000; Jordan and Devi, 1999). In
the case of interactions between DORs and KORs, the heterodimers were found to have
reduced affinities for highly selective KOR or DOR agonists. Cells co-expressing KOR
and DOR also exhibited synergistic effects on agonist signaling. Additionally,
dimerization was found to affect the trafficking of these receptors, as etorphine-induced
trafficking of DOR was greatly reduced in cells expressing KOR/DOR dimers. Etorphine
causes DOR to be internalized, but when DOR/KOR dimers were expressed, the DOR
remained at the cell surface (Jordan and Devi, 1999). MOR/DOR dimers have also been
demonstrated to have altered selective agonist binding affinities (George et al., 2000). In
cells expressing MOR/DOR dimers, low doses of DOR selective ligands produced a
significant increase in the binding of a MOR selective agonist, which enhanced MOR-
mediated signaling (Gomes et al., 2004). Additionally, MOR/DOR dimers constitutively
recruit beta-arrestin 2, which results in alterations in ERK1/2 signaling (Rozenfeld and
Devi, 2007). More recently, MOR/KOR heterodimers expression in spinal cords of
Sprague-Dawley rats was found to be dependent on the estrous cycle stage of females and
was also less expressed in males (Chakrabarti et al., 2010). Opioid receptors have also
been shown to dimerize with distantly related GPCRs (Table 1.3). These receptor
heterodimers exhibit properties that are quite distinct from those of each individual
18
receptor. Unfortunately, the influence of SNPs on heterodimers formation has not been
studied. This lack of study is likely due to the lack of data on SNPs in other receptors
and the functional consequences of these differences.
Table 1.3: Opioid receptor dimers with related G protein-coupled receptors
This table lists the known opioid receptor heterodimers that have been discovered and the
proposed function of the interaction.
Dimer Function Reference
MOR and serotonin
receptor 5-HT(1A)
MOR-induced ERK1/2
phosphorylation was desensitized
with 5-HT(1A) activation
(Cussac et al., 2012)
MOR and α2A-adrenergic Activation of either receptor leads
to an increase in signaling, but
activation of both leads to a
decrease in signaling
(Jordan et al., 2003)
Apelin receptor and KOR Agonist stimulation of either
receptor resulted in higher
ERK1/2 phosphorylation
(Li et al., 2012)
Chemokine CXCR2 and
DOR
Antagonists of CXCR2 enhanced
the effects of DOR agonists
(Parenty et al., 2008)
Opioid receptor-like 1 and
MOR
Reduced potency of mu-agonist to
inhibit adenylate cyclase
(Wang et al., 2005)
Substance P receptor (NK1)
and MOR
Co-internalization with mu-
agonist treatment
(Pfeiffer et al., 2003)
Somatostatin (SST2A)
receptor and MOR
Mu-agonist treatment induced
phosphorylation and
desensitization of both receptors
(Pfeiffer et al., 2002)
19
1.2.5 Endogenous Opioid Peptides
Endogenous opioid peptides include enkephalins, dynorphins, endorphins, and
nociceptin (Bloom, 1983). Early studies that identified and characterized these peptides
suggested the possibility that they were derived from larger precursor proteins (Snyder
and Childers, 1979), which have now been identified and cloned (Chavkin et al., 1983;
Chretien et al., 1979; Mollereau et al., 1996). The enkephalins, endorphins, and
dynorphins contain the same four amino acid residues at their amino-termini (Try-Gly-
Gly-Phe). This sequence was thought to be necessary for binding to opioid receptors
(Snyder and Childers, 1979); however, more recently identified opioid peptides, such as
endomorphins, do not contain this sequence but still bind opioid receptors (Akil et al.,
1998). The endogenous peptides have not been found to be selective to binding to the
different opioid receptors, but there are differences in their binding affinities for the
opioid receptors (Akil et al., 1998; Dhawan et al., 1996). Table 1.4 lists the endogenous
opioid peptides, their precursor proteins, and their binding affinities.
20
Table 1.4: Endogenous opioid peptides
Precursor proteins and resulting peptides are listed along with the relative differences in
binding affinities of the peptide family for the opioid receptor subtypes. Adapted from
(Corbett et al., 2006).
Precursor Gene Peptide Binding Affinity
Proopiomelanocortin POMC Endorphins
α-endorphin
β-endorphin
γ-endorphin
δ-endorphin
MOR, KOR, DOR
Unknown Unknown Endomorphins
endomorphin-1
endomorphin-2
MOR
Preproenkephalin Penk Enkephalins
met5-enkephalin
leu5-enkephalin
DOR MOR, KOR
Preprodynorphin Pdyn Dynorphins
dynorphin A
dynorphin B
α-neoendorphin
β-neoendorphin
KOR MOR, DOR
Prepronociceptin PPNOC Nociceptin NOR
21
1.3 Neurobiology of Opioid Exposure
Historically, addiction of any kind was thought to be a problem of weak-willed
people. The general consensus was that the addicted person was not trying hard enough
to break his or her bad habit. However, with the sustained scientific efforts of the last 40
years, “addiction” has begun to be recognized as a neuropsychiatric disease.
Unfortunately, much of the social stigma associated with addiction still exists to this day
(Hyman, 2007). Opioid addiction is triggered by a chemical substance that activates a
specific set of receptors in the brain. These same receptors are also acted upon by
endogenous ligands to regulate normal neurobiological processes. Since not all people
that are exposed to opioid drugs develop addiction, it is difficult to conclude if the
activation of these receptors is the sole underlying cause of the disease. Twin, family,
and adoption studies have provided evidence that addiction is a complex genetic disorder
that can also be influenced by environmental factors, such as parental support, home
environment, level of education, and socioeconomic status (Lachman, 2006). This
complexity suggests that there are additional mechanisms that help to determine whether
or not a particular individual will develop addiction to an opioid drug.
1.3.1 Mechanisms of Opioid Addiction
Addiction to opioids is thought to be mediated by activation of MORs in areas of
the brain that mediate reward, such as the ventral tegmental area (VTA) and the nucleus
accumbens (NAc), which are the major constituents of the mesolimbic dopaminergic
pathway (Le Merrer et al., 2009). The direct injection of morphine or endomorphins into
these brain areas has been observed to mediate self-administration and conditioned place
preference in animal models (Tang et al., 2005; van Ree and de Wied, 1980; Zangen et
al., 2002). The potency of these exogenous opioid drugs leads to an over-activation of
the dopaminergic pathway that reinforces the drug-taking behavior. Unfortunately, this
explanation only pertains to the psychological and behavioral foundations of opioid
addiction and does not address the biological adaptations that may contribute to this
22
condition. However, these behavioral studies have illustrated the central role of the MOR
in mediating the rewarding effect of these drugs. In a study by David et al. (2008), wild-
type mice and δ-opioid receptor (DOR) knockout mice self-administered morphine
injected into the VTA, but MOR knockout mice did not. Along with the initial
characterization of the MOR knockout mouse (Matthes et al., 1996), these results directly
implicate the MOR as an essential component of the behavioral response to opioid
exposure.
Interestingly, almost all people who receive opioid drugs over a prolonged period
of time will develop tolerance and physical dependence. Although the mechanisms that
underlie the development of these changes are not clear, many studies hypothesize that
the regulation of MORs at the cellular and molecular levels may be involved (Bailey and
Connor, 2005; Harrison et al., 1998).
1.3.2 Animal Models of Addiction-Like Behavior
Animal studies have been crucial in understanding the biology and pathobiology
of drug addiction. In contrast to clinical studies, the subject population can be more
easily controlled for variables. Animal models often focus on the ability of the drugs to
directly control the animal’s behavior. Animal studies have demonstrated that the
rewarding effect is not dependent on preexisting conditions. Exposure to the drug alone
is sufficient to motivate drug-taking behavior (Lynch et al., 2010). There are two major
approaches to assessing addiction-like behavior; conditioned place preference (CPP) and
self-administration.
The CPP procedure exposes animals to a distinct environment in the presence of
drug. If the rodents find this substance positively reinforcing, they will show preference
for that environment when later given a choice (Tzschentke, 1998). CPP is widely used
to study the tolerance and sensitization to the rewarding effects of drugs induced by pre-
treatment regimens. This method can also be used to model certain aspects of relapse to
drug use, as in extinction/reinstatement procedures; however, this aspect is more often
investigated using self-administration paradigms (Myers et al., 2012; Tzschentke, 2007).
23
A more direct procedure to evaluate the reinforcing properties of a drug is to test
whether animals will work (usually lever press) to obtain the drug. This model
traditionally entails training an animal to self-administer a drug during daily sessions,
usually 1-3 hours in length. Most self-administration procedures use a fixed ratio (FR)
schedule of reinforcement. In this assessment, the animal must perform a fixed number
of responses to receive an intravenous drug infusion. If the animal finds the drug
rewarding, the number of responses that an animal will perform to obtain an infusion
generally increases. This is clearly seen in progressive ratio studies where the number of
responses required for the infusion is increased until the animal no longer responds
(Moser et al., 2011). Results from these kinds of studies have revealed that drugs can
serve as positive reinforcers and that there is correspondence between humans and
animals in terms of drugs that are self-administered and the pattern of drug intake. For
example, drugs that are abused by humans typically maintain responding in animals,
whereas drugs that do not maintain responding in animals are usually not abused by
humans (Collins et al., 1984). Similar patterns of drug intake have been reported in
humans and animals for opioids and other abused drugs (Griffiths, 1980).
1.3.3 Opioid Receptor Knockout Studies
Each gene encoding a receptor or peptide that is involved in the endogenous
opioid system has been knocked out in mice either singly or in combination (Gaveriaux-
Ruff and Kieffer, 2002).
The generation of a MOR knockout (KO) mouse confirmed the importance of the
MOR in mediating the analgesic and rewarding properties of opioid drugs. These mice
appeared normal morphologically, did not differ in weight when compared to wild-type
littermates, bred normally, and showed no changes in maternal behavior (Matthes et al.,
1996). However, MOR KO pups exhibited less attachment behavior when separated
from their mothers and were less responsive to their own mother’s cues (Moles et al.,
2004). The loss of MOR expression did not appear to significantly change other
components of the endogenous opioid system, nor were other components compensating
24
for this loss (Matthes et al., 1996; Sora et al., 1997). Tail flick, tail immersion, and hot
plate tests were used to confirm that MOR KO mice were insensitive to the analgesic
effects of morphine and other agonists (Kitanaka et al., 1998; Matthes et al., 1996; Sora
et al., 1997). Other responses to opioid drugs including respiratory depression and
slowed intestinal transit were also abolished in the absence of MORs (Dahan et al., 2001;
Matthes et al., 1998; Roy et al., 1998; Roy et al., 2001). Importantly, MOR KO mice
were insensitive to the rewarding effects of morphine and heroin in conditioned place
preference tests (Contarino et al., 2002; Matthes et al., 1996; Sora et al., 2001). A few
studies also found that self-administration of morphine and withdrawal precipitated by
naloxone was also abolished in these mice (Becker et al., 2000; Matthes et al., 1996; Sora
et al., 2001).
MOR KO mice have also provided a great deal of insight into the rewarding
properties of other drugs of abuse. Conditioned place preference for the active ingredient
of marijuana (Δ9-tetrahydrocannabinol) and nicotine were reduced when MORs were not
expressed (Berrendero et al., 2002; Ghozland et al., 2002). The self-administration of
cocaine and ethanol were also abolished in MOR KO mice (Mathon et al., 2005; Roberts
et al., 2000). Taken together, these data implicate the MOR in processing the rewarding
effects of other substances, not just opioid drugs. This also indicates that the endogenous
opioid system is involved in the regulation of reward processes.
Delta opioid receptor (DOR) knockout mice studies revealed that these mice
displayed higher levels of anxiety than MOR or kappa opioid receptor (KOR) knockouts
(Filliol et al., 2000). In a study by Roberts (2001), DOR KO mice were exposed to
alcohol in a two-bottle choice test. Naïve mutant mice and wild-type mice were
indistinguishable in their alcohol consumption. However, when tested in an operant
paradigm, the KO mice self-administered more alcohol than the wild-type mice.
Additionally, the elevated anxiety-like behavior in the DOR KO mice returned to wild-
type levels after alcohol administration. Therefore, the DOR may be an important factor
to explain the relationship between emotional states and the initiation and maintenance of
drug abuse. Interestingly, DOR-deficient mice do not develop tolerance to morphine,
25
which indicates that it may be involved in some kind of adaptive response to chronic
morphine exposure (Zhu et al., 1999).
Kappa and mu opioid receptors have largely been shown to oppose each other’s
effects. The KOR is involved in pain control, particularly visceral pain. KOR-deficient
mice showed no obvious change in the perception of mechanical or thermal pain;
however, they did show an enhanced response to peritoneal acetic acid injections. The
administration of kappa agonists typically leads to dysphoria. When KOR KO mice were
exposed to the kappa agonist, U 50,488H, conditioned place aversion was almost absent.
These mutant mice also displayed an attenuation of naloxone-precipitated withdrawal
syndrome when chronically treated with morphine (Simonin et al., 1998). This suggests
that the KOR participates in adaptations to long-term exposure to opioid drugs.
All three subtypes of opioid receptors appear to be potentially involved in some
aspect of addiction. A summary of the opioid receptor knockout mice studies is
presented in Table 1.5.
26
Table 1.5: Summary of opioid receptor knockout mice studies
Summary of responses to drugs and spontaneous behaviors observed in opioid receptor
knockout mice. Adapted from Gaveriaux-Ruff and Kieffer, 2002.
Knockout Drug Response Behavior in absence of drugs
MOR Morphine Decreased analgesia,
reward and dependence
Decreased locomotion
Increased thermal nociception
Decreased anxiety and depression
δ agonists Decreased analgesia,
reward and dependence
THC, alcohol Decreased reward
DOR δ agonists Decreased analgesia Increased locomotion
Increased anxiety, and depression
Morphine Decreased tolerance
Alcohol Increased intake
THC No change in reward
KOR κ agonists Decreased analgesia and
dysphoria
No change in locomotion
Increased visceral nociception
No change in anxiety or depression
Morphine Decreased Dependence
THC Decreased dysphoria
1.3.4 Physiological and Psychological Adaptations to Opioid Exposure
Our current understanding of opioid pharmacology suggests that activation of the
MOR by agonists, either endogenous or exogenous, is the most effective way to modulate
pain signals. However, DOR- and KOR-specific ligands have also demonstrated pain
modulation, but not to the same degree as MOR agonists.
27
How these drugs cause the development of tolerance, physical dependence, and
addiction is still poorly understood. Tolerance is the physiological process of adaptation,
where increasing amounts of the same drug are needed to achieve the same therapeutic
benefit. Physical dependence refers to the adaptive process whereby cessation of drug
use causes the onset of unpleasant withdrawal symptoms. Physical dependence can be
tested by treating individuals with an opioid antagonist like naloxone. One of the severe
side effects of opioid drugs is the development of addiction or substance dependence.
Addiction can be summarized as the inappropriate use of a drug despite negative
consequences (i.e. loss of job, criminal activity, poor health). The concern over
“creating” addicts has been shown to influence the prescribing habits of physicians,
leading to under-treatment of severe pain. For this reason, much effort has been focused
on a better understanding of these side effects and attempting to synthesize opioid
agonists that provide analgesia, but do not lead to tolerance, physical dependence, or
addiction. These efforts have resulted in an extensive library of agonists, antagonists, and
mixed agonists-antagonists; however, no compound has been developed that relieves pain
with no side effects.
Prolonged exposure to opioid agonists has been shown to cause pronounced
changes in the structure of neurons and the synaptic organization of the brain (Robinson
and Kolb, 2004). These changes are long lasting and may contribute to the characteristics
of addiction, including drug craving, compulsive drug use, and analgesic tolerance. For
example, in the ventral tegmental area chronic morphine exposure via subcutaneous
morphine pellets caused reductions in the overall size of the neurons in the mesolimbic
dopaminergic reward pathway. This long-term exposure also caused less branching and
fewer spines on the dendrites of medium spiny neurons in the shell of the nucleus
accumbens and on pyramidal cells in the prefrontal and parietal cortex (Robinson and
Kolb, 1999). The significance of these morphological changes is based on the
understanding that memory, learning, and other behavioral functions are associated with
the reorganization of synaptic connections in relevant neural circuits (Lamprecht and
LeDoux, 2004). Therefore, exposure to opioid drugs may be altering or creating neural
connections that potentiate drug-seeking behaviors. However, the susceptibility for
28
addiction is highly variable in individuals. Twin, family, and adoption studies have
shown that addiction is a complex genetic disorder that can be influenced by
environmental factors, such as home environment, parental support, socioeconomic
status, and education level (Lachman, 2006). This variable susceptibility has also been
documented in rats. Certain strains, such as Lewis, are more susceptible to drugs of
abuse than other strains, such as Fischer 344 (Suzuki et al., 1988). In Lewis rats,
morphine self-administration caused decreases in the branching and size of dendrites of
pyramidal cells in the motor cortex (Ballesteros-Yanez et al., 2007), but not in the Fischer
344 rats (Ballesteros-Yanez et al., 2008). When these two strains were compared prior to
morphine administration, Fischer 344 rats had neurons with shorter dendrites containing
fewer spines and branches when compared to neurons in Lewis rats (Ballesteros-Yanez et
al., 2008). These studies imply that significant changes to the structure of dendrites in
response to long-term opioid agonist exposure may contribute to susceptibility to
addiction.
Apart from its general inhibitory effects on dendritic structure, morphine has also
been shown to decrease neurogenesis in the hippocampus of adult rats (Eisch et al., 2000)
and to inhibit long-term potentiation (LTP) of excitatory synapses on CA1 neurons in the
hippocampus (Williams et al., 2001). Additionally, morphine has been shown to inhibit
the release of γ-aminobutyric acid (GABA) from neurons in the VTA (Nugent et al.,
2007). The exact role of neurogenesis in the hippocampus is not definitively known, but
recent evidence suggest that it plays an important role in integrating memories of events
that occur closely in time (Deng et al., 2010). Therefore, the inhibition of neurogenesis
by chronic morphine treatment may have deleterious effects on the function of this brain
region. The role of the MOR in mediating this effect was confirmed in a separate study
where it was found that the numbers and maturation of neurons in the granule cell layer
was increased in MOR knockout mice (Harburg et al., 2007). LTP is important for
synaptic plasticity, which has been implicated as a potential cellular mechanism that the
brain uses to encode activity- or experience-dependent events, such as learning and
memory (Cooke and Bliss, 2006). Presently, there is no clear relationship between LTP
and neurogenesis. Inhibition of LTP in the hippocampus and VTA may lead to abnormal
29
patterns of neural activity with unknown behavioral consequences. Thus, mechanisms
that are responsible for the structural and synaptic plasticity of the brain may play
important roles in the development of addiction. However, the specific mechanisms
linking opioid agonist activity to neuronal alterations observed with chronic opioid
exposure remain unknown.
1.3.5 Regulation of MOR
As GPCRs, MORs are regulated by the same mechanisms that regulate these
receptors, including endocytosis, recycling, and degradation (Drake et al., 2006;
Marchese et al., 2008). These regulatory mechanisms are usually initiated in response to
agonist receptor activation (Gainetdinov et al., 2004). Activated MORs undergo
internalization (or endocytosis) in response to most agonist drugs in most experimental
systems (Johnson et al., 2005). In general, agonist binding to MOR leads to downstream
signaling events mediated by Gαi/Gαo proteins. This now activated receptor can serve as
a target of G protein-coupled receptor kinases, which phosphorylate receptors at specific
threonine and serine residues. Phosphorylated receptors are then capable of binding to
arrestins that bind to clathrin and AP2, the clathrin-adaptor protein, which promotes
internalization of receptors by clathrin-coated pits. This process is known as homologous
desensitization (Gainetdinov et al., 2004). Following internalization, MORs are recycled
back to the surface during resensitization, while other opioid receptors, such as DORs,
are targeted to lysosomes for degradation and down-regulation (Tanowitz and von
Zastrow, 2003). Using chimeric MORs and DORs, a specific sequence present in the C-
terminal tail of the MOR was found to be both necessary and sufficient for recycling as
opposed to degradation. More recent studies have localized this sequence to the last 17
residues in the C-terminal tail called the MOR regulatory sequence (MRS) (Hislop et al.,
2009). The MRS promotes MOR recycling after short-term agonist exposure. The
essential trafficking of the MOR is controlled by the MRS, as mutant receptors lacking
this sequence are rapidly down-regulated. Some 3’ splice variants of the MOR lack the
MRS, which may account for increased tolerance to drugs that normally cause recycling
30
of the receptor (Pasternak et al., 2004). However, the MOR also undergoes agonist-
stimulated ubiquitination that promotes the sorting of receptors from the endosome
limiting membrane to the lumenal compartment. This ubiquitination of the MOR
functions independently and downstream of the ‘recycling versus degradation’ decision
of the MRS. More importantly, the ubiquitin-directed step is not required to direct
internalized MORs to lysosomes or to initiate their proteolysis, but does play a role in the
destruction of the MOR’s opioid binding site (Hislop et al., 2011). However, the
prototypical opioid agonist, morphine, does not cause endocytosis of MORs in human
embryonic kidney (HEK) 293 cells that have been stably transfected with the receptor
(Keith et al., 1996). Interestingly, the efficacy of morphine to produce internalization is
highly dependent on the system and brain region being examined (Johnson et al., 2005).
Brain slices from transgenic mice expressing epitope-tagged MORs in neurons of the
locus ceruleus showed no morphine-induced internalization of MORs (Arttamangkul et
al., 2008) and neither did MORs heterologously expressed in neurons of the hippocampus
(Bushell et al., 2002). In contrast, primary cultures of dissociated rat striatal neurons
showed rapid endocytosis of MORs in response to morphine (Haberstock-Debic et al.,
2005). These studies suggest that the expression of cellular components that regulate
MORs, such as GRKs and arrestins, are likely cell-type specific. For example, GRK2 has
been shown to phosphorylate MORs in HEK293 cell culture models (Schulz et al., 2004),
but has not been clearly shown in vivo (Johnson et al., 2005). However, a recent study on
the effect of beta arrestin on MOR regulation has shed some light on the reason for
morphine’s poor internalization. Mouse embryonic fibroblasts that were deficient in beta
arrestin 1, beta arrestin 2 or both were assessed for their ability to regulate MOR when
treated with morphine or DAMGO. DAMGO was shown to recruit both beta arrestin 1
and beta arrestin 2 to the MOR and either was sufficient to promote internalization.
DAMGO was also shown to promote ubiquitination of the MOR, which did not occur in
the absence of beta arrestin 1. In contrast morphine recruited beta arrestin 2 to the MOR,
but not beta arrestin 1. This interaction was sufficient to promote internalization of the
MOR, but not ubiquitination of the MOR. Taken together these results imply that beta
arrestin 1 is important in the ubiquitination of the MOR (Groer et al., 2011).
31
Despite these agonist-specific differences on MOR internalization, all agonists
produce similar physiological responses short-term (euphoria and analgesia) and
adaptation (tolerance and physical dependence) long-term. A seminal study from the lab
of Marshall Nirenberg attempted to correlate the development of tolerance and physical
dependence in animals with the specific effects that opioid agonists produce in cells.
This study showed that repeated administration of an opioid agonist on neurons
diminished their responsiveness to the drug as measured by the progressive dampening of
agonist-mediated cyclic adenosine monophosphate (cAMP) inhibition. When the agonist
was removed, the levels of cAMP rebounded above baseline (Sharma et al., 1975). This
suggested that receptor desensitization and subsequent cellular compensatory
mechanisms may underlie the development of tolerance and physical dependence.
Although numerous studies have continued to explore the mechanisms of MOR
regulation in an attempt to better understand the cellular context of tolerance, physical
dependence, and addiction; no clear mechanistic explanation for these adaptations has
been found (Bailey and Connor, 2005).
1.4 GPCR Interacting Proteins
The GPCR family is composed of a large and multifaceted group of cell surface
proteins that transduce extracellular signals into intracellular responses (Hill, 2006).
GPCR signaling is mediated by the activation of heterotrimeric guanosine triphosphate
(GTP)-binding proteins (G proteins), which act as secondary messengers to activate or
inhibit other proteins creating a cascade of events that produce a specific cellular
response (Hamm and Gilchrist, 1996). The versatility of these receptors is illustrated by
the varied physiological events they mediate, including sensory recognition (vision, taste,
and smell), endocrine regulation, and complex behavior phenomena (Gainetdinov et al.,
2004). In the central nervous system, these receptors serve as important targets for a
number of neuromodulatory drugs, such as psychotics and opioid analgesics (Hill, 2006).
Mounting evidence indicates that GPCRs bind to other proteins to form multiprotein
signaling complexes or signalplexes (Kabbani and Levenson, 2007; Milligan, 2005). The
32
interacting proteins have the potential to mediate previously unknown downstream
effects or to alter known mechanisms of receptor signaling and regulation (Bockaert et
al., 2010). For example, neuronal calcium sensor-1 (NCS-1) was shown to interact with
the D2 dopamine receptor (D2R) and interfere with its phosphorylation, which prevented
its endocytosis and desensitization (Kabbani et al., 2002). Since the MOR is known to
mediate the analgesic and rewarding properties of opioid drugs, proteins that interact with
the MOR likely play important roles in the development of tolerance, physical
dependence, and addiction to opioid drugs (Milligan, 2005). Identifying and
characterizing these proteins may provide insight into the mechanisms that underlie these
phenomena.
1.4.1 MOR Interacting Proteins
A comprehensive list of proteins that have been shown to interact with the MOR
is shown in Table 1.6. Many of these proteins affect the trafficking of the MOR by
promoting endocytosis (synaptophysin, PLD2, and M6a), helping to recycle and sort
internalized receptors (filamin A and GASP), or interfering with internalization
(RanBP9). Others affect the signaling properties of the MOR by inhibiting activation of
G-proteins (CaM and periplakin) or by inhibiting the ability of the MOR to inhibit cAMP
levels (PCK1) (Georgoussi et al., 2012). A novel signaling outcome for the MOR was
found when it was discovered that the MOR interacts with STAT5A. STAT5A is a
transcription factor and a member of the Signal Transducers and Activators of
Transcription family. When the MOR was activated, downstream transcriptional
activation of STAT-responsive reporter genes was detected in this study (Mazarakou and
Georgoussi, 2005). Although the role of these MORIPs has not been extensively studied
in terms of addiction, the identity of these proteins has contributed to the overall
knowledge of the MOR life cycle and downstream signaling events.
33
Table 1.6: Previously identified MORIPs
The location of the interaction (if known), a brief summary of the function of the
interaction and the original reference that identified the interaction are listed.
Interacting Protein Location
on MOR
Function Reference
Alpha 2A adrenergic
receptor
ND Receptor signaling (Jordan et al.,
2003)
Beta-arrestin 1
Beta-arrestin 2
ND Receptor desensitization and
endocytosis
(Bohn et al., 2000)
(Molinari et al.,
2010)
Calmodulin (CaM) IL3 Interferes with activation of G
proteins
(Wang et al., 1999)
Cannabinoid receptor
(CB-1)
ND Inhibition of signaling (Rios et al., 2006)
Delta opioid receptor
(DOR)
ND Modulation of signaling and ligand
binding
(Gomes et al.,
2000)
Filamin A C-tail Regulation of receptor recycling
and degradation
(Onoprishvili et
al., 2003)
Gai2/Gai3 IL3, C-tail Receptor signaling
(Georgoussi et al.,
1997)
Glycoprotein M6a ND Endocytosis and recycling of
receptor
(Wu et al., 2007)
G protein-regulated
inducer of neurite
outgrowth
IL-3 Regulates receptor distribution in
lipid rafts
(Ge et al., 2009)
Heat shock protein
HLJ1
C-tail ND (Ancevska-Taneva
et al., 2006)
Kappa opioid receptor ND Sex-dependent antinociception (Chakrabarti et al.,
2010)
Opioid receptor-like 1
receptor (ORL1)
C-tail Inhibits receptor signaling (Wang et al., 2005)
34
Periplakin C-tail Interferes with activation of G
proteins
(Feng et al., 2003)
Phospholipase D2
(PLD2)
C-tail Regulates receptor endocytosis (Koch et al., 2003)
Protein kinase Ci
(PKCi)
C-tail Negatively regulates receptor
desensitization
(Guang et al.,
2004)
Ran binding protein 9
(RanBP9)
C-tail Negatively regulates receptor
internalization
(Talbot et al.,
2009)
Regulator of G-protein
signaling 4 (RGS4)
C-tail Signaling and scaffolding (Georgoussi et al.,
2006)
RGS9-2 ND Receptor signaling and endocytosis (Garzon et al.,
2005a; Garzon et
al., 2005b)
Somatostatin receptor ND Internalization and desensitization (Pfeiffer et al.,
2002)
Spinophilin ND Modulates receptor signaling and
endocytosis
(Charlton et al.,
2008)
STAT5A C-tail Regulates receptor mediated
transcription
(Mazarakou and
Georgoussi, 2005)
Substance P receptor
(NK-1)
ND Receptor internalization and
resensitization
(Pfeiffer et al.,
2003)
Synaptophysin IL3 Modulates receptor signaling and
endocytosis
(Liang et al., 2007)
Wntless (WLS), GPR
177
IL2 Wnt secretion (Jin et al., 2010)
Of particular interest is the discovery of an interaction between MORs and
spinophilin, a scaffolding protein enriched in dendritic spines. Overexpression of
spinophilin in rat pheochromocytoma 12 (PC12) cells allowed the morphine-induced
endocytosis of the MOR within 30 minutes, an effect that was not seen in control cells.
In agreement with this finding, the loss of spinophilin expression in spinophilin KO mice
35
was found to increase measures of MOR signaling by delaying receptor internalization.
More importantly, in behavioral studies, spinophilin KO mice exhibited greater
sensitivity to the rewarding properties of morphine and developed a higher degree of
physical dependence (Charlton et al., 2008). This was the first study to show that the
behavioral response of an animal to opioid drugs could be affected by altering the
expression of a MOR interacting protein (MORIP). As more interactions and
multiprotein complexes are identified, researchers should be able to propose functional
roles for new associations, identify novel therapeutic targets, and devise more effective
strategies for encountering pathologies where the functionality of MORs is relevant
(Georgoussi et al., 2012). Thus, the continued identification and characterization of
MORIPs will be necessary to get a better understanding of the variety of functions that
are mediated by the MOR and the roles that these proteins may play in the pathogenesis
and treatment of complex neuropsychiatric diseases (Bockaert et al., 2010).
1.4.2 Identifying Candidate MORIPs
Many well-characterized methods exist to identify potential GPCR interacting
proteins. The conventional yeast two-hybrid approach was first introduced by Fields and
Song (1989) as a method for identifying an interaction between two proteins. This
technique relies on the reconstitution of the GAL4 yeast protein (a transcription factor),
which contains two distinct and separable functional domains: a DNA-binding domain
(BD) and a transcription-activating domain (AD). To test for an interaction, one protein
is fused with the BD and another is fused to the AD. Plasmid constructs are then
transformed into yeast cells that have been modified to have certain nutritional
requirements. If the proteins form an interaction, the binding and activating domains will
come into close proximity to one another and reconstitute the functional activity of the
GAL4 protein. This protein can then activate transcription and translation of the yeast
lacZ gene, which encodes for the enzyme β-galactosidase. This enzyme catalyzes the
chemical conversion of 5-bromo-4-chloro-3-indolyl-β-galactopyranoside to galactose and
5-bromo-4-chloro-3-hydroxyindole, which is visualized as a blue pigment when oxidized
36
to 5,5’-dibromo-4,4-dichloro-indigo. This method has been utilized as a screening
platform by the development of various cDNA libraries fused to the AD of the GAL4
protein. The selected library can then be tested against any ‘bait’ protein fused to the BD
of GAL4 (Miller and Stagljar, 2004). This approach is limited because the protein
interactions must take place within the nucleus of yeast cells. This technical issue makes
it difficult to use this conventional method with highly hydrophobic or membrane-bound
proteins (Lentze and Auerbach, 2008). In order to overcome these limitations, a
membrane-based yeast two-hybrid method was developed to enable the ability to detect
interactions between proteins that occur at the plasma membrane (Iyer et al., 2005;
Lentze and Auerbach, 2008). This method utilizes a split-ubiquitin molecule where the
N-terminal half (fused to a transcription factor) is fused to one protein and the C-terminal
half is fused to another protein. If an interaction occurs between the two proteins, the
ubiquitin molecule will be reconstituted and lead to the cleavage of the transcription
factor that is fused to the N-terminal half of the split-ubiquitin molecule.
1.5 Rationale and Hypothesis
Although opioid analgesics are efficacious for the treatment of pain, their use is
limited due to the side effects that they produce. The most undesirable side effect that
these drugs produce is the development of addiction. Unfortunately, the precise cellular
and molecular mechanisms that cause this effect are not yet known. What is known is
that these drugs activate the MOR and knockout studies have implicated the MOR as the
single opioid receptor subtype that mediates both the analgesic and rewarding properties
of these drugs. A growing body of evidence suggests that MORs are part of large
signaling complexes containing various MOR interacting proteins. Previously identified
MORIPs have been found to alter the regulation and signaling properties of the MOR.
Therefore, we hypothesize that identifying and characterizing novel MORIPs will help us
to better understand the cellular and molecular mechanisms that underlie the addiction
process.
37
In the following chapters, we examine the identification and validation of
interactions between the MOR and several novel MORIPs (Chapter 2), the functional
significance of the interaction between the MOR and novel MORIPs, seven in absentia
homologs 1 and 2 (Chapter 3), and the significance of the interactions between the MOR
and several novel MORIPs with opioid drug exposure (Chapter 4). The final chapter
discusses the potential relevance of the MOR-MORIP interactions in relation to MOR
regulation, possible implications for addiction, and directions for future research.
38
Chapter 2
Identification of MOR Interacting Proteins
2.1 Introduction
Most clinically relevant opioid agonists provide analgesia by activating MORs
(Berrettini et al., 1994; Inturrisi, 2002). MOR activation produces a euphoric effect in
many individuals, which is considered the primary motivational reward for abusing
opioid drugs (Bailey and Connor, 2005). The importance of the MOR in analgesia and
opioid dependence has been demonstrated by MOR knockout mice which show
significantly reduced sensitivity to both the analgesic and rewarding properties of opioids
(Contarino et al., 2002; Matthes et al., 1996). Like most GPCRs, the MOR is regulated
by multiple mechanisms, such as receptor desensitization, internalization, degradation,
and recycling (Petruzzi et al., 1997; Raehal and Bohn, 2005). The desensitization and
trafficking of MORs has been shown by a number of studies to represent key aspects in
the development of opioid tolerance and dependence (Bailey and Connor, 2005; Corbett
et al., 2006; von Zastrow et al., 2003; Waldhoer et al., 2004). Therefore, understanding
the mechanisms that regulate MOR signaling and trafficking is crucial for determining
the physiological basis of opioid dependence and enhancing opioid receptor
pharmacology for the treatment of addiction and pain.
A number of studies have shown that MOR desensitization and receptor
trafficking can increase the rewarding properties of opioid drugs, while reducing the
development of opioid tolerance and addiction-like behaviors (Berger and Whistler,
39
2011; Finn and Whistler, 2001; He et al., 2002; He et al., 2009; Martini and Whistler,
2007; von Zastrow et al., 2003; Whistler et al., 1999). However, the specific molecular
mechanisms that regulate these processes are largely unknown. Elucidating the
mechanisms that regulate MOR signaling and trafficking is critical for determining the
cellular response to opioid agonist drugs and for opening new avenues of investigation
into the pharmacotherapy of pain management.
Evidence indicates that GPCR signaling is modulated by proteins that bind to
form mulitcomponent protein assemblies, or signalplexes, involving interactions between
themselves, other GPCRs, and with other interacting proteins (Kabbani and Levenson,
2007; Milligan, 2005). A number of proteins have been identified and shown to affect
MOR biogenesis, trafficking, and signaling. For example, β-arrestin, calmodulin, and
synaptophysin have all been found to bind to the third intracellular loop of the MOR
(Georgoussi et al., 2011). Arrestins have been shown to be key components of MOR
desensitization because they can block the receptor’s interaction with G-proteins and can
promote recycling of the receptor through interactions with the clathrin assembly
machinery and clathrin-dependent endocytosis (Bohn et al., 2000; Molinari et al., 2010).
Calmodulin is believed to compete with G-protein for binding to the receptor and may
serve as an independent second messenger molecule released in response to MOR
stimulation (Wang et al., 1999). Synaptophysin is a major integral membrane
glycoprotein found in synaptic vesicles that interacts with a variety of nerve terminal
proteins such as dynamin I and adaptor protein 1 (AP-1). It is believed that
synaptophysin recruits dynamin to the plasma membrane to facilitate fusion of clathrin-
coated vesicles so that MOR can be endocytosed (Liang et al. 2007). The complete list of
40
currently known MOR interacting proteins along with a short description of the
functional significance of each interactor is shown in Table 1.6 of Chapter 1.
To better understand the potential role of MORIPs in the MOR life cycle, we have
performed conventional and modified yeast two-hybrid (Y2H) studies designed to
identify novel constituents of the MOR signalplex. Previous interaction screens for
MORIPs have primarily utilized the third intracellular loop (IL3) or the C-terminal tail
(Ctail) of the MOR as bait (Georgoussi et al., 2012). Previous studies in our laboratory
identified several D2 dopamine receptor interacting proteins, using the second
intracellular loop as bait in a conventional yeast two-hybrid screen (Kabbani and
Levenson, 2007). Therefore, we utilized the second intracellular loop as well as the
entire MOR to screen human brain cDNA libraries in order to expand the growing list of
MORIPs. Using these approaches, we have identified ten novel MOR binding partners.
Functional characterization of these interacting proteins will serve to further our
understanding of the mechanisms regulating MOR-mediated signaling and may help
elucidate the underlying molecular basis of cellular response to opioid agonist drugs.
2.2 Experimental Procedures
2.2.1 Membrane Yeast Two-Hybrid Screen
A modified split-ubiquitin yeast two-hybrid (MYTH) screen was performed in
collaboration with Igor Stagljar (University of Toronto) as described previously (Iyer et
al., 2005; Kittanakom et al., 2009; Stagljar et al., 1998). Briefly, the full-length human
41
MOR (transcript MOR-1) cDNA was cloned into the bait vector pCCW-STE
(Dualsystems Biotech AG, Switzerland) and the human fetal brain cDNA library was
cloned into the prey vector pRP3-N (Dualsystems). These constructs were then
sequentially transformed into S. cerevisiae reporter strain THY.AP4. This transformation
yielded 6 x 106 transformants/μg DNA on synthetic dropout (SD) agar plates (-Trp/-Leu/-
His/-Ade; Clontech, Palo Alto, CA) containing 3-amino-1,2,4-triazole (3AT). The
identity of the putative MORIPs was determined by recovering the prey plasmid from the
transformed yeast colonies. The cDNA clone in the plasmid was sequenced using
primers designed against the regions flanking the cloning site. These sequences were
then analyzed using Basic Local Alignment Search Tool (BLAST) to determine the
identity of the clone.
2.2.2 Conventional Yeast Two-Hybrid Screen
Two conventional yeast two-hybrid screens were performed as described
previously (Lin et al., 2001; 2005) using either the second intracellular loop (IL2; amino
acids 166-187) or the C-terminal tail (C-tail; residues 361-420) of the MOR as bait to
screen a fetal human brain cDNA library (Clontech). The MORIL2 and MOR C-tail
were cloned into the yeast GAL4 DNA binding domain expression vector pAS2-1
(Clonetech), while the human fetal brain cDNA library was provided in the GAL4
activation domain vector pACT2 (Clontech). Bait and prey plasmids were successively
transformed into yeast strain MaV103. Transformation of the yeast with MORIL2 and
the fetal human brain library produced approximately 2 x 106
transformants/μg DNA on
42
quadruple dropout plates (-Leu/-Trp/-His/-Ura; Clontech) containing 3AT.
Transformation of the yeast with MOR C-tail and the fetal human brain library yielded no
colonies on transformation efficiency plates; therefore the number of transformants/μg
DNA could not be determined. Interactions were assayed for β-galactosidase (β-gal)
activity via the nitrocellulose lift method (Lin R, 2005). cDNAs were extracted from
yeast colonies, sequenced, and subjected to BLAST analysis to determine their identities.
2.2.3 Mapping of Binding Regions
To map sites of interaction between the MOR and the newly identified MORIPs,
each MOR intracellular loop (IL) was tested for interaction with individual MORIPs
using the traditional Y2H method. MOR IL domains (IL1, amino acids 97-102; IL2,
amino acids 166-187; IL3, amino acids 259-282; and C- tail residues 361-420) were
separately ligated into pAS2-1 (Clontech) and assayed for interaction with candidate
MORIP cDNA clones in pACT2. Bait and prey plasmids were simultaneously co-
transformed into S. cerevisiae strain MaV103 and interactions were assayed for β-gal
activity as described above.
2.2.4 Glutathione S-transferase Pulldown Assay
MORIP-GST (glutathione S-transferase) fusion proteins were constructed by
separately fusing cDNAs encoding human AUP1 (residues 1-410), human DOK4
(residues 1-326), human CSN5 (residues 1-335), or the second intracellular loop of the
43
mu-opioid receptor (MOR-IL2; residues 166-187) to GST in the expression vector
pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). The polymerase chain reaction
(PCR) primers used for this cloning can be found in Table 2.1. cDNAs encoding the
MOR-IL2 (residues 166-187), SIAH1 (residues 1-233) and RanBP9 (residues 170-381)
were subcloned into the pET30a expression vector (Novagen, Madison, WI) containing
an S-tag. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the
ZYP-5052 auto-induction media as described previously (De Cotiis et al., 2008; Studier,
2005). MORIP-GST fusion proteins bound to glutathione sepharose beads (GE
Healthcare, Piscataway, NJ) were used to pull down S-tagged proteins from bacterial
lysates as previously described (Jin et al., 2010; Lin et al., 2001). Eluted proteins were
separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) filter for
western blot analysis. The filter was probed with a horseradish peroxidase (HRP)-
conjugated anti-S-tag antibody (1:5000 dilution, Novagen, Madison, WI), and
immunoreactivity detected by enhanced chemiluminescence with an ECL Plus kit (GE
Healthcare).
44
Table 2.1: PCR primers used for cloning MORIPs
This table lists the PCR primers that were used to clone MORIPs into pET-30a to
produce S-tagged proteins or into pGEX-4T-1 to produce GST-fusion proteins for use in
GST pull down experiments.
Construct Direction PCR primer sequence
SIAH2 S-tag Forward GAT ATC ATC AGG AAC CTG GCT ATG GAG AAG GTG
Reverse CTC GAG GGC CAA CCC CAT TCA TCC CAG GT
SIAH1 S-tag Forward GAT ATC ATG AGC CGT CAG ACT GCT ACA GCA TTA CC
Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC ATT GAT GCC
CSN5-GST Forward GGA TCC GTA AAG TTG CGT CTT GGT TGT
Reverse GAA TTC TGT CTT TCA GGT AAA GTA CTT CTC AGA GAC TGT
DOK4-GST Forward GGA TCC ATG GCG ACC AAT TTC AGT GAC ATC GTC AAG
Reverse GAA TTC CTG TGG TCA CTG GGA TGG GGT CTT
AUP1-GST Forward GAA TTC ATG GAG CTT CCC TCA GGG CCG
Reverse CTC GAG GTT CCT TTG AGC TCA GTC AGC CTC
MORIL2 S-tag Forward GGG GAA TTC GAT CGA TAC ATT GCA GTC TGC CAC CCT
MORIL2 GST Reverse GGG CTC GAG TTT GGC ATT TCG GGG AGT ACG GAA ATC
RanBP9-GST Forward GGA TCC GCC GTG GAC GAA CAA GAG ACG
Reverse CTC GAG GGT CTG CCA TTC TCC TCG ATC
2.2.5 Cell Culture
Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG-
tagged mu-opioid receptor (HEK-MOR), delta opioid receptor (HEK-DOR), or kappa
opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium
45
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418.
HEK-MOR and HEK-DOR cell lines were generously provided by Dr. Mark van
Zastrow (University of California San Francisco). HEK-KOR cells were a gift from Dr.
Lee-Yuan Liu-Chen (Temple University School of Medicine).
2.2.6 Co-immunoprecipitation and Western blot Analysis
HEK-MOR, DOR, or KOR cells (stably expressing FLAG-tagged MOR, DOR, or
KOR, respectively) were separately transfected with constructs encoding full-length
MORIPs sub-cloned in the pCMV-Tag3B expression vector (Stratagene, La Jolla, CA)
containing a myc-tag. Transfections were carried out using Effectene transfection
reagent (Qiagen, Valencia, CA). Cells were cultured for 24 hours in DMEM and then
lysed in 1X lysis buffer (20 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1
mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,
1% Triton X-100 and 1mg/mL leupeptin) supplemented with protease inhibitor cocktail
(Pierce, Rockford, IL). MOR, DOR, and KOR were immunoprecipitated from total cell
lysates using a polyclonal rabbit anti-FLAG antibody (Sigma, St. Louis, MO) coupled to
Protein-G Dynabeads (Invitrogen). Western blot analysis of immunoprecipitated
complexes was performed by using a monoclonal mouse anti-myc antibody (1:5000
dilution; Millipore, Billerica, MA) and a rabbit anti-mouse HRP-conjugated secondary
antibody (1:20,000 dilution; Jackson ImmunoResearch, West Grove, PA).
Immunoreactivity was detected by ECL Plus.
46
2.2.7 Immunofluorescence Microscopy
Immunohistochemistry was performed by transfecting HEK-MOR as described
for the co-immunoprecipitation. Cells were then split onto collagen coated coverslips
(BD Biosciences) and allowed to grow overnight. After 18 hours, cells were fixed with
4% paraformaldehyde and 5% sucrose. The cells were immunostained with monoclonal
mouse anti-myc antibodies (1:1000 dilution; Millipore) and rabbit anti-MOR antibodies
(1:500; AB5511, Millipore). After this initial staining, the cells were washed with 1X
PBS, and exposed to FITC conjugated donkey anti-rabbit antibodies (1:500 dilution;
Jackson ImmunoResearch) and rhodamine conjugated donkey anti-mouse antibodies
(1:500 dilution; Jackson ImmunoResearch). Images were obtained with a confocal
microscope (Zeiss LSM 510 Meta, Carl Zeiss Inc., Thornwood, NY), and digital images
were captured and imported with the LSM 5 image browser (Carl Zeiss, Inc.).
2.3 Results
2.3.1 Identification of Novel MOR Interacting Proteins
In the MYTH screen, 104 positive clones were obtained from the 6 x 106
colonies
screened. The putative MOR interacting proteins identified in this screen can be found in
Table 2.2. Sequence analysis identified GPR177 (WLS) (Jin et al., 2010), a putative
multi-pass membrane-spanning protein that is the mammalian ortholog of Drosphila
Wntless/Evi/Sprinter (reviewed in Hausmann et al., 2007). WLS plays an important role
in Wnt protein secretion, a process that is inhibited by the opioid agonist morphine (Jin et
47
al., 2010). Members of the Wnt pathway, such as WLS, are of particular interest because
they have been shown to affect neuronal development (Salinas and Zou, 2008). Many of
these Wnt-mediated effects mirror the effects induced by opioid agonist activity, but with
opposite outcomes. For example, opioid drugs have been shown to inhibit neurogenesis
in the adult rat hippocampus (Eisch et al., 2000), whereas Wnt proteins have been
demonstrated to promote neurogenesis in the adult rat hippocampus (Lie et al., 2005).
Vesicle-associated membrane protein-A (VAPA or VAP33) plays a role in endoplasmic
reticulum to Golgi transport (Peretti et al., 2008; Wyles et al., 2002). The number and
activity of opioid receptors at the cell surface are important in determining their capacity
to modulate downstream signaling. The receptor number at the cell surface can be
regulated through the biosynthesis pathway, including transcription, translation, protein
folding, and transport (Chen et al., 2006a; Chen et al., 2006b; Petaja-Repo et al., 2000).
Although the general consensus is that total opioid receptor protein does not change in
response to most opioid drugs (Christie, 2008; Johnson et al., 2005; Patel et al., 2002;
Stafford et al., 2001). This is not always the case. In a study using rat nucleus
accumbens, acute morphine treatment was shown to cause an increase in the amount of
epitope-tagged MORs in neuronal processes (Haberstock-Debic et al., 2003). Therefore,
proteins involved in the biosynthetic pathway may play an important role in responses to
opioid drugs. Connexin 37 (Cx37), and voltage-gated potassium channel Kv5.1 were
also identified in the MYTH screen (Table 2.1). These proteins play a role in, gap
junction formation (Haefliger et al., 1992), and regulation of resting membrane potential
(Kramer et al., 1998), respectively. Gap junctions mediate electrical transmission
between neurons by providing a low resistance pathway for the spread of electrical
48
currents and small metabolites (Bennett, 1997), while voltage-gated potassium channels
help to repolarize neurons after an action potential has been generated. Changes in
electrical transmission can contribute to synaptic plasticity in the form of long-term
potentiation (strengthening of synapses) or long-term depression (weakening of synapses)
(Dacher and Nugent, 2011). Thus, these two proteins may be important in the synaptic
plasticity observed with opioid addiction.
Table 2.2: Putative MORIPs identified in MYTH screen
ADD3 Adducing gamma subunit
GJA4 Gap junction protein, alpha 4
GPR177 Orphan g-protein coupled receptor
KCNF1 Potassium channel subunit
SLC31A2 Putative copper transporter
SLC39A13 Putative zinc transporter
SLC9A9 Putative Na+/H
+ exchanger
VAP33 VAMP-associated protein A
WHSC2 Wolf-Hirschhorn syndrome candidate 2
YIPF3 Yipl domain family, member 3
In the conventional yeast two-hybrid screen using the second intracellular loop
(IL2) of the MOR as bait to screen a fetal human brain cDNA library, 20 positive clones
were obtained from the 2 x 106 colonies screened. Sequence analysis identified several
clones encoding components involved in the ubiquitin pathway and in signal transduction
(Table 2.3). These included COP9 subunit 5 (CSN5), a protein proposed to regulate
49
exosomal protein sorting in both a deubiquitinating activity-dependent and -independent
manner (Liu et al., 2009), ancient ubiquitous protein 1 (AUP1), a protein that promotes
ubiquitination of misfolded proteins (Mueller et al., 2008), and seven in absentia
homologs 1 and 2 (SIAH1 and SIAH2), known really interesting new gene (RING) finger
E3 ubiquitin ligases (Hu et al., 1997a). The ubiquitin pathway has been shown to
mediate synaptic plasticity, learning and memory, and neurogenesis (Bingol and Sheng,
2011; Tuoc and Stoykova, 2010). Thus, these proteins were of interest for further study.
Additionally, four putative scaffolding proteins, docking protein 4 (DOK4), docking
protein 5 (DOK5), zyxin, and Ran binding protein 9 (RanBP9) were also identified as
candidate mu-opioid receptor interacting proteins (Table 2.3). Scaffolding proteins bind
and bring together two or more signaling proteins. In doing so, these proteins can direct
the flow of information by activating, coordinating, and regulating signaling events
(Buday and Tompa, 2010). Drugs of abuse can alter MOR signaling (Bailey and Connor,
2005; Christie, 2008), so these types of proteins are of interest for further study.
50
Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens
MORIP NAME BAIT RESIDUES
AUP1 Ancient ubiquitous protein 1 IL2 321-410
CSN 5 Constitutive photomorphogenic signalosome 9
subunit 5
IL2 25-318
DOK4 Downstream of kinase 4 IL2 65-289
DOK5 Downstream of kinase 5 IL2 40-306
PLAGL1 Pleiomorphic adenoma gene-like 1 IL2 28-165
PLXNA4 Plexin A4 C-tail Non-coding
RanBP9 RAN binding protein 9 IL2, C-tail 88-366
SIAH1 Seven in absentia homolog 1 IL2 1-233
SIAH2 Seven in absentia homolog 2 IL2 122-324
Zyxin Zyxin IL2 203-483
In the conventional yeast two-hybrid screen using the C-terminal tail of the MOR
as bait to screen a fetal human brain cDNA library, 7 positive clones were obtained in the
screen. The transformation efficiency plates did not produce yeast colonies, so the
estimation of transformation efficiency could not be determined. Sequence analysis
identified RanBP9, which was identified in the screen using MORIL2, and plexin A4, a
protein that is involved with axonal guidance (Haklai-Topper et al., 2010) (Table 2.2).
However, the portion of plexin A4 pulled out in the screen was part of the non-coding
region, and was not pursued further.
51
2.3.2 Mapping of Binding Regions
The split-ubiquitin screen used the entire coding region of MOR as bait; therefore,
this approach provided no information regarding the physical location of MORIP binding
sites on the MOR. To map the binding site of the MORIPs on the MOR, the
conventional yeast two-hybrid system was used to interrogate interaction of individual
MORIPs with each of the MOR intracellular loops and the C-terminal cytoplasmic tail
(Ctail). Each of the MORIPs identified in the split-ubiquitin screen was found to interact
with only the second intracellular loop of the MOR (Figure 2.1). This result was
somewhat unexpected, as previously discovered MORIPs have been found to bind to IL3,
IL2, and the Ctail. Therefore, it is a little unsual that all of the MORIPs discovered in the
MYTH screen interacted only with the second intracellular loop. However, this data does
suggest that the second intracellular loop of the MOR is an important site for protein-
protein interactions.
The MORIPs identified in the screen using MORIL2 as bait were also tested,
because some GPCR interacting proteins have been shown to interact with more than one
intracellular region of GCPRs (Cen et al., 2001; Georgoussi et al., 2006). Each of the
MORIPs from the conventional screens interacted, as expected, with the second
intracellular loop (IL2) of the MOR (Figure 2.1). Two of the MORIPs, CSN5 and
RanBP9, also interacted with the first intracellular loop (IL1), while AUP1, CSN5,
DOK4 and RanBP9 were found to interact with the C-terminal tail of the receptor. This
is consistent with RanBP9 being pulled out in the screen using the MOR Ctail as bait.
The binding site for Cx37 was not tested in this assay. These results suggest that the
52
MORIPs identified interact with specific domains of the MOR in the yeast system, and
that the second intracellular loop of the receptor is an apparent hot-spot for protein-
protein interactions.
Figure 2.1: Mapping of MORIP binding regions
Portions of each MORIP were tested in a directed Y2H assay for interaction with each of
the intracellular loops (IL) and carboxyl-terminus (C-tail) of the MOR. Empty bait
vector (pACT2) was used as a negative control. A positive interaction is indicated by the
production of a blue colony in the β-galactosidase assay.
53
2.3.3 Validation of MOR/MORIP interaction by GST Pulldown
To further validate direct interaction between newly identified MORIPs and the
MOR, we tested the ability of selected MORIP-GST fusion proteins to associate with S-
tagged MORIL2 in a pull-down assay. Western blots containing lysates from bacteria
expressing an S-tagged MORIL2 cDNA produced an immunoreactive band of ~13 kDa
when probed with anti-S-tag antibodies (Figure 2.2). This band corresponds to the
expected size of the MORIL2 encoded by the cDNA construct. The same band was
detected by pull-down after the bacterial lysate was incubated with either the CSN5-GST,
AUP1-GST, or DOK4-GST fusion proteins, but not when the lysate was absorbed onto
beads alone or GST-coated beads. We also utilized the pull-down assay to test the ability
of RANBP9, SIAH1, and zyxin to associate with a MORIL2-GST fusion protein. As
shown in Fig. 2.2, S-tagged RANBP9 cDNA produced an immunoreactive band of ~34
kDa, S-tagged SIAH1 cDNA produced a band of ~32 kDa, and S-tagged Zyxin was
detected at ~85 kDa when probed with anti-S-tag antibody. These bands correspond in
size to the predicted sizes of the protein fragments encoded by the respective cDNA
constructs, and were not detected when the lysates were absorbed onto beads alone or
GST-coated beads.
54
Figure 2.2: GST pull-down of MORIPs and MOR
GST pull-down assays were performed to interrogate the interaction between selected
full-length MORIPs and the MORIL2 domain. In the top three panels, MORIP-GST
fusion proteins were used to pull down the S-tagged MORIL2. In the bottom three
panels, MORIL2-GST fusion proteins were used to pull down S-tagged MORIPs. Pull-
down products were purified on glutathione beads, separated by SDS-PAGE, and probed
on Western blots using HRP-conjugated anti-S-tag antibodies. S-tagged MORIPs or
MORIL2 domains produced in bacteria are shown in lysate lanes (Ly), while uncoated
glutathione sepharose beads (Beads) or GST-coated glutathione sepharose beads (GST)
incubated with S-tagged proteins served as negative controls. PD indicates pull-down
lanes.
55
2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured Mammalian Cells
The interaction between full-length MOR and selected MORIPs was verified
using co-immunoprecipitation (co-IP) experiments. To demonstrate an interaction in cell
culture, the ability of an anti-FLAG antibody to co-IP MORIPs from lysates prepared
from HEK 293 cells stably expressing FLAG-tagged MORs (HEK-MOR) was tested.
Probing Western blots with anti-myc antibodies revealed immunoreactive bands of the
expected sizes in lysate lanes (L) prepared from HEK-MOR cells transiently transfected
with myc-tagged AUP1, CSN5, Cx37, DOK4, VAPA cDNAs (Figure 2.3B). Anti-
SIAH1, anti-SIAH2, and anti-WLS antibodies detected immunoreactive bands of the
expected sizes in lysate lanes for SIAH1, SIAH2, and WLS, respectively, in
untransfected cells. In co-IPs where no anti-FLAG antibody was added (mock, M), no
such bands were detected; however, bands migrating at similar molecular weights were
detected in the IP lanes. Y2H and co-IP experiments have recently confirmed an
interaction between RANBP9 and the MOR (Talbot et al., 2009). Taken together, these
results support the view that the MORIPs identified in our Y2H screens interact with full
length MOR in the context of cultured mammalian cells.
56
Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR
(A) Amino acid sequence comparison between MOR, DOR, and KOR IL2 domains. (B)
Co-immunoprecipitation of full length MORIPs with MOR, DOR, or KOR. Flag-tagged
MOR, DOR, or KOR was immunoprecipitated from HEK-MOR, HEK-DOR, or HEK-
KOR cells, respectively, using rabbit anti-flag antibodies. Mock immunoprecipitations
were performed with Protein-G beads coated with non-specific rabbit IGG. Blots were
probed with either anti-MORIP antibodies for the presence of endogenously expressed
MORIPS (SIAH1, SIAH2 and WLS) or with anti-myc antibodies for transiently
transfected myc-tagged MORIPs. Lysate lanes (L) contain 5% of the total protein
compared to the mock (M) and immunoprecipitation (IP) lanes.
The Y2H and pull-down data indicate that each of the MORIPs identified in the
screens interacts with the second intracellular loop of the MOR. Sequence comparisons
indicate that the second intracellular loops of the MOR, DOR and KOR exhibit a high
57
degree of amino acid sequence homology (Figure 2.3A). Among the opioid receptor
proteins, the second intracellular loop exhibits 85-90% amino acid sequence similarity,
with the highest homology within the N-terminal portion of the loop (Figure 2.3A).
Because of this homology, we used co-IPs to determine whether any of the MORIPs also
interacted with other opioid receptor family members. To do this, we tested the ability
of anti-FLAG antibodies to co-IP MORIPs from lysates prepared from HEK-DOR and
HEK-KOR cells (stably expressing FLAG-tagged DORs and KORs, respectively). As
shown in Figure 2.3B, AUP1-myc, Cx37-myc, SIAH1, SIAH2, VAPA-myc, and WLS
were able to interact with both DOR and KOR. However, neither CSN5-myc nor DOK4-
myc showed interaction with DOR or KOR, despite the fact that the proteins were
expressed in the transfected cells, as indicated by the presence of immunoreactive bands
of the appropriate size in the DOR and KOR lysate lanes (Figure 2.3B). Together, these
results suggest that many of the MORIPs we identified are also capable of interaction
with the DOR and KOR, at least within the context of transfected mammalian cells. The
interaction of these proteins with each of the opioid receptor family members will depend
in large part on whether an opioid receptor family member and a particular opioid
receptor binding protein are co-expressed within the same cell (and cellular
compartment) within the nervous system.
2.3.5 Immunofluorescence Microscopy
Immunofluorescence microscopy was performed to determine the cellular
location of MORIPs and MOR in HEK-MOR cells. Due to the lack of available
58
antibodies to selected MORIPs or their use in immuohistochemistry, myc-tagged proteins
were transfected into HEK-MOR cells. Cells were double labeled with mouse anti-myc
and rabbit anti-FLAG antibodies, donkey anti-mouse FITC-conjugated, and donkey anti
rabbit rhodamine-conjugated secondary antibodies to label the MORIPs green and MORs
red. If the MOR and the MORIP signals overlap (orange or yellow), it suggests that the
proteins occupy the same space within the cell, and therefore are able to interact.
However, the observed staining of the MOR is not entirely at the membrane, as would be
expected (Figure 2.4). This may be due the overexpression of the MOR in these cells or
to non-specific staining by the rabbit anti-MOR antibodies. Therefore, conclusions cannot
be made in regards to the possible co-localization of the MOR with these MORIPs. The
immunofluorescence does allow the approximate intracellular location of the MORIPs to
be determined. DOK4 was observed primarily near the plasma membrane (Figure 2.4),
which is consistent with a previous report (Uchida et al., 2006). AUP1, SIAH1, and
CSN5 were all predominantly expressed in the cytosol (Figure 2.4). As with DOK4, the
cellular locations of AUP1, SIAH1, and CSN5 are consistent with previous reports (Hu
and Fearon, 1999; Kato et al., 2002; Luo et al., 2006).
59
Figure 2.4: Co-localization of MOR and MORIPs
MOR was labeled with rabbit anti-MOR antibody and donkey anti-rabbit FITC. Myc
fusion proteins were visualized using mouse anti-myc antibody and donkey anti-mouse-
rhodamine. Images are confocal. Overlay shows the merging of the labels and is
indicated by a yellow color.
2.4 Discussion
GPCR signaling is modulated by proteins that bind to form signalplexes,
involving interactions between themselves, other GPCRs, and with other interacting
proteins. As a GPCR, the MOR is expected to have an extensive signalplex (Kabbani
and Levenson, 2007; Milligan, 2005). Utilization of conventional and modified yeast
two-hybrid (Y2H) methods identified ten novel and two previously reported components
of this complex. Directed Y2H and GST-PD studies were used to confirm interaction
60
and map the binding site for several MORIPs to the second intracellular loop of MOR.
Many MORIPs were demonstrated to interact with full length MOR in the context of
cultured human cells. Due to high sequence homology in intracellular loop two, many
MORIPs also interact with the KOR and DOR, suggesting that these proteins may be
universal regulators of opioid function
Previous conventional Y2H screens have identified many of the known MOR
interactors including periplakin, protein kinase C I (PKCI), phospholipase D2 (PLD2),
synaptophysin, glycoprotein M6a, filamin A, and DnaJ-like 1 protein (Hlj) (Georgoussi et
al., 2012). The conventional Y2H presented here was unique in that it utilized the
second intracellular loop of MOR, a region of the receptor that was previously unstudied
in terms of interacting proteins. This screen yielded six novel interactors and two
previously reported MORIPs (wntless and Ran binding protein 9). In addition, a
modified split-ubiquitin Y2H screen identified four interactors of full length MOR. The
advantage of this type of screen is that full length membrane proteins can be used as bait,
and the screen is able to identify membrane-localized interactors which may be missed in
the conventional Y2H screen.
Although these screens were successful in discovering novel MORIPs, the Y2H
method can result in false negative and false positive results. False positive results are
usually eliminated during subsequent analysis, such as GST pull-down and co-IP. False
negative results can occur for a variety of reasons. For example, if the interaction
depends on posttranslational modifications, such as glycosylation or phosphorylation,
these modifications may not occur properly or at all in the yeast system. Therefore, these
interactions would not be detected in the Y2H. Since the fusion proteins must be targeted
61
to the nucleus, hydrophobic proteins (such as membrane bound proteins) or proteins with
strong targeting signals may not reach the nucleus to activate the reporter gene and would
also be missed in the Y2H. Additionally, very weak or transient interactions are not
usually detected using this method and would likely be missed in a screen. These
disadvantages in using the Y2H method are important, because they help explain why
more interacting proteins were not found.
These studies have shown via directed Y2H that several MORIPs are capable of
interacting with multiple loops of the MOR. This is not surprising as some previously
identified interactors of MOR and DOR have been shown to be able to associate with
more than region of the receptor. Regulators of G protein signaling 4 protein (RGS4) and
beta arrestin have been demonstrated to interact with both the third intracellular loop and
the C-terminal tail of the DOR (Cen et al., 2001; Georgoussi et al., 2006).
Heterotrimeric G proteins (Gi) bind to the MOR at the third intracellular loop and the C-
terminal tail (Georgoussi et al., 1997).
The MORIPs identified in this and other studies are functionally diverse, but
many share common functions including protein processing, cellular localization, signal
regulation, receptor desensitization, ubiquitination, scaffolding, WNT secretion, and axon
guidance. Several of the novel MORIPs identified in the current screens belong to the
ubiquitin proteasome pathway. Seven in absentia homolog 1 and 2 (SIAH 1 and 2) are
RING finger E3 ubiquitin ligases that mediate ubiquitination and subsequent proteasomal
degradation of target proteins (Hu et al., 1997a). COP9 subunit 5 (COP9S5 or CSN5) is
one subunit of the large COP9 signalosome (CSN) which is responsible for the
deneddylation and activation of Cullin ring E3 ubiquitin ligases that ubiquitinate targets
62
for degradation (Choo et al., 2011). These MORIP/MOR interactions are surprising
considering that MOR is typically downregulated by lysosomal degradation and not the
proteasome. However, ubiquitination of MOR does occur and is required for trafficking
events that downregulate receptor ligand binding (Hislop et al., 2011). Since the E3
ligase for ubiquitination of MOR is unknown, it will be important to determine whether
SIAH1, SIAH2, or CSN5 promotes ubiquitination of the opioid receptors. An alternative
hypothesis is that MOR can act to scaffold these proteins for ubiquitination of other
MORIPs. In fact, synaptophysin, a previously identified MORIP is a target of both
SIAH1 and SIAH2 (Wheeler et al., 2002). There is also mounting evidence that the
ubiquitin pathway is involved in a variety of neuronal activities, including dendritic
morphogenesis and synaptic plasticity (Bingol and Sheng, 2011; Tuoc and Stoykova,
2010; Yang et al., 2008). Similarly, chronic opioid exposure has been shown to lead to
changes in dendritic arborization and synaptic plasticity (Robinson and Kolb, 2004). To
date there is no direct evidence to connect these observations.
Two proteins from the downstream of kinase (DOK) family of adaptor proteins
were also identified as interactors of MORIL2. This family of proteins has a broad range
of functions. In the nervous system, DOK4 has been shown to be required for axon
myelination and the regulation of GDNF-dependent neurite outgrowth via the activation
of the Rap1-ERK1/2 pathway (Uchida et al., 2006). DOK5 was demonstrated to interact
with TrkB and TrkC neurotrophin receptors and is involved in the activation of the
MAPK pathway induced by neurotrophin stimulation (Shi et al., 2006). Like opioid
receptors, neurotrophins have a strong link to pain perception, reward, and synaptic
plasticity (Pardon, 2010; Pezet and McMahon, 2006). For example, BDNF (ligand of
63
TrkB) has been implicated in substance dependence as well as antinociception (Ghitza et
al., 2010; Marcol et al., 2007; Obata and Noguchi, 2006; Ren and Dubner, 2007). It will
certainly be of interest to determine whether there is functional interplay between the
MOR and neurotrophic factors in MAPK-mediated signaling, pain processing, reward,
and memory formation.
The results presented in this chapter highlight the use of multiple screening methods to
expand our knowledge of proteins that interact with, and may contribute to the regulation of
opioid receptor-mediated signaling in brain. Identification of the full panorama of MORIPs
represents a critical step in understanding the normal regulation of the receptor life-cycle, as well
as how receptor signaling or trafficking may be hijacked in response to drugs of abuse. The
MORIPs identified in this and other studies may also represent key players involved in other
opioid-mediated processes such as neural development, nociception, aversion, and synaptic
plasticity. As the list of MORIPs grows, and their contributions to receptor function become
better understood, it is possible that some of these proteins will also become targets for new drug
development to prevent and/or treat opioid addiction.
64
Chapter 3
Functional Analysis of the MOR/SIAH Interaction
3.1 Introduction
In Chapter 2, two E3 ligases, SIAH1 and SIAH2, were identified as novel
MORIPs. Since the identity of ubiquitin ligases that regulate the MOR are unknown,
these proteins were chosen for further study.
E3 ligases are the last enzymes utilized by the cell to modify proteins with
ubiquitin. Ubiquitin is attached to lysine residues of substrate proteins covalently and
requires the action of a cascade of enzymes. First, an ubiquitin-activating enzyme (E1)
forms an ATP-dependent high energy thiol ester bond with ubiquitin. Then the activated
ubiquitin is transferred to an ubiquitin-conjugating enzyme (E2). Some E2s are capable
of ubiquitinating the substrate directly; however, this is usually done by an ubiquitin
ligase (E3) and often confers substrate specificity (Myung et al., 2001).
Seven in absentia was first described as a gene required for the specification of R7
cell fate in the Drosophila melanogaster eye (Carthew and Rubin, 1990). Three years
later, the murine homologues were discovered and designated Siah (seven in absentia
homolog) and were found to exist as three genes, Siah-1a, Siah-1b, and Siah-2 (Della et
al., 1993). The first report and cloning of a human Siah gene suggested a role for Siah in
apoptosis and tumor suppression (Nemani et al., 1996). The human SIAH1 is
homologous to the mouse Siah1a, whereas the human SIAH2 is homologous to the mouse
Siah2. Humans do not have a homologus Siah1b gene (Hu et al., 1997a). The first report
65
of the involvement of Siah proteins in the ubiquitin-proteasome pathway was found when
the deleted in colorectal cancer (DCC) protein was shown to be regulated by Siah and
that Siah interacted with ubiquitin-conjugating enzymes (Hu et al., 1997b). Since these
initial studies, SIAH1 and SIAH2 have been shown to be RING (Really Interesting New
Gene)-finger E3 ligases capable of interacting with a variety of proteins and targeting
them for degradation via the ubiquitin-proteasome pathway.
Knockout mice have been generated for each of the Siah proteins. Knock out of
Siah1a (analogous to knock out of human SIAH1) causes postnatal growth retardation
and premature death, with few surviving beyond 3 months. Surviving males are infertile
and females are subfertile (Dickins et al., 2002). In contrast, Siah2 knockout mice do not
exhibit growth retardation or early mortality and both males and females are fertile, but
have abnormal bone marrow. Knock out of both Siah1 and Siah2 in combination results
in pups dying within hours of birth (Frew et al., 2003).
Both SIAH1 and SIAH2 have a protein-protein interacting domain in the C-
terminal region (amino acids 90-282 for SIAH1; amino acids 116-325 for SIAH2) and
the RING-finger (catalytic domain) in the N-terminal region (amino acids 1-89 for
SIAH1; amino acids 1-115 for SIAH2) (House et al., 2003; Polekhina et al., 2002). It has
been shown that these two domains can be separated, where mutations in the RING-
finger do not abrogate the binding of the proteins to their binding partners. The Siah
proteins have been shown to have auto-ubiquitinating activity and can form homo- and
heterodimers with each other and other E3 ligases (Hu and Fearon, 1999). They mediate a
variety of different pathways by regulating proteins via the ubiquitin-proteasome
pathway. Some of the reported targets of Siah-mediated degradation include
66
adenomatous polyposis coli protein (APC) (Liu et al., 2001), kinesin like DNA binding
protein (Kid) (Germani et al., 2000), synaptophysin (Wheeler et al., 2002), group 1
metabotropic glutamate receptor (Ishikawa et al., 1999), Pard3A (Famulski et al., 2010),
and beta-catenin (Dimitrova et al., 2010). SIAH2 has been reported to interact with Vav,
a GDP-exchange factor for Rac/Rho, but does not cause its degradation. SIAH2 was
shown to negatively regulate Vav-induced JNK activation (Germani et al., 1999). SIAH1
interacts with BAG1, an Hsp70/Hsc70-binding protein that modulates cell proliferation
and death, but does not cause its degradation. In fact, BAG1 inhibits the activity of
SIAH1 in p53-mediated cell cycle arrest (Matsuzawa et al., 1998). Therefore, not all
Siah-binding proteins are targets for Siah-mediated degradation.
In addition to the roles the Siah proteins play in protein-protein interactions and in
Siah-mediated degradation, these proteins have been implicated as players in
neurological disease and in addiction. Specifically, SIAH2 has been demonstrated to be
elevated in the blood of patients with psychosis and this elevation corresponded to greater
symptom severity (Bousman et al., 2010). SIAH1 has been shown to exhibit decreased
expression in the brains of patients with a history of alcohol abuse and/or dependence
although the functional consequence of this change was not pursued (Sokolov et al.,
2003).
The role of ubiquitin has largely been characterized to function as a sorting signal
for lysosomal degradation of GPCRs. Ubiquitin is best known to target agonist-activated
GPCRs to lysosomes for degradation via the endosomal-sorting complex required for
transport (ESCRT) pathway (Marchese et al., 2008). This process is important for
disposing of irreversibly activated receptors and for ridding cells of receptors after
67
chronic agonist stimulation (Marchese et al., 2008; Trejo et al., 1998). Interestingly,
delta opioid receptors are ubiquitinated following agonist stimulation, but ubiquitin is not
required for lysosomal sorting, indicating an additional undiscovered role for ubiquitin in
GPCR regulation (Hislop et al., 2009). More recent studies have demonstrated a role for
ubiquitination in the sorting of MORs from the endosome limiting membrane to the
lumenal compartment, but not in determining the recycling or degradation of the
receptors by the lysosome or proteasome (Hislop et al., 2011).
The identity of the ubiquitin ligases that mediate these responses is largely
unknown for the opioid receptors. However, an E3 ligase, atrophin-interacting protein 4
(AIP4), has been reported for the DOR. AIP4 was shown to control the down-regulation,
measured as loss of ligand binding, of DORs without affecting the delivery of the
receptor to lysosomes. This effect required the direct ubiquitination of receptors (Hislop
et al., 2009). The identity of E3 ligases that regulate either the KOR or the MOR have not
yet been identified.
Ubiquitination can serve as a signal for many cellular processes, including
degradation, trafficking, signal transduction, and endocytosis (Hicke and Dunn, 2003;
Marchese et al., 2008; Myung et al., 2001). Many of these same cellular processes
regulate the MOR and have been shown to play a role in the development of addiction
(Tuoc and Stoykova, 2010). E3 ligases are believed to confer substrate specificity for
ubiquitination; therefore, I investigated the functional role of the interaction between the
MOR and SIAH1. SIAH1 was chosen for further study over SIAH2, due to its reported
role in the ubiquitination and degradation of the metabotrophic glutamate receptor 1
(mGluR1), a GPCR (Moriyoshi et al., 2004). SIAH2 has no reported role in GPCR
68
regulation. Using overexpression and siRNA knockdown experiments, I show that
SIAH1 appears to form an interaction with SIAH2, and that cooperation between the Siah
proteins affects MOR ubiquitination and MOR protein levels.
3.2 Experimental Procedures
3.2.1 Mapping of binding sites
SIAH1 truncation (TR) deletion mutants (SIAH1-TR, residues 91-282; SIAH1-
TR2; residues 91-157, and SIAH1-TR3; residues 151-217) were ligated into pGEX-4T-1
to create GST fusion proteins. The PCR primers used in the cloning of the mutants can be
found in Table 3.1. The MORIL2-S-tag fusion protein was constructed as described in
Chapter 2. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the
ZYP-5052 autoinduction media as described previously (De Cotiis et al., 2008; Studier,
2005). SIAH1 deletion mutant GST fusion proteins were used to pull down S-tagged
MORIL2 using glutathione sepharose beads (GE Healthcare) as previously described
(Lin et al., 2001). Eluted proteins were separated by SDS-PAGE and transferred to
polyvinylidene fluoride (PVDF) filters for western blot analysis. The filter was probed
with a horseradish peroxidase (HRP)-conjugated anti-S-tag antibody (1:5000 dilution,
Novagen, Madison, WI), and immunoreactivity was detected by enhanced
chemiluminescence with an ECL Plus kit (GE Healthcare).
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Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants
This table lists the PCR primers that were used to clone SIAH1 mutants into pGEX-4T-1
to produce GST-fusion proteins for use in GST pull down experiments.
Construct Direction Sequence
SIAH1-TR-GST
Amino Acids 91-282
Forward GGA TCC AAT TCA GTA CTT TTC CCC TGT
AAA
Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC
ATT GAT GCC
SIAH1-TR2-GST
Amino Acids 91-157
Forward GAA TTC AAT TCA GTA CTT TTC CCC TGT
AAA TAT
Reverse CTC GAG TAG GGT TGT AAT GGA CTT ATG
SIAH1-TR3-GST Forward GAA TTC AAG TCC ATT ACA ACC CTA CAG
Amino Acids 151-217 Reverse CTC GAG AAA ATT TTC AGC TTG CTT
3.2.2 Cell culture and transfections
Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG-
tagged mu-opioid receptor (HEK-MOR), delta-opioid receptor (HEK-DOR), or kappa-
opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418. Full-
length SIAH1 and SIAH1 deletion mutant (SIAH1-TR, residues 91-282) were cut from
pGEX-4T-1 with EcoRI and XhoI restriction endonucleases and ligated into pCMV-
tag3B to create myc-tagged constructs. These constructs were transfected into HEK-
MOR cells using Effectene per manufacturer instructions (Qiagen, Valencia, CA).
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3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome
Human embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged
mu-opioid receptor (HEK-MOR) were treated for 30 minutes, 1 hour, 2 hours, and 4
hours with 50 μM cyclohexamide (protein synthesis inhibitor; Sigma) or 30 μM MG132
(proteasome inhibitor; Sigma). HEK-MOR cells were also treated for 30 minutes, 1 hour,
2 hours, 4 hours, and 6 hours with 50 μM of chloroquine (lysosome inhibitor; Sigma).
After the specified time point cells were collected and lysed in lysis buffer (20 mM
Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM sodium fluoride, 5 mM tetrasodium
pyrophosphate, 10mM β-glycerophosphate, 1% NP-40, 1 mM Pefabloc, 1 mM DTT, 1.2
mg/ml protease inhibitors). Crude cell lysates were run on SDS-PAGE and transferred to
PVDF membrane. To visualize the MOR blots were incubated with rabbit anti-FLAG
antibodies (1:500 dilution; Sigma) and goat anti-rabbit HRP-conjugated antibodies
(1:20,000 dilution; Jackson ImmunoResearch).
3.2.4 Detection of ubiquitinated MOR using TUBEs
HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc as
described above. After 24 hours cells were lysed in lysis buffer containing 200 μg/mL of
tandem ubiquitin binding entities fused to GST (TUBEs; Life Sensors, Malvern, PA).
TUBEs contain multiple ubiquitin binding domains, which recognize and bind to
ubiquitin and protect the bound proteins from degradation via the proteasome. Cell lysate
was allowed to nutate for 30 minutes before the addition of glutathione sepharose beads
(Invitrogen, Grand Island, NY). Eluted proteins were resolved on SDS-PAGE and
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transferred to PVDF membrane for western blotting. Blots were probed with rabbit anti-
MOR antibodies (1:5000 dilution AB5511; Millipore, Billerica, MA) and goat anti-rabbit
HRP conjugated antibodies (1:20,000 dilution; Jackson ImmunoResearch).
3.2.5 siRNA knockdown of SIAH proteins
HEK-MOR, HEK-DOR, or HEK-KOR cells were cultured in 100 cm2 plates to
50% confluency and then transfected with 120 pmol of Stealth siRNA for either SIAH1
(5’-GAUGCAUCAGCAUAAGUCCAUUACA-3’) or SIAH2 (5’-
ACUGGGUGAUGAUGCAGUCAUGUUU-3’) (Invitrogen) using RNAiMAX per
manufacturer instructions (Invitrogen). Cells were incubated for 72 hours and then
harvested in lysis buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM
sodium fluoride, 5 mM tetrasodium pyrophosphate, 10mM β-glycerophosphate, 1% NP-
40, 1 mM Pefabloc, 1 mM DTT, 1.2 mg/mL protease inhibitors) with 50μg/mL of
MG132 (Sigma, St. Louis, MO). MG132 was added to prevent protein degradation via
the proteasome during lysis. Western blotting was performed to confirm knockdown of
proteins.
3.2.6 Co-immunoprecipitation and western blot analysis
Crude cell lysate was prepared as described in siRNA knockdown of SIAH
proteins. Protein G magnetic agarose beads (GE Healthcare) incubated with polyclonal
rabbit anti-FLAG antibodies (Sigma) were used to immunoprecipitate receptors from
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HEK-MOR, HEK-DOR, and HEK-KOR cell lysate. Immunoprecipitations (IPs) using
rabbit immunoglobulin g were performed as negative controls. IPs were resolved by
SDS-PAGE and transferred to PVDF membranes. For the detection of ubiquitin, blots
were first treated with 0.5% gluteraldehyde (as recommended by the antibody
manufacturer) (Sigma), then immunoblotted using mouse anti-ubiquitin antibody (1:5000
dilution of VU-1, LifeSensors) and rabbit anti-mouse HRP conjugated secondary
antibody (1:20,000 dilution, Jackson ImmunoResearch). The amount of endogenous
SIAH1 in crude lysate was determined by resolving lysates by SDS-PAGE and
immunoblotting using goat anti-SIAH1 (1:5000 dilution, Abnova, Taipei City, Taiwan)
antibody and bovine anti-goat HRP conjugated secondary antibody (1:20,000 dilution,
Jackson ImmunoResearch). The amount of SIAH2 in crude lysate was determined by
resolving lysates by SDS-PAGE , treating blots with Pierce Western Blot Enhancer Kit
per manufacturer instructions (Pierce), and immunoblotting using goat anti-SIAH2
antibody (1:500 dilution, N-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
bovine anti-goat secondary antibody (1:20,000 dilution, Jackson ImmunoResearch). The
amount of opioid receptors in crude lysate was determined by immunoblotting with rabbit
anti-FLAG antibody (1:5000 dilution, Sigma) and goat anti-rabbit HRP conjugated
antibody (1:20,000 dilution, Jackson ImmunoResearch). To control for loading, blots
were also probed for GAPDH (1:5000 dilution, Sigma) using chicken anti-GAPDH
antibodies and donkey anti-chicken antibody (1:20,000 dilution, Jackson
ImmunoResearch) or tubulin (1:10,000 dilution, Novus) using mouse anti-tubulin
antibodies and goat anti-mouse HRP conjugated antibody (1:20,000 dilution, Jackson
ImmunoResearch).
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3.2.7 Quantitation of Western blots
All blots were run in duplicate or triplicate. Blots were scanned using a back-lit
scanner and quantitation was performed using ImageJ software (Abramoff et al., 2004).
Expression was normalized to total protein for siRNA experiments (as measured by
GAPDH), averaged between replicates, and subjected to a two-tailed Student’s t-test.
3.3 Results
3.3.1 Mapping of the MOR binding site on SIAH1
In Chapter 2, the binding site for SIAH1 on the MOR was found to be the second
intracellular loop. However, the location where MORIL2 binds to SIAH1 has not been
determined. The SIAH1 protein can be divided into an N-terminal region (residues 1-
89), which includes the RING domain (residues 40-75), and the C-terminal region (amino
acids 90-282), which is involved in substrate binding (Hu and Fearon, 1999). The RING
domain is the catalytic domain of the protein and is required for ubiquitination of target
proteins (Hu and Fearon, 1999). I therefore hypothesized that the C-terminal region of
SIAH1 would bind to the second intracellular loop of the MOR. To determine a more
specific binding region, SIAH1 deletion mutants were made in the C-terminal region of
the protein (Figure 3.1B). As expected, the C-terminal region but not the N-terminal
region of SIAH1 was required for SIAH1 to bind to the MOR (Figure 3.1A). Upon the
truncation of the C-terminal region, amino acids 91-157 (SIAH1-TR2-GST) were able to
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pull down MORIL2, while amino acids 151-282 (SIAH1-TR3-GST) were no longer able
to pull down MORIL2 (Figure 3.1A). This indicates that the region of the C-terminus
that includes amino acids 91-157 is required for the interaction between SIAH1 and
MORIL2.
Figure 3.1: Mapping of SIAH1 binding site
(A) Deletion mutants of SIAH1 were used in GST pull down experiments to localize the
MOR binding site on SIAH1. SIAH1-TR-GST (a. a 91-282), SIAH1-TR2-GST (a. a 91-
157), and SIAH1-TR3-GST (a. a 151-217) were used to pull down S-tagged MORIL2.
Pull-down products were purified on glutathione beads, separated by SDS-PAGE, and
probed on western blot using HRP-conjugated anti-S-tag antibodies. S-tagged MORIL
produced in bacteria are shown in the lysate lane (LY), while uncoated glutathione
sepharose beads (BEADS) or GST-coated beads (GST) incubated with S-tagged
MORIL2 served as controls. (B) Map of constructs. The green represents the N-terminal
region, which includes the RING-finger domain in gold (RF). The pink represents the C-
terminal region, which includes the substrate binding domain in pink (SBD).
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3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin
As an E3 ligase, SIAH1 has the ability to conjugate ubiquitin to its binding
partners. I wanted to determine if SIAH1 changes the ubiquitination of the MOR. To
test this, HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc.
SIAH1-TR-myc has the RING domain (catalytic domain) removed and should not be
able to participate in ubiquitination, but retains the ability to bind to the MOR. Tandem
ubiquitin binding entities (TUBEs) fused to GST were used to pull down the total
ubiquitinated protein from cell lysates. HEK-DOR cells were mock transfected and used
as a negative control to demonstrate the specificity of the MOR antibody (Figure 3.2 A).
MOR protein modified with ubiquitin often resolves as a broad high molecular weight
band or smear of 75 -250 kDa on western blot (Groer et al., 2011). Due to this broad
band, quantitation cannot be performed for this kind of analysis and is not performed by
other researchers in the field. In HEK-MOR cells transfected with empty vector, the pull
down lane shows ubiquitinated MOR as an immunoreactive band between 100 and 200
kDa (Figure 3.2, top panel, lane 2). Unexpectedly, when SIAH1 was overexpressed, the
intensity of the immunoreactive band between 100 and 200 kDa decreased slightly
(Figure 3.2 A, lane 4). This indicates that overexpression of SIAH1 may cause a
reduction in the amount of MOR that is ubiquitinated. In contrast, when the RING
mutant was overexpressed, the intensity of this band increased (Figure 3.2 A, lane 6).
To confirm the results seen with the TUBEs, I next immunoprecipitated the MOR
and probed the eluted material for ubiquitin. If SIAH1 is an E3 ligase for the MOR, the
overexpression of SIAH1 would be expected to cause the amount of ubiquitinated MOR
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to increase. Although not as robust as the TUBEs experiment, the amount of MOR that is
ubiquitinated is slightly decreased when SIAH1 is overexpressed as compared to mock
transfected cells (Figure 3.2B, top panel, lanes 3 and 5). Conversely, when the RING
mutant SIAH1 is overexpressed, there is a slight increase in the amount of ubiquitinated
MOR (Figure 3.2B, top panel, lane 7). Without the ability to quantitate these results, it is
difficult to determine the significance of the effect of SIAH1 on ubiquitination of the
MOR. However, SIAH1-TR overexpression did cause a noticeable increase in the
ubiquitination of the MOR in the TUBEs experiment, which indicates that the RING-
finger is not responsible for the increase in the observed ubiquitination. Therefore,
SIAH1 is likely not an E3 ligase for MOR.
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Figure 3.2: SIAH1 decreases the ubiquitination of the MOR
(A) HEK-MOR cells were transfected with empty vector (pCMV-tag3B), SIAH1 with a
myc tag (SIAH1-myc), SIAH1 RING mutant with a myc tag (SIAH1-TR-myc). The
amount of MOR eluted from the beads was determined by immunoblotting with anti-
MOR antibodies and is shown in the IP lanes. HEK-DOR cells were used as a control to
show that the TUBEs do not cause non-specific banding patterns. (B) HEK-MOR cells
were transfected as in (A) and cell lysate was subjected to immunoprecipitation with anti-
FLAG antibodies to IP the MOR and then immunoblotted with mouse anti-ubiquitin
antibodies. IPs using rabbit IgG was used as a negative control (M). Crude cell lysates
were immunoblotted to show that the constructs were expressed in the cells used in pull
down or immunoprecipitation experiments (Bottom panels A and B). Blots are
representative of three separate experiments.
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3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid receptors
Since the overexpression of SIAH1 caused a decrease in the amount of detectable
ubiquitinated MOR, I expected that knocking down endogenous SIAH1 would result in
increased ubiquitination of MOR. HEK-MOR cells were transfected with siRNA against
SIAH1. Crude cell lysate was immunoblotted to detect the amount of SIAH1 and
GAPDH (Figure 3.3B). Knockdown of SIAH1 resulted in an 80% reduction in the
amount of endogenous Siah1 protein (Figure 3.3C). To determine the ubiquitination
level of the MOR, MOR was immunoprecipitated from lysates of cells that were
transfected with scrambled or SIAH1 siRNA, subjected to SDS-PAGE, and
immunoblotted with anti-ubiquitin antibodies. As expected, Siah1 knockdown resulted in
an increase in the amount of detectable ubiquitinated MOR (Figure 3.3A). These results
are consistent with observations made by overexpression of SIAH1-TR-myc, which
suggests that SIAH1-TR-myc acts as a dominant negative mutant. It is possible that this
assay is not measuring MOR ubiquitination, but the ubiquitination of another MORIP.
This assay is indirect as any protein that immunoprecipitates with the MOR and is also
ubiquitinated may be observed on the blot. However, this assay can be used to determine
whether or a particular protein is a putative E3 ligase or not. If SIAH1 is an E3 ligase for
the MOR, its knockdown should cause the ubiquitination of the MOR to decrease, not
increase. Therefore, SIAH1 most likely is not an E3 ligase for the MOR.
The opioid receptors show considerable similarity in the second intracellular loop
(Chapter 2, Figure 2.3A) and SIAH1 has been shown to co-immunoprecipitate with all
three receptors (Chapter 2, Figure 2.3B). Therefore, it seems plausible that SIAH1 might
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affect the ubiquitination of DOR and/or KOR. To investigate this possibility, siRNA
against SIAH1 was transfected into HEK-DOR cells and HEK-KOR cells. Siah1 protein
was knocked down by 94% in HEK-DOR cells and by 82% in HEK-KOR cells (Figures
3.3B and 3.3C). As with the MOR, knockdown of SIAH1 caused an increase in the
detectable ubiquitination of DOR and KOR (Figure 3.3A). These results indicate that
SIAH1 is likely not an E3 ligase for either DOR or KOR.
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Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and
KOR
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH1 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors
were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with
mouse anti-ubiquitin. The ubiquitinated opioid receptors yield immunoreactive smears
between 75-200 kDa. IPs using normal rabbit IgG were used as a negative control
(Mock). (B) Western blots showing the amount of SIAH1 in lysate was used to confirm
the knockdown, while GAPDH was used as a loading control. (C) Western blot bands
were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a
paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
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3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but increased
ubiquitination of DOR and KOR.
Another E3 ligase, SIAH2, was found to interact with all three opioid receptors
(Chapter 2). Since SIAH1 did not seem to be a candidate E3 ligase for any of the opioid
receptors, I wanted to determine if SIAH2 had any effect on the ubiquitination of the
opioid receptors. To test this HEK-MOR, HEK-DOR, and HEK-KOR cells were
transfected with siRNA targeting SIAH2.
Crude cell lysate was immunoblotted to detect the amount of SIAH2, and
GAPDH (Figure 3.4B). Knockdown of SIAH2 resulted in a 98% reduction in the amount
of endogenous Siah2 protein (Figure 3.4C). To determine the ubiquitination level of the
MOR, MOR was immunoprecipitated from lysates of cells that were transfected with
scrambled or SIAH2 siRNA, subjected to SDS-PAGE, and immunoblotted for ubiquitin.
SIAH2 knockdown resulted in a slight decrease in the amount of detectable ubiquitinated
MOR (Figure 3.4A). These results are more consistent with SIAH2 being an E3 ligase
involved in MOR ubiquitination because the ubiquitination of MOR would be expected
to decrease. Therefore, SIAH2 may be a putative E3 ligase of the MOR.
Siah2 protein was knocked down by 96% in HEK-DOR cells and 97% in HEK-
KOR cells (Figuress 3.4B and 3.4C). In contrast to the MOR, knockdown of SIAH2
caused an increase in the detectable ubiquitination of DOR and KOR (Figure 3.4A). If
SIAH2 is an E3 ligase for the DOR and/or KOR, the knockdown would be expected to
result in a decrease in the ubiquitination of DOR. Therefore, these results indicate that
SIAH2 is most likely not an E3 ligase for this receptor. However, if the results of SIAH1
and SIAH2 knockdown in HEK-DOR and HEK-KOR cells are considered together, they
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suggest that the Siah proteins may compensate for each other and therefore, cannot be
ruled out as putative E3 ligases for these receptors.
Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases
ubiquitination of DOR and KOR
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH2 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors
were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with
mouse anti-ubiquitin. IPs using rabbit IgG were used as negative controls (Mock). The
ubiquitinated opioid receptors yield immunoreactive smears between 75-200 kDa. (B)
Western blots showing the amount of SIAH2 in cell lysate was used to confirm the
knockdown, while GAPDH was used as a loading control. (C) Western blot bands were
quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired
Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
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3.3.5 The MOR undergoes degradation via the proteasome and lysosome
The degradation of the MOR has been demonstrated to occur primarily through
the lysosomal pathway, but has also been demonstrated to occur via the proteasome
pathway in overexpression cell culture systems. However, both pathways have been
observed in to occur in overexpression cell culture systems (Chaturvedi et al., 2001;
Hislop and von Zastrow, 2011). Since E3 ligases can target proteins to either pathway in
the HEK-MOR cells, I wanted to determine if the MOR was capable of undergoing
degradation via the proteasome and/or the lysosome. This was tested by treating HEK-
MOR cells with cyclohexamine, MG132, or chloroquine for varying amounts of time. A
protein that undergoes proteasomal degradation should accumulate in the cell as the
length of proteasome inhibitor, MG132, exposure increases. Likewise, a protein that
undergoes lysosomal degradation should accumulate in the cell as the length of lysosomal
inhibitor, chloroquine, exposure increases. Cyclohexamide, a protein synthesis inhibitor,
was used to determine the half-life of the MOR. The levels of a protein that are
degraded (lysosomal or proteasomal) will decrease in the cell overtime if protein
synthesis is inhibited. The MOR has previously been shown to resolve on Western blot
as two major bands, one at 50 kDa and one at 75 kDa due to varying amounts of
glycosylation (Huang and Liu-Chen, 2009). Interestingly, only the low molecular weight
(50 kDa band) form of the MOR was observed to change with either cyclohexamide
(Figure 3.5A) or MG132 (Figure 3.5B) treatment. The half-life of the low molecular
MOR band is short, as this band is no longer detectable after 1 hour of cyclohexamide
treatment (Figure 3.5A; 50 kDa band). This result suggests that the low molecular weight
84
form of MOR is either subject to degradation or further post-translational modification
(perhaps maturing into high molecular weight form of MOR). It is likely that a portion
of low molecular weight MOR is subject to proteasomal degradation because the levels
of this protein species increased with lengthening MG132 exposure (Figure 3.5B; 50 kDa
band). The high molecular weight form of the MOR (75 kDa band) did not change with
either cyclohexamide or MG132 treatment (Figures 3.5A and B) indicating that this
protein species is stable and not degraded within the time points measured. Therefore, no
conclusions on the involvement of the proteasome in degradation of the high molecular
weight form of MOR can be made from this experiment.
Contrary to the results seen with the proteasome block, both the low molecular
weight band (50 kDa band) and the high molecular weight band (75 kDa band) of MOR
increased over time with chloroquine treatment (Figure 3.5C). Thus, it appears that
lysosomal degradation has a more prominent role than proteasomal degradation for the
MOR.
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Figure 3.5: Inhibitor treatment of HEK-MOR cells
HEK-MOR cells were treated with (A) 50 μM cyclohexamide, a protein synthesis
inhibitor, (B) 30 μM MG132, a proteasome inhibitor, or (C) 50 μM of chloroquine, a
lysosomal inhibitor for 30 minutes, 1 hour, 2 hours, and 4 hours. Cells treated with
chloroquine were also treated for 6 hours. Cells were treated with vehicle (0) as a
control. After treatment cells were lysed and crude cell lysate was resolved. Blots are
representative of three individual experiments.
3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels
Since both the proteasomal and lysosomal degradation pathways can be mediated
by ubiquitination, I wanted to determine the effects of overexpressing SIAH1 on MOR
protein levels in HEK-MOR cells. Although it appears that SIAH1 does not ubiquitinate
the MOR, it may still play a role in its degradation. If SIAH1 is involved in MOR
degradation by either pathway, overexpression of the protein would be expected to cause
the amounts of detectable MOR to decrease. HEK-MOR cells were transfected with full-
length SIAH1 fused to myc using increasing microgram concentrations of the SIAH1-
myc construct or with vector as a negative control. Transfecting increasing amounts of
SIAH1-myc resulted in decreasing amounts of both the low molecular weight (50 kDa
86
band) and the high molecular weight (75 kDa band) species of MOR protein with
increasing concentrations of SIAH1-myc (Figure 3.6). These results indicate that SIAH1
may regulate MOR protein levels in mammalian cells.
Figure 3.6: SIAH1 induces MOR degradation
HEK-MOR cells were transfected with increasing amounts of SIAH1-myc DNA. Vector
DNA (V) was added to make the total amounts of DNA transfected equal. 2.0 μg of
vector DNA was transfected as a negative control. The amount of MOR-FLAG, SIAH1-
myc, and GAPDH in cell lysates was determined by immunoblotting with anti-FLAG,
anti-myc, and anti-GAPDH antibodies, respectively. GAPDH was used as a loading
control. Blot images are representative of three experiments.
3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but decreases DOR and
KOR protein
Since the overexpression of SIAH1 caused a decrease in MOR protein levels, I
expected that knocking down endogenous SIAH1 would result in increased MOR protein
levels. To test this, siRNA against SIAH1 was transfected into HEK-MOR cells. The
reduction in Siah1 protein levels caused a 16-fold increase in the amount of low
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molecular weight MOR (50 kDa band) (Figures 3.7A and D), while no significant change
was detected for the high molecular weight version of MOR (75 kDa band) (Figures 3.7A
and 3.7C). This suggests that SIAH1 may play a role in the regulation of MOR protein
levels.
Since SIAH1 had been shown to co-immunoprecipitate with all three receptors,
and knockdown of SIAH1 caused an increase in ubiquitination of the DOR and KOR, it
seems possible that SIAH1 might affect DOR and/or KOR protein levels. To investigate
this possibility, siRNA against SIAH1 was transfected into HEK-DOR cells and HEK-
KOR cells. In contrast to the results for MOR, the levels of both the DOR low molecular
weight protein (45 kDa band) (Figures 3.7A and D) and the high molecular weight DOR
(55 kDa band) decreased with knockdown of SIAH1 (Figuress 3.7A and 3.7C). The
DOR is also differentially glycosylated and the observation of multiple bands is due to
this modification (Petaja-Repo et al., 2000). The levels of KOR low molecular weight
protein (45 kDa) (Figuress 3.7A and 3.7D) and high molecular weight protein (55 kDa)
(Figures 3.7A and 3.7C) also decreased, as was the case with DOR. Like the MOR and
the DOR these differences in molecular weight are due to glycosylation (Chen et al.,
2006). Therefore, these results indicate that SIAH1 may play a role in the regulation of
DOR and KOR protein levels.
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Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of
receptor in cell lysate. (B) Western blot showing the levels of SIAH1 protein to confirm
the knockdown. GAPDH was used as a loading control. (C) High molecular weight
bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data
was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
(D) Low molecular weight bands on Western blots were quantitated as in (C).
89
3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but increases DOR and
KOR protein
Since SIAH2 knockdown resulted in decreased ubiquitination of the MOR, I
wanted to determine if SIAH2 was involved in degradation of the receptor. To test this,
HEK-MOR cells were transfected with siRNA targeting SIAH2. The reduction in Siah2
protein levels caused a significant decrease in the amount of both the low molecular
weight MOR (band of 50 kDa) (Figures 3.8A and 3.8D) and the high molecular weight
MOR (band of 75 kDa) (Figures 3.8A and 3.8C). However, if SIAH2 is the E3 ligase
that targets the MOR for degradation, the knockdown of Siah2 protein should result in an
increase in MOR protein levels. Therefore, SIAH2 may be a putative E3 ligase of the
MOR, but its tagging of the MOR with ubiquitin may not result in degradation of the
MOR.
In contrast to the results for MOR, the ubiquitination of DOR and KOR increased
with SIAH2 knockdown. Therefore, I wanted to determine if this ubiquitination lead to
degradation of these receptors. Knockdown of Siah2 protein, caused the levels of the
DOR low molecular weight protein (45 kDa band) (Figures 3.8A and 3.8D) to increase
significantly, but did not significantly change the high molecular weight DOR (55 kDa
band) protein levels (Figures 3.8A and3.8C). Contrary to the results seen in HEK-DOR
and HEK-MOR cells, the levels of the KOR low molecular weight protein (45 kDa band)
(Figures 3.8A and 3.8D) and the high molecular weight KOR (55 kDa band) both
significantly increased with knockdown of SIAH2 (Figures 3.8A and 3.8C). Together
these results suggest that SIAH2 may play a role in the degradation of DOR and KOR.
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Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of
receptor in cell lysate. (B) Western blots showing the levels of SIAH1 protein to confirm
the knockdown. GAPDH was used as a loading control. (C) High molecular weight
bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data
was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
(D) Low molecular weight bands on Western blots were quantitated as in (C).
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3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells
If SIAH1 is an E3 ligase for the MOR, its overexpression would be expected to
cause an increase in the amount of ubiquitinated receptor; however, this was not the case.
In Chapter 2, SIAH2 was also identified as an interactor with the MOR. The Siah1 and
Siah2 proteins share more than 85% amino acid identity in the C-terminal region (amino
acids 80-324) and 77% overall identity. Essentially, they only differ from one another in
the length and sequence of the extreme N-terminus, which includes a portion of the ring
domain (Hu et al., 1997b). Therefore, the antibodies used in this study were to epitopes
in the N-terminus of the proteins. There is evidence that SIAH1 and SIAH2 can form
heterodimers with each other and in some cases regulate each other’s function (Depaux et
al., 2006; Hu and Fearon, 1999). Additionally, the Siah proteins have been shown to
interact with some of the same proteins, such as ALL-1 fused gene on chromosome 4
(Af4) (Bursen et al., 2004; Oliver et al., 2004), phospholipase C eplison (Yun et al.,
2008) and alpha synuclein (Szargel et al., 2009). Since SIAH2 was also found to be a
MOR interactor, I wanted to determine if SIAH1 and SIAH2 interact in HEK-MOR cells
as this may affect ubiquitination and degradation studies of MOR. Due to the lack of
commercial antibodies that can immunoprecipitate either Siah protein, HEK-MOR cells
were transfected with SIAH2-myc. Anti-myc antibodies were used to immunoprecipitate
SIAH2-myc and anti-SIAH1 antibodies were used to detect SIAH1 in eluted proteins via
western blot. As shown in the IP lane of Figure 3.9, SIAH2-myc was able to co-
immunoprecipitate with SIAH1. These results indicate that SIAH1 and SIAH2 may
interact with each other in HEK-MOR cells.
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Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2
HEK-MOR cells were transfected with SIAH2-myc. Myc-tagged SIAH2 was
immunoprecipitated using mouse anti-myc antibodies. Mock immunoprecipitations were
performed with Protein-G beads coated with normal rabbit IgG. Blot was probed with
goat anti-SIAH1 antibodies. Lysate lane contains 5% of the total protein compared to the
mock (M) and immunoprecipitation lands (IP).
3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells
After determining that SIAH1 and SIAH2 can interact in HEK-MOR cells, I
wanted to determine if either Siah protein had an effect on the protein level of the other.
Knockdown of SIAH1 resulted in a two-fold increase in Siah2 protein in HEK-MOR,
HEK-DOR, and HEK-KOR cells (Figures 3.10A and 3.10B). This result was somewhat
unexpected, as SIAH2 has been shown to be capable of regulating SIAH1 levels, but the
opposite has not (Depaux et al., 2006). However, my results suggest that SIAH1
regulates SIAH2 levels.
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Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH1 (to
confirm knockdown), SIAH2, and GAPDH (loading control). (B) Western blots were
quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired
Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
Although SIAH1 appears to regulate the levels of SIAH2 protein, the knockdown
of SIAH2 did not result in an increase in SIAH1 protein levels in any of the cell lines
tested (Figures 3.11A and 3.11B). Again this was unexpected as SIAH2 has been shown
to regulate SIAH1 levels (Depaux et al., 2006). However, it does not appear that SIAH2
regulates the protein level of SIAH1.
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Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels
HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against
SIAH2 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH2 (to
confirm knockdown), SIAH1, and GAPDH (loading control). (B) Western blots were
quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired
Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the ubiquitination of
the MOR and an increase in MOR protein levels
Knockdown of an E3 ligase involved in the ubiquitination and subsequent
degradation of the MOR would be expected to cause both a decrease in ubiquitination of
the MOR and an increase in MOR protein levels. Since neither SIAH1 nor SIAH2
knockdown alone had that effect on the MOR, I wanted to determine the effect of a
double knockdown on protein levels and ubiquitination. HEK-MOR cells were
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transfected with siRNA targeting SIAH1 and SIAH2 in combination. There were
difficulties in knocking both proteins in combination; therefore the experiments were
performed in duplicate and no statistical analysis was performed. However, it is clear
that SIAH1 protein was knocked down, as was SIAH2 (Figure 3.12B). Due to the lack of
replicates, the effect of the double knockdown on MOR protein levels cannot be
determined. Interestingly, the detection of ubiquitinated MOR was abolished when both
SIAH1 and SIAH2 were knocked down in combination (Figure 3.12A). When these
results are considered with the results of the knockdown of the Siah proteins singly, they
indicate that the Siah proteins may cooperate to regulate the levels of MOR protein and
the ubiquitination the MOR.
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Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation
HEK-MOR cells transfected with siRNA against SIAH1 and SIAH2 in combination
(DKD) or scrambled siRNA as a control (SC). (A) Crude cell lysate was
immunoprecipitated with rabbit anti-FLAG antibodies and probed with mouse anti-
ubiquitin antibodies to detect ubiquitinated MOR. (B) Cell lysate was immunoblotted
with rabbit anti-FLAG antibodies, goat anti-SIAH1 and goat anti-SIAH2 to detect MOR,
SIAH1, and SIAH2, respectively. GAPDH was used as a loading control.
3.4 Discussion
The functional relevance of the SIAH1/MOR interaction was investigated using a
series of experiments designed to test the hypothesis that SIAH1 is the E3 ligase that
mediates the ubiquitination and subsequent degradation of the MOR. If this hypothesis is
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correct, overexpression of SIAH1 should result in a decrease in MOR protein levels and
MOR ubiquitination. In line with this hypothesis, when myc-tagged SIAH1 was
transfected in increasing concentrations, the amount of MOR decreased. However, this
observed decrease was found to be different for the two species of MOR detected on
Western blots. The high molecular weight species (band of 75 kDa) was not as sensitive
to the effect of SIAH1 overexpession as the low molecular weight species (band of 50
kDa). These differences in molecular weight have been reported to be attributable to the
amount of N-linked glycosylation present on the receptor as the predicted size of the
unmodified MOR protein is approximately 43 kDa. These two species of the receptor
correspond to highly glycosylated plasma membrane associated receptors (60-100 kDa)
and slightly glycosylated or unglycosylated intracellularly associated (40-60 kDa)
receptors (Huang and Liu-Chen, 2009). These results indicate that SIAH1 may be acting
on the immature form of the MOR, possibly at the endoplasmic reticulum or some other
intracellular location.
Treatment of HEK-MOR cells with the proteasome inhibitor, MG132, shows that
the intracellular MOR (low molecular weight, 50 kDa band) is sensitive to degradation
via the proteasome, but that the plasma membrane MOR is not (high molecular weight,
75 kDa band) within the time frame tested. These results are consistent with recent
evidence showing that the plasma membrane MOR is degraded via the lysosome and that
ubiquitination does not serve as a signal for degradation via the proteasome (Hislop et al.,
2011). When HEK-MOR cells were treated with the lysosomal inhibitor, chloroquine,
both species of the receptor increased over time. Taken together, these results suggest
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that the immature, intracellular form of the MOR is sensitive to proteasomal degradation
and that this degradation may be mediated by SIAH1.
Contrary to the proposed hypothesis, overexpression of myc-tagged SIAH1
caused a decrease in the amount of ubiquitinated MOR in both the TUBEs experiments
and MOR immunoprecipitation experiments, while overexpression of a RING-deficient
mutant (SIAH1-TR-myc) caused an increase in detectable ubiquitinated MOR. Although
these results are contradictory to the hypothesis that SIAH1 is an E3 ligase for MOR,
they are confirmed by the SIAH1 siRNA knockdown experiments in HEK-MOR cells
and suggest that the RING domain is not required for this increase in ubiquitination of the
MOR. A summary of results from the overexpression and siRNA knockdown
experiments is shown in Table 3.2. The siRNA knockdown of SIAH1 and the
overexpression of myc-tagged RING-deficient SIAH1 show similar results which is
consistent with the previous observation that the RING domain mutant functions as
dominant negative (Hu and Fearon, 1999). Both the knockdown of SIAH1 and the
overexpression of SIAH1-TR-myc resulted in an increase in the amount of ubiquitinated
MOR, which is not in line with SIAH1 playing a role as an E3 ligase which conjugates
ubiquitin to the MOR.
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Table 3.2: Summary of SIAH overexpression and knockdown experiments
For experiment column, upward arrows indicate overexpression, whereas downward
arrows indicate siRNA knockdown. For effect on Siah proteins, ubiquitination, and
protein levels, upward arrows indicate an increase and downward arrows indicate a
decrease. ND indicates that the experiment was not performed.
Cell Line Experiment Effect on Siah
proteins
Effect on
ubiquitination
Effect on
protein levels
HEK-MOR SIAH1 ↑ ND ↓ Both species ↓
HEK-MOR SIAH1-TR ↑ ND ↑ ND
HEK-MOR SIAH1↓ SIAH2 ↑ ↑ Low species ↑
HEK-MOR SIAH2 ↓ No change in
SIAH1
↓ Both species ↓
HEK-MOR SIAH1 ↓ and
SIAH2 ↓
Not applicable ↓ Low species ↑
HEK-DOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓
HEK-DOR SIAH2 ↓ No change in
SIAH1
↑ Low species ↑
HEK-KOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓
HEK-KOR SIAH2 ↓ No change in
SIAH1
↑ Both species ↑
The knockdown of SIAH1 caused a significant increase in the amount of another
MORIP, SIAH2, which is an E3 ligase that can interact with SIAH1 and MOR in these
cells. One could argue that the results obtained in the SIAH1 knockdowns could be due
to the upregulation of SIAH2. This is partially supported by the observation that MOR
ubiquitination and protein expression levels are reduced in SIAH2 knockdowns in HEK-
MOR cells, a result that is opposite from those obtained from SIAH1 knockdown.
However, when both SIAH1 and SIAH2 were knocked down in combination in HEK-
MOR cells, the amount of detectable ubiquitinated MOR was abolished.
In summary, the results obtained from the knockdown of either SIAH1 and
SIAH2 alone are not consistent with the hypothesis that one of these individually acts as
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both an E3 ligase and an inducer of degradation for the MOR. On the contrary, a
combinatorial knockdown of SIAH1 and SIAH2 did provide results that would be
consistent with the hypothesis that these two proteins may work in concert to ubiquitinate
MOR. Unfortunately, no conclusions can be drawn about the effect of the double
knockdown on the levels of MOR protein as not enough replicates were able to be used
for statistical analysis.
The effect of Siah protein knockdown on the amount of low and high molecular
MOR species is unexpected. SIAH1 knockdown caused the amount of the low molecular
weight (50 kDa band), intracellular MOR to increase significantly but did not
significantly affect the high molecular weight (75 kDa band), plasma membrane-
associated form of MOR (Huang and Liu-Chen, 2009). This suggests that SIAH1 may
have a role in the regulation of intracellular MOR protein levels, but does not
significantly affect MOR at the plasma membrane. In contrast, SIAH2 knockdown
caused a decrease in both species. The knockdown of SIAH2 may allow the activity of
SIAH1 to increase, but this increased activity is not due to increases in Siah1 protein
levels, as the amount of Siah1 protein did not increase as shown via Western blot
analysis. Alternatively, SIAH2 may regulate another protein that is involved in
regulation of MOR, resulting in decreased levels of MOR protein. However, the results
of the double knock down experiments indicate that the Siah proteins are working
together, as there is a reduction in the ubiquitination of MOR. These results also indicate
that SIAH2 may participate in ubiquitination of the MOR, but that SIAH1 is required for
degradation of the receptor. Models for the role of the Siah proteins in the ubiquitination
and regulation of the MOR are discussed in more detail in Chapter 5.
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Knockdown of either SIAH1 or SIAH2 in HEK-DOR or HEK-KOR cells resulted
in an increase in the ubiquitination of DOR or KOR, respectively. These results may
indicate that both Siah proteins play a role in the ubiquitination of DOR and KOR.
Additionally, SIAH1 knockdown caused a decrease in the amount both low and high
molecular weight DOR (45 kDa and 55 kDa bands) and KOR (45 kDa and 55 kDa
bands). While SIAH2 knock down caused significant increase in the amount of low
molecular weight DOR both species of KOR were increased. As has been demonstrated
with the MOR, the DOR and KOR also exist as differentially glycosylated species. For
the DOR and KOR, the 45 kDa protein has been shown to remain in an intracellular
compartment and is not transported to the cell surface (Chen et al., 2006b; Petaja-Repo et
al., 2000), whereas the 55 kDa protein is the fully mature receptor found at the plasma
membrane (Petaja-Repo et al., 2001). Without performing a double knockdown of
SIAH1 and SIAH2 in these cell lines, there are several interpretations that fit the data
collected from knockdown of SIAH1 or SIAH2 in DOR-HEK and KOR-HEK cells.
These interpretations are discussed in more detail in Chapter 5. One explanation is that
SIAH1 and SIAH2 could possibly have an overlapping role in receptor ubiquitination.
Knockdown of either protein could result in upregulation or overactivation of the other
which would lead to the observed increase in receptor uiquitination. Indeed as with all
cell lines observed knockdown of SIAH1 caused an increase in SIAH2 levels. Another
possibility is that another unknown protein is involved in the ubiquitination of DOR and
KOR and that the SIAH proteins play a role in regulating this unknown protein. Double
knockdowns of both Siah proteins were attempted for both DOR and KOR cell lines, but
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were unsuccessful. Optimization of the double siRNA transfection in these cell lines may
clarify the role these E3 ligases play in the MOR life cycle.
These interactions between the Siah proteins and the opioid receptors are
surprising considering that opioid receptors are typically downregulated by lysosomal
degradation and not the proteasome (Marchese et al., 2008). However, ubiquitination of
plasma membrane-bound MOR does occur in response to receptor activation and is
required for trafficking events that downregulate receptor ligand binding (Hislop et al.,
2011). This is the first study to address basal (unstimulated) ubiquitination of the MOR.
My results indicate that SIAH1 and SIAH2 may play a role in the ubiquitination the
intracellular form of the MOR under unstimulated conditions. The exact location of the
the low molecular weight, intracellular MOR is not well established, but it is suspected
that some resides within the biosynthetic pathway, possibly in the endoplasmic reticulum
(ER) (Huang and Liu-Chen, 2009). As a glycoprotein, the MOR must be properly folded
and modified within the ER before being translocated to the plasma membrane. If the
MOR misfolds, it is subjected to endoplasmic reticulum-associated degradation (ERAD).
ERAD requires poly-ubiquitination and retro-translocation of the misfolded protein from
the ER lumen to the ER membrane to the cytosol. Once in the cytosol, these proteins are
subjected to degradation via the proteasome (Feldman and van der Goot, 2009).
Interestingly, misfolded delta opioid receptors have been shown to be transported to the
cytoplasmic side of the ER membrane, where they are deglycosylated and conjugated
with ubiquitin before being degraded by the proteasome (Petaja-Repo et al., 2001). It
seems reasonable to assume that the other opioid receptors may be treated similarly. If
Siah proteins are involved in the biosynthesis of MORs, then they may regulate the
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number and activity of opioid receptors at the cell surface, which is important in the
modulation downstream signaling (Chen et al., 2006b; Petaja-Repo et al., 2000).
Presently, the precise intracellular locations of the Siah proteins are not known.
Evidence continues to mount suggesting that the ubiquitin pathway is involved in
a variety of neuronal activities, including dendritic morphogenesis and synaptic plasticity
which have been implicated in the development of addiction (Robinson and Kolb, 2004;
Tuoc and Stoykova, 2010; Yang et al., 2008). Thus, it will be interesting to determine
how the SIAH proteins regulate the opioid receptors in cells where the both the receptors
and the SIAH proteins are expressed at endogenous levels. Additionally, tissue specific
or conditional knockouts of SIAH1 or SIAH2 could be tested in addiction paradigms to
determine whether this regulation plays a role in the neuronal changes observed in
addiction.
Chapter 4
Effect of Opioid Exposure on MORIPs
4.1 Introduction
The mu-opioid receptor (MOR), which belongs to the Class A family of G protein
coupled receptors (GPCRs), mediates a variety of functions in the body, including
modulation of pain perception, cell proliferation, cell survival, neural development, and
synaptic plasticity (Christie, 2008; Tegeder and Geisslinger, 2004). This receptor couples
preferentially to Gi/o types of G proteins and therefore inhibits adenylyl cyclase activity
and regulates a number of other signaling intermediates such as mitogen activated kinase
(MAPK), phospholipase C (PLC), and various ion channels. The MOR is activated by
endogenous and exogenous opioids, the latter of which include some of the most
effective analgesics, but also the most highly addictive drugs of abuse.
Abuse of drugs or addiction is an abnormal behavior characterized by compulsive,
out-of-control drug use despite negative consequences. The development of these
behavioral abnormalities occurs gradually and progressively during repeated exposure to
the abused drug. In some cases, these abnormalities can persist for a long period of time
after the cessation of use (Nestler, 2004). Thus, drug addiction is seen as a form of drug-
induced plasticity (Russo et al., 2010). Multiple studies have shown that repeated
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exposure to an addictive drug can alter the types or the levels of genes/proteins expressed
in the brain. In addition, these alterations mediate the function of individual neurons and
related neural circuits which may be responsible for the observed behavioral
abnormalities seen in addicted individuals (Nestler, 2001). Therefore, the identification
of the genes/proteins associated with drug dependence is vital to understanding the
molecular mechanisms underlying addiction.
Many different techniques have been utilized to investigate the gene/protein
expression changes associated with drug exposure and addiction. Proteomic approaches
allow for the study of these changes without the need for a prior hypothesis. Since drug
exposure and addiction involve a large array of interacting proteins, it is well suited for
this type of analysis. However, the lack of a hypothesis, also presents problems. Without
a hypothesis, any found changes have little meaning unless further studies are
implemented. One reason for this is that changes observed in the proteome may not be a
direct consequence of the drug itself, but could represent downstream changes that are
caused by but are far removed from the cellular pathways that are directly affected. For
example, a drug might act directly on the mesolimbic dopaminergic tract. Changes in
proteins in the prefrontal cortex may result as an indirect effect of altered signaling from
the mesolimbic pathway. While this might give you information about the disease state
overall, it provides little evidence as to how the drug itself is working on its receptors.
Another approach is to use a candidate gene(s) approach, where genes/proteins with
known functions or interactions associated with genes or proteins that have already been
shown to be involved in drug exposure or abuse are interrogated for expression changes
during drug exposure or in addiction models.
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The first proteins shown to interact functionally with the MOR were G proteins,
which modulate canonical G protein-dependent signaling. However, it is now clear that
modulation of MOR signaling and regulation are far more complex (Georgoussi et al.,
2012). Using approaches such as yeast-two hybrid screens, immunoprecipitation,
immunofluorescence, or bioluminescence resonance energy transfer in live cells, it has
been shown that a diverse group of other interacting partners can participate in the
regulation of virtually every aspect of MOR activity. Observations from these studies
have indicated that the MOR can form mulitcomponent protein assemblies, or
signalplexes, involving interactions between themselves, other GPCRs, and with other
interacting proteins. These interactions likely contribute to the process of signaling
downstream of the receptor (Alfaras-Melainis et al., 2009; Georgoussi et al., 2012;
Kabbani et al., 2002; Milligan, 2005).
Few previously identified mu-opioid receptor interacting proteins have been
investigated with regards to altered expression when cells or animals are treated with
opioid drugs. Spinophilin was found to have decreased expression with acute but not
with chronic exposure to morphine in the nucleus accumbens of mice. Additionally,
spinophilin knockout mice exhibited decreased morphine analgesia, but increased
tolerance, physical dependence, and place conditioning to morphine (Charlton et al.,
2008). Acute morphine administration has been demonstrated to cause an increase in
RGS9-2 protein levels in the nucleus accumbens and spinal cord of C57BL/6 mice.
However, mice chronically treated with morphine, showed decreases in RGS9-2 proteins
levels in the same brain regions (Zachariou et al., 2003). These studies suggest that
MORIP expression levels can change with drug exposure and that these changes are
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likely to have functional consequences, which may contribute to the development of
addiction. Therefore, investigation of MORIP expression upon drug exposure may lead
to the discovery of additional molecular components that may be involved in the
development of the addicted state.
In this chapter, we examine the effect of drug exposure on the SIAH1/MOR
interaction and the expression of selected MORIPs in specific brain regions of morphine
treated mice. We demonstrate that the SIAH1/MOR interaction is altered in response to
morphine and that changes in the expression of several MORIPs may be correlated with
morphine exposure.
4.2 Experimental Procedures
4.2.1 Cell culture and drug treatment
Human neuroblastoma cells (SYH5Y) were cultured in Dulbecco's Modified
Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human
embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged mu-opioid
receptor (HEK-MOR), FLAG-tagged delta-opioid receptor (HEK-DOR), or FLAG-
tagged kappa-opioid receptor (HEK-KOR) were maintained in DMEM supplemented
with 10% FBS and 400 μg/mL G418. HEK-MOR and HEK-DOR cell lines were
generously provided by Dr. Mark van Zastrow (University of California San Francisco).
HEK-KOR cells were a gift from Dr. Lee-Yuan Liu-Chen (Temple University School of
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Medicine). For drug treatment, either 10 μM morphine or 10 μM [D-Ala2, NMe-Phe
4,
Gly-ol5] (DAMGO) was added to cell culture media and cells were incubated for
specified time periods.
4.2.2 Co-immunoprecipitation and Western blot analysis
Human neuroblastoma cells (SHSY-5Y) were exposed to either 10 μM morphine
or 10 μM DAMGO for 30 minutes and 1 hour, then lysed in 1X lysis buffer (20 mM Tris-
HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium
pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100 and
1mg/mL leupeptin) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL).
The MOR was immunoprecipitated from total cell lysates using a polyclonal rabbit anti-
MOR antibody (AB 1580, Millipore, Billerica, MA) coupled to Protein-G Dynabeads
(Invitrogen, Grand Island, NY). Human embryonic kidney cells (HEK) stably transfected
with FLAG-tagged MOR, DOR, or KOR (HEK-MOR, HEK-DOR, HEK-KOR,
respectively) were treated for 1 hour and 21 hours with 10μM morphine. Cells were
collected and lysed in 1X lysis buffer supplemented with protease inhibitor cocktail. The
opioid receptors were immunoprecipitated using polyclonal rabbit anti-FLAG antibody
(Sigma, St. Louis, MO) bound to Protein-G Dynabeads (Invitrogen). Western blot
analysis of immunoprecipitated complexes was performed by using a polyclonal goat
anti-SIAH1 antibody (1:5000 dilution; Abnova, Taipei City, Taiwan) and a bovine anti-
goat HRP-conjugated secondary antibody (1:20,000 dilution; Jackson ImmunoResearch).
Blots were also immunoblotted using Guinea pig anti-MOR antibodies (1:5000 dilution;
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Neuromics, Ednia, MN) or monoclonal mouse anti-FLAG antibodies (1:5000 dilution;
F1804; Sigma) as a positive control and donkey anti-Guinea pig horseradish peroxidase
(HRP)-conjugated antibodies or goat anti-mouse-HRP conjugated secondary antibodies
(Jackson ImmunoResearch). Mock immunoprecipitations using normal rabbit IgG (Santa
Cruz Biotechnology) were used as negative controls. Immunoreactivity was detected by
ECL Plus.
4.2.3 Animal Care and Drug Treatment
All mouse studies were performed in collaboration with Tom Ferraro (University
of Pennsylvania) in the research laboratories at the Veteran’s Administration Medical
Center (VAMC) in Coatsville, PA, and were approved by the University of Pennsylvania
Institutional Animal Care and Use Committee. C57BL/6J mice were bred in-house,
maintained on a 14hr/10hr light/dark schedule and had food and water available ad
libitum throughout the course of the experiment.
Female mice, 8-9 weeks of age, were implanted sub-cutaneously with a 25 mg
morphine (n=5) or placebo (n=4) pellet. Morphine and placebo pellets were obtained
from the NIDA Drug Supply Program. Mice were anesthetized with ketamine and
zylazine (80 and 10 mg/kg, ip, respectively, then euthanized by cervical dislocation under
carbon dioxide anesthesia four days (96 hr) after pellet implantation. Brains were
harvested and dissected on ice under 5x magnification into 6 regions including prefrontal
cortex, nucleus accumbens, dorsal striatum, midbrain, hippocampus, and cerebellum.
Specimens were frozen on dry ice and stored at -80C.
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4.2.4 Tissue Preparation and Protein Analysis
Frozen brain tissue obtained from drug or sham treated mice was utilized for protein
expression analysis. Fresh whole-brain tissue from untreated mice was obtained for co-IP
experiments. All tissue was suspended in lysis buffer (50mM Tris-HCl, 1mM EDTA, 150mM
NaCl, 1% NP40, 0.25% deoxycholate, 5mM NaF, 2mM NaVO3) containing protease inhibitors
(cOmplete MINI EDTA free, Roche), homogenized using a microcentrifuge pestle for 2 minutes,
sonicated using a probe sonicator, then centrifuged at 13,000 RPM to remove cellular debris.
For protein expression analysis, samples were analyzed by electrophoresis on 10% SDS-
containing polyacrylamide gels, followed by Western blotting. In addition to antibodies utilized
for co-IP, guinea pig anti-MOR (GP10106, Neuromics, Edina, MN) and Rabbit anti-DOK4
(SAB1300112, Sigma-Aldrich) antibodies were also used to probe brain lysates. For quantitative
analysis of MORIP expression in drug treatment studies, lysates from sham and morphine-treated
mice (20 μg/brain region) were analyzed in quadruplicate. To rule out transfer artifacts, samples
from each brain region were loaded in a different order on each of the four blots. Blots were
scanned using a back-lit scanner and quantitation was performed using IMAGEJ software
(Abramoff et al., 2004). Expression was normalized to total protein (as measured by Ponceau
stain), averaged between replicates, and subjected to a two-tailed Student’s t-test.
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4.3 Results
4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid receptors
In an effort to better understand the effect of the MOR/SIAH1 interaction on the
regulation of the MOR under stimulated conditions, I wanted to investigate the effect of
opioid agonists on MOR/SIAH1 complex formation. To determine whether opioid drugs
promote MOR/SIAH1 complex formation, human neuroblastoma cells (SHSY-5Y) were
exposed to either 10 μM DAMGO or 10μM morphine for 30 minutes or 1 hour. These
two drugs were chosen because of their different effects on the trafficking and signaling
of the MOR (Groer et al., 2011). DAMGO causes robust internalization and recycling of
the MOR, while morphine causes markedly slow internalization and no recycling of the
MOR (Christie, 2008). The doses chosen have been shown to saturate receptors and
activate downstream signaling as measured by adenylyl cyclase activity. The treatment
times represent acute exposure that should theoretically capture internalization and
downstream events (such as recycling and ubiquitination), but should not be long enough
to capture degradation of the MOR (Christie, 2008).
To investigate the effects of acute DAMGO and morphine exposure on the
MOR/SIAH1 interaction, SH-SY5Y cells were treated with drug for the designated time
periods. The MOR was immunoprecipitated from equal microgram concentration of total
protein in the lysate (900 μg), resolved by SDS-PAGE, and immunoblotted using goat
anti-SIAH1 antibodies. As seen in figure 4.1A, DAMGO caused an increase in the
amount of SIAH1 that co-immunoprecipitated with the MOR in SH-SY5Y cells after 1
hour of exposure when compared to DMSO treated cells. In contrast, morphine did not
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cause a change in the amount of co-immunoprecipitated SIAH1 at any time point tested
(Figure 4.1B). These results indicate that DAMGO induced MOR signaling or
internalization enhances the MOR/SIAH1 interaction, which in turn could result in
altered ubiquitination of the receptor.
Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex
formation
(A) SHSY-5Y cells were treated with 10 μM DAMGO for 30 minutes and 1 hour. After
the designated time period cells were lysed and the MOR was immunoprecipitated from
900 μg of total protein using rabbit anti-MOR antibodies bound to Protein G beads.
Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1 antibodies. (B)
SHSY-5Y cells were treated with 10 μM morphine for 30 minutes and 1 hour. The MOR
was immunoprecipitated from 900 ug of total protein using rabbit anti-MOR antibodies
bound to Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat
anti-SIAH1 antibodies. (A and B) Mock IPs using normal rabbit IgG were used as
negative controls. Crude cell lysate (Ly) was used as a positive control and represents
approximately 10% of protein used in IPs.
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The SHSY cells express both MOR and DOR, which have been shown to
heterodimerize and affect each other’s trafficking and function (Prather et al., 1994). I
therefore wanted to assess opioid drug effects on the interaction of SIAH1 with the
individual opioid receptors in the HEK cell models each expressing a single opioid
receptor subtype. Although, acute morphine exposure did not alter the interaction of
SIAH1 and MOR in SHSY cells, this agonist will act at all three receptors (albeit with
different affinities) and is a known clinically relevant and addictive substance (Neil,
1984). DAMGO is a selective agonist for MOR and would not be useful for the DOR and
KOR studies. HEK-DOR, HEK-KOR, and HEK-MOR cells were exposed to 10 μM of
morphine for 1 hour and 21 hours. For this experiment both acute (1hr) and chronic
effects (21hr) were measured as there were no changed in the MOR/SIAH1 interaction
with acute exposure of morphine in the SHSY cells. Chronic treatment (21hr) has
previously been determined to allow for degradation of these receptors and, therefore
may be a more likely time point to observe a change in the OR/SIAH1 interaction (Afify,
2002; Chen et al., 2006b). Similar to the results with morphine treatment in SHSY-5Y
cells, the MOR/SIAH1 interaction did not change with 1 or 21 hours of treatment in
HEK-MOR cells (Figure 4.2, lanes 11-13). The DOR/SIAH1 interaction, increased with
both acute and chronic morphine treatment (Figure 4.2, lanes 3-5), whereas the
KOR/SIAH1 interaction did not change. These results indicate that acute morphine
exposure may only alter the interaction of SIAH1 with the DOR. However, the IP lanes
appear to have a double band for SIAH1 when immunoprecipitated with DOR and KOR.
This double band could be due to a number of things, such as degradation of SIAH1 or
posttranslational modification. Taken together these results suggest that morphine may
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have a selective effect on the DOR/SIAH1 interaction by promoting the formation of this
complex.
Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation
(A) HEK-DOR, HEK-KOR, and HEK-MOR cells were treated with 10 μM morphine for
1 hour and 21 hours. After the designated time period cells were lysed and the DOR,
KOR, or MOR was immunoprecipitated using rabbit anti-FLAG antibodies bound to
Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1
antibodies. Mock IPs using normal rabbit IgG were used as negative controls. Crude cell
lysate (Ly) was used as an additional positive control and represents approximately 5% of
protein used in IPs.
4.3.2 Interaction of MOR and MORIPs in Rodent Brain
To determine whether the interaction between the MOR and MORIPS may occur
in vivo, we analyzed the expression of MOR and several MORIPS in various mouse brain
regions. Not all commercially available antibodies were reliable when used to detect
MORIPs in the brain. For example, antibodies available for SIAH2, zyxin, and DOK5
were not able to detect proteins of the proper size in brain lysates. Other antibodies, such
as AUP1 could detect the protein in cell lysates, but yielded many non-specific bands in
brain lysates. Therefore, only MORIPs whose commercially available antibodies
detected a protein of the correct molecular weight in brain lysates were chosen for further
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study. As shown in Fig. 4.3A, the MOR and its interacting partners DOK4, SIAH1,
VAPA, and WLS were each expressed in cerebellum, hippocampus, nucleus accumbens,
midbrain, prefrontal cortex, and dorsal striatum. Co-expression of the MOR and each of
the MORIPs in all brain regions tested is consistent with the idea that the MOR and its
binding partners have the potential to interact in multiple brain regions. To verify
interaction, the MOR was immunoprecipitated from a whole mouse brain lysate, and
immunocomplexes probed for the presence of several MORIPs. As shown in Fig. 4.3B,
SIAH1, VAPA, and WLS were able to co-IP with MOR but not with rabbit-IgG alone,
suggesting that these MORIPs are capable of interacting with MOR in mouse brain.
DOK4 was not tested due to crossreactivity of the antibody with rabbit IGG used for
immunoprecipitations.
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Figure 4.3: MORIP expression and interaction with MOR in mouse brain
(A) Expression of MOR and MORIPS in mouse brain regions. Western blots containing
lysates prepared from mouse cerebellum (C), hippocampus (H), midbrain (M), nucleus
accumbens (N), prefrontal cortex (P), and striatum (S) were probed with Guinea pig anti-
MOR, rabbit anti-DOK4, goat anti-SIAH1, mouse anti-VAPA, and chicken anti-WLS
antibodies. (B) Co-immunoprecipitation. The MOR was immunoprecipitated from
whole mouse brain lysates using rabbit anti-MOR antibodies. Immunocomplexes were
probed for the presence of SIAH1, VAPA, or WLS using MORIP-specific antibodies.
Lysate lanes (L) contain 5% of the total protein prepared from whole mouse brain lysate
compared to the mock (M, rabbit IGG) and immunoprecipitation (IP) lanes. Experiments
performed by Jessica Petko at The Pennsylvania State University College of Medicine.
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4.3.3 The Effect of Morphine on MORIP Expression
To determine whether chronic morphine exposure alters the protein levels of
MORIPs, we performed Western blot analysis of selected MORIPs (DOK4, SIAH1, and
WLS) in brain regions thought to be involved in response to opioid drug exposure. MOR
levels are not reported for this experiment, as the antibody lot utilized resulted in high
background and very light bands that were unable to be quantified. Mice were implanted
subcutaneously with either saline (sham n=4) or morphine (n=5) pellets for 96 hours,
then euthanized, and the brains dissected.
As shown in Figure 4.4, no significant changes were detected in any of the
MORIPs tested in either cerebellum or nucleus accumbens. However, in response to
morphine exposure, DOK4 levels were decreased by ~50% in hippocampus (p=0.02) and
midbrain (p=0.003). In morphine-treated animals, SIAH1 expression was reduced by
48% in hippocampus (p=0.008) but showed a 55% increase in midbrain (p=0.03) (Figure
4.4). WLS levels were decreased by 49% in midbrain (p=0.006) and 39% in striatum
(p=0.02) following drug exposure (Figure 4.4). Although every effort was made to
ensure equal loading of proteins, some samples consistently contained higher amounts of
total protein than others (e.g., sham versus morphine treated samples in hippocampus).
Despite these differences in protein loading, our data suggest that morphine treatment
may lead to significant alterations in MORIP protein expression in various regions of
mouse brain.
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Figure 4.4: MORIP expression in brain regions of morphine treated mice
Mice were treated for 96 hours with either morphine-containing (n=5) or placebo (n=4)
pellets. Animals were sacrificed, brain regions dissected, and Western blots of selected
MORIPs probed with MORIP-specific antibodies. Each panel contains a representative
blot for a MORIP in the specified brain region (n=4 blots/MORIP/brain region). Total
protein was quantitated by Ponceau stain of the blot prior to antibody probing. Bar graphs
represent the average pixel density (as determined by IMAGEJ) of four blots for each
brain region normalized to total protein and placebo treatment. Data was analyzed using
a two-tailed Student's t-test. Error is expressed as standard error of the mean. * indicates
a statistically significant difference (p<.05) between sham and morphine treatment.
Experiments performed by Jessica Petko at The Pennsylvania State University College of
Medicine.
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4.4 Discussion
My results indicate that the strength of the interaction between SIAH1 and the MOR
changes with acute exposure to DAMGO, but not with acute or chronic morphine treatment. In
line with this finding, DAMGO has been shown to cause ubiquitination of mu opioid receptors
that peaks at 1 hour of agonist stimulation (Groer et al., 2011). An acute treatment with
DAMGO causes robust internalization of the MOR within 30 minutes which may make MOR
available in the cytosol for an interaction with SIAH1. In contrast, morphine causes slow
internalization and no ubiquitination of the MOR which could explain why no increase in
SIAH1/MOR interaction was detected. Alternatively, the differences seen in MOR/SIAH1
complex formation could be due to proteins that are brought to the signaling complex during
activation of the MOR. For example, DAMGO stimulation of MOR brings β-arrestin 2 to the
MOR signaling complex, but morphine does not. Additionally, loss of β-arrestin 2 prevents the
DAMGO-stimulated ubiquitination of the MOR (Groer et al., 2011). Degradation of the MOR in
response to agonist (DAMGO or morphine) requires several hours of agonist binding (Afify,
2002; Chen et al., 2006b). Although DAMGO was not studied under chronic conditions, no
change in the MOR/SIAH1 interaction was detected with morphine. Together, these results may
indicate that SIAH1 may be involved in agonist stimulated ubiquitination (by DAMGO) of the
MOR as well as unstimulated ubiquitination (chapter 3).
Morphine was used to determine the effects of acute and chronic receptor activation on
the interaction of the individual opioid receptors and SIAH1. With chronic morphine treatment,
the interaction between SIAH1 and DOR increased while the KOR/SIAH1 interaction did not
change. These results are interesting as morphine has a lower binding affinity for DOR and
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KOR and likewise only weakly activates these receptors (Keith et al., 1996; Trescot et al., 2008).
While DOR and KOR can be ubiquitinated in response to selective agonists, nothing is known
about the ubiquitination these receptors in regards to morphine stimulation (Henry et al., 2011;
Li et al., 2008). Morphine does not cause ubiquitination of MOR, but this does not rule out this
possibility for DOR or KOR. In addition, little is known about the trafficking of these receptors
in response to morphine exposure. Therefore, we cannot make any conclusions as to whether
trafficking events are changing the DOR or KOR interaction with SIAH1.
The study of MORIP levels in morphine treated mice, utilized the inbred strain
C57BL/6J. This strain has been found to be more sensitive to the rewarding effects of morphine
than other strains, such as DBA/2J, in self-administration and conditioned place preference
paradigms (Elmer et al., 2010). However, our study did not use a reward paradigm (self-
administration or conditioned place preference), but was designed to detect differences between
those individuals exposed to morphine and naïve individuals. Although behavior was not
measured in this study, 96 hour morphine treatment is known to lead to changes in dendritic
morphology and neurogenesis associated with synaptic plasticity and addiction (Eisch et al.,
2000). Additionally, these changes are found to occur, regardless of the method of
administration (pellet, experimenter, or self-administration) (Robinson and Kolb, 2004).
Therefore this kind of study can be a useful tool when combined with behavioral paradigms to
tease out which changes are due to the effect of the drug and which may be due to behavior.
An inbred strain of mouse was utilized in this analysis, because it should control for
genetic variability between animals as they should be homozygous at almost all loci. Even with
this homogeneity of mice, there are some differences in the level of MORIPs detected within
treatment groups. The explanation for this interanimal variability is unknown. However, it may
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result from genetic variation, such as single nucleotide polymorphisms (SNPs) between animals.
According to the Mouse Genome Informatics database, the C57BL/6J strain can have SNPs in
SIAH1 (Siah1a of mouse), DOK4, and WLS. If these SNPs are found in some individuals and
not others in this study, it may help to explain the observed variation in the level of expression of
a particular MORIP in a mouse. However, no studies have investigated the role of single
nucleotide polymorphisms or splice variants in SIAH1, DOK4, or WLS in regards to the
expression of these proteins.
Knockout mouse experiments have shown that MOR is the primary mediator of the
analgesic and rewarding properties of opioid drugs. Since MORIPs regulate the functional
output of the MOR, they represent potential candidates for proteins that may contribute to the
underlying molecular mechanisms leading to drug addiction. Protein expression analysis of
selected brain regions from morphine-treated mice indicate that the expression of DOK4, SIAH1,
and WLS are altered in distinct brain regions following chronic morphine exposure. Morphine
has been shown to decrease WNT secretion in a cell culture system, and that WLS shifts from a
predominantly cytosolic to a more plasma membrane-associated distribution in cultured cells as
well as rat striatal neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The
decrease in WLS expression observed in midbrain and striatum of mice after morphine
administration could possibly lead to long term decreases in WNT secretion within the CNS. It
is possible that decreased Wnt secretion could be responsible for many of the observed effects of
chronic opioid exposure including decreased neurogenesis and decreased dendritic spine density
that are known to be affected by WNT signaling (Eisch et al., 2000; Liao et al., 2005).
The implications of decreased SIAH1 expression in the hippocampus and its increased
expression in the midbrain could be enhanced by looking at MOR levels. As mentioned in the
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results this was attempted, but changes in antibody performance (change of Lot number) did not
allow the completion of this analysis. Although the functional relevance of changes in DOK4
and SIAH1 expression after morphine exposure is currently unknown, it is of interest that these
changes in protein expression were observed within specific brain regions, rather than
throughout the entire brain. Thus the changes in protein expression we detected may play an
important role in the response to opioid drug exposure mediated by these brain regions. In the
future, it should become possible to examine the role of these MORIPs in behavioral aspects of
opioid addiction using animal models of opioid self-administration.
In summary the findings presented in this chapter indicate that while the non-
internalizing agonist morphine does not affect the MOR/Siah1 interaction, the internalizing
agonist DAMGO enhances this interaction. This indicates that although the SIAH proteins
appear to be involved in a basal level of ubiquitination and receptor degradation (as described in
chapter 3), there may also be a role for SIAH1 in agonist stimulated MOR regulation. In
addition, several newly identified MORIPS including SIAH1 are co-expressed with and interact
with MOR in the mouse brain. Finally, several MORIPs display altered expression in the mouse
brain during chronic exposure to morphine. This finding not only suggests a role for these
proteins in the drug adaptation process, but also validates the interaction screen/candidate gene
approach to identifying potential players in development of addiction.
Chapter 5
Overall Discussion
The overall aim of this thesis was to characterize novel mu opioid receptor
interacting proteins (MORIPs) in an attempt to provide new understandings into the
cellular and molecular mechanisms that underlie the development of opioid addiction.
This goal was founded upon several lines of evidence, including the central role of the
MOR in mediating both the analgesic and rewarding effects of opioid agonists (Matthes
et al., 1996), the growing body of evidence suggesting that G protein-coupled receptors
(GPCRs) are part of large signaling complexes whose protein constituents have not all
been identified (Kabbani and Levenson, 2007), and that we still do not have a clear
understanding of the basic biological underpinnings of this disease. In working towards
this aim, several novel MORIPs were discovered, the function of the MOR/SIAH1/2
interaction was explored, and changes in MORIP protein levels were detected in
morphine treated mice.
Possible roles of Novel MORIPs in morphine exposure
Utilization of traditional and modified yeast two-hybrid (Y2H) screens identified
10 novel and two previously discovered components of the MOR signaling complex.
Some of these MORIPs were shown to interact with other opioid receptors, which
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suggests that they may be universal regulators of opioid function. The conventional Y2H
was unique as it utilized the second intracellular loop of the MOR, which had not
previously been studied in terms of interacting proteins. Several of the novel MORIPs
identified in these screens have been shown to share common cellular functions that may
correlate with changes that have been observed in the development of addiction, such as
synaptic plasticity and neurogenesis. A summary of the cellular function of MORIPs
found in this study and in previous studies is depicted in Figure 5.1.
Figure 5.1: MOR interaction map
Web of newly identified and previously known MOR interacting proteins. MORIPs were
grouped based on previously characterized functional properties. MORIPs identified in
the current study are depicted by green shaded circles, while previously identified
MORIPs are represented by white circles. Many of the newly identified MORIPs also
interact with DOR and KOR (see Chapter 2 Results).
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Since MORIPs have the potential to regulate the functional output of the MOR, they
represent potential candidates for proteins that may contribute to the underlying molecular
mechanisms leading to drug addiction. Protein expression analysis of selected brain regions
from morphine-treated mice indicate that the expression of DOK4, SIAH1, and WLS are altered
in distinct brain regions following chronic morphine exposure. Morphine has been shown to
decrease WNT secretion in a cell culture system, and WLS shifts from a predominantly cytosolic
to a more plasma membrane-associated distribution in cultured cells as well as rat striatal
neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The decrease in WLS
expression observed in midbrain and striatum of mice after morphine administration could
possibly lead to long term decreases in WNT secretion within the CNS. The midbrain contains
the ventral tegmental area (VTA) nuclei that are the main sources of dopamine (DA) in the brain,
whereas other regions, such as the nucleus accumbens, striatum, and prefrontal cortex are the
target regions for DA released from the VTA. It is widely accepted that addictive drugs cause
functional changes these brain regions. These functional changes may manifest as changes in
neuron firing, strengthening or weakening of synapses, changes in neuron morphology, and
neurogenesis (Nestler, 2004; Nugent et al., 2007; Robinson and Kolb, 2004; Russo et al., 2010).
Coincidentally, dendritic spine density and neurogenesis are known to be affected by WNT
signaling (Eisch et al., 2000; Liao et al., 2005). Therefore, it has been hypothesized that
decreased WNT secretion could be responsible for many of the observed structural and
functional effects of chronic opioid exposure (Jin et al., 2010).
In the nervous system, DOK4 has been shown to be required for axon myelination
(Blugeon et al., 2011) and the regulation of growth derived neurotropic factor (GDNF)-
dependent neurite outgrowth (through activation of the Rap1-ERK1/2 pathway) (Uchida et al.,
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2006). Neurite outgrowth has been proposed as a possible mechanism for plasticity in the
hippocampus (Ring et al., 2006). A hypothetical explanation for the decreased levels of DOK4
in the hippocampus is that the reduction in DOK4 protein may contribute to reduced neurite
outgrowth and consequently reduced plasticity in this brain region. Determining the function of
the MOR/DOK4 interaction in relation to neuronal morphology, MAPK-mediated signaling, and
other processes associated with MOR activation and opioid addiction may clarify the
consequences of reduced DOK4 in response to morphine exposure in mice.
The expression of SIAH1 mRNA has been found to be decreased in the middle temporal
gyrus (includes the hippocampus) of patients with a history of alcohol abuse (Sokolov et al.,
2003). Our study found a decrease in SIAH1 protein levels in the hippocampus of morphine
treated mice. The method of morphine treatment performed in this study has previously been
shown by an independent group to cause structurally changes in the hippocampus that are
associated with the development of addiction (Liao et al., 2005; Nestler, 2004). Additionally,
other proteins involved in the ubiquitin proteasome pathway have been shown to be altered in
addiction (Bingol and Sheng, 2011; Tuoc and Stoykova, 2010). Based on the results from
Chapter 3, it is tempting to speculate that SIAH levels play an important role in the addiction
process by direct regulation of MOR ubiquitination, trafficking, and/or degradation. Further
characterization of the roles of SIAH E3 ligases in ligand-mediated MOR regulation will be
crucial to determining whether this speculation may be possible.
Although the functional relevance of changes in WLS, DOK4 and SIAH1 expression
after morphine exposure is currently unknown, it is of interest that these changes in protein
expression were observed within specific brain regions, rather than throughout the entire brain.
Thus, the changes in protein expression we detected may play an important role in the response
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to opioid drug exposure mediated by these brain regions. In the future, it should become
possible to examine the role of these MORIPs in behavioral aspects of opioid addiction using
animal models of opioid self-administration. Knockout animals of WLS, SIAH1, or DOK4
could be used in self-administration paradigms to determine the possible role that they play in
the addiction process. This type of approach allows for the measurement of drug seeking, taking,
and reinstatement. The affect on these addiction-like behaviors due to the loss of a particular
MORIP, has already been used for spinophilin (a previously identified MORIP). The loss of
spinophilin in mice resulted in a greater sensitivity to the rewarding properties of morphine and
the development of a higher degree of physical dependence (Charlton et al., 2008). The use of a
similar approach for WLS, SIAH1, and DOK4 may be able to clarify the roles of these proteins
in behavior. Additionally, the role of degradation could be explored through the use of
lysosomal and proteasomal inhibitors in conjunction with a self-administration paradigm with
knockout animals.
The DOR/SIAH1/2 and KOR/SIAH1/2 interactions
The MOR is known to form functional dimers with the other two major opioid
receptors (Chakrabarti et al., 2010; Gomes et al., 2000), and all three of these receptors
have been demonstrated to interact with both SIAH1 and SIAH2 (Chapter 2). Thus, it
was important to investigate the role of the Siah proteins in the regulation of the DOR
and KOR. MOR will be discussed in more detail; therefore, DOR and KOR will be
discussed first. In chapter 3, SIAH1 and SIAH2 were knocked down in HEK-DOR and
HEK-KOR cells. The results from those experiments yield several possible models for
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the function of the Siah proteins in the basal (unstimulated) regulation of DOR and KOR.
Two of these models will be discussed below.
In the first model, both SIAH1 and SIAH2 are capable of conjugating ubiquitin to
the DOR and the KOR (Figure 5.2A). In other words, the Siah proteins may compensate
for the loss of the other. This ubiquitination of DOR and KOR by the Siah proteins leads
to different fates for the receptors. Ubiquitination by SIAH2 appears to signal for
degradation of these receptors, as knockdown of SIAH2 caused an increase in the protein
levels of both DOR and KOR. In contrast, ubiquitination by SIAH1 does not appear to
signal for degradation, as knockdown of SIAH1 caused a decrease in the protein levels of
both DOR and KOR. In the second model, the Siah proteins regulate the ability of
another E3 ligase to conjugate ubiquitin to DOR and KOR. SIAH 1 may regulate
SIAH2, SIAH2 may regulate SIAH1, or the regulation may require a heterodimers of
SIAH1/SIAH2, which regulate a third E3 ligase. This E3 ligase would then conjugate the
DOR or KOR with ubiquitin and signal for degradation or trafficking (Figure 5.2B). This
model predicts increased ubiquitination with SIAH1 or SIAH2 knockdown. Consistent
with this model, knockdown of either SIAH1 or SIAH2 resulted in increased receptor
ubiquitination. Without the benefit of double knockdown experiments in HEK-DOR and
HEK-KOR cells, it is difficult to discern which model may best represent what is
occurring in these cells with regards to DOR and KOR regulation.
The experiments performed in this dissertation did not look at the effects of
opioid agonist stimulation in combination with SIAH1 or SIAH2 knockdown. However,
the effect of acute and chronic morphine treatment on DOR/SIAH1 and KOR/SIAH1
complex formation was examined. Although morphine weakly binds and activates DOR
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and KOR, chronic morphine treatment resulted in an increase in the formation of
DOR/SIAH1 complexes. Therefore, it is possible that SIAH1 may play an additional role
in the regulation of DOR and/or KOR stimulated with agonist. Experiments utilizing
DOR- and KOR-specific agonists in combination with knockdown of SIAH1 and SIAH2
may help clarify the roles of the Siah proteins in receptor regulation.
Lastly, it will be important to determine the functional consequences of the
ubiquitination of these receptors both basally and upon activation. My results suggest that
ubiquitination in the absence of SIAH1 allows for receptor degradation, whereas the
absence of SIAH2 does not. Since both the lysosomal and proteasomal pathways can
degrade the DOR and KOR, it will be important to determine which pathway is being
used (Chaturvedi et al., 2001; Henry et al., 2011; Li et al., 2008). This could be
accomplished by blocking the lysosome and the proteasome with inhibitors in
combination with the knockdown of SIAH1 and SIAH2.
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Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR
cells
(A) SIAH1 and SIAH2 are capable of ligating ubiquitin to the DOR and/or KOR. This
ubiquitination signal has three possible consequences, degradation by the lysosome,
degradation by the proteasome, or a trafficking event. (B) SIAH1 and SIAH2 or a
SIAH1/SIAH2 heterodimer may regulate another E3 ligase, which acts to ubiquitinate
DOR and/or KOR. This ubiquitination signal has three possible consequences,
degradation by the lysosome, degradation by the proteasome, or trafficking of the
receptor(s).
Model for the Interaction between the MOR and the Siah proteins
The discovery that SIAH1 and SIAH2 interact with the MOR was surprising,
considering that the MOR is usually downregulated by lysosomal degradation and not via
the proteasome. However, ubiquitination of the MOR does occur and has been shown to
be required for trafficking events that downregulate receptor ligand binding after agonist
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stimulation (Hislop et al., 2011). Additionally, this ubiquitination has been shown to be
ligand dependent, as DAMGO but not morphine, will cause ubiquitination of the MOR
(Groer et al., 2011).
The work presented in this dissertation suggests a previously unrecognized role
for ubiquitination in the regulation of unstimulated MOR expression via interaction with
SIAH1 and SIAH2. The combined overexpression data and Siah proteins knockdowns,
yield several possible models for their role in MOR regulation. In both models, SIAH1
acts to regulate the levels of SIAH2 protein, either directly (Figure 5.3A) or indirectly
(Figure 5.3B). This is consistent with the observation that Siah2 proteins levels increased
with SIAH1 knockdown in all cell lines tested. Both models also suggest that SIAH2 is
an E3 ligase for the MOR, as SIAH2 knockdown resulted in a decrease in receptor
ubiquitination. However, SIAH2 knockdown did not result in increased levels of MOR
protein, suggesting that SIAH2-mediated ubiquitination of the MOR alone is not
sufficient for degradation of the receptor. It is possible that another protein is required to
escort the ubiquitinated MOR to the site of degradation (lysosome or proteasome). It is
tempting to speculate that this shuttle protein may be another novel MORIP, such as
AUP1 or CSN5. The effect of the knockdown of these proteins on ubiquitination and
receptor protein levels has not yet been investigated and may shed some light on the role
of these ubiquitin pathway proteins in the MOR life cycle. Another alternative is that
SIAH1 may serve a dual role as the SIAH2 regulator and a shuttle protein. The double
knockdown of both SIAH1 and SIAH2 yielded results that suggest that both SIAH1 and
SIAH2 are required for the ubiquitination of the MOR.
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Figure 5.3: Simplified model of Siah protein-mediated MOR degradation
(A) SIAH1 inhibits SIAH2, which ubiquitinates MOR. Degradation via either the
lysosomal or proteasomal pathway requires a shuttle protein to bring the ubiquitinated
MOR to the site of degradation. (B) SIAH1 may inhibit another protein that is
responsible for activation of SIAH2, which ubiquitinates the receptor. Either the
lysosomal or proteasomal degradation requires a shuttle protein to bring the ubiquitinated
MOR to the site of degradation.
Treatment of SH-SY5Y cells with DAMGO and morphine, showed different
effects on the SIAH1/MOR interaction. DAMGO has been shown to cause rapid
internalization, recycling, and ubiquitination of the MOR, while morphine treatment
results in much slower internalization and no recycling or ubiquitination of receptors
(Christie, 2008; Hislop et al., 2011). The interaction between SIAH1 and the MOR was
strengthened with acute DAMGO treatment, but did not change with either acute or
chronic morphine treatment. This observation indicates that DAMGO, but not morphine
promotes the formation of MOR/SIAH1 complexes. One hypothesis is that the increased
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interaction detected between MOR and SIAH1 with DAMGO stimulation, results in the
ubiquitination of the MOR. In contrast to the role of SIAH1 in unstimulated MOR
regulation, DAMGO stimulated MOR regulation suggests that SIAH1 may be the
putative E3 ligase responsible for the ubiquitination of the MOR observed by Groer and
colleagues (2011). Unfortunately, probing the Western blots from these experiments
with anti-ubiquitin antibodies was unsuccessful. An alternative explanation is that
DAMGO treatment increases SIAH2 activity resulting in MOR ubiquitination and that
the ubiquitination signals SIAH1 to form an interaction with the MOR. Determining the
affect of DAMGO treatment on the MOR/SIAH2 interaction was attempted, but was
unsuccessful. Unfortunately re-probing the Western blots from these experiments with
the anti-SIAH2 antibody did not produce bands. In the future it will be important to
determine the effect of DAMGO and morphine treatment on the MOR/SIAH2
interaction. In addition, acute and chronic DAMGO treatment of cells in which the Siah
proteins have been knocked down may clarify the roles of the Siah proteins in regulation
of agonist stimulated MOR.
Interestingly, not all MOR species were sensitive to the effects of SIAH1, as only
the low molecular weight species (50 kDa band) significantly changed. This low
molecular weight, intracellular species has been postulated to represent MOR in the
biosynthetic pathway (Huang and Liu-Chen, 2009). Therefore, it is possible that some of
this MOR species is associated with the endoplasmic reticulum or another intracellular
compartment. Proteins destined for insertion into the plasma membrane enter the
endoplasmic reticulum (ER) in an unfolded state and generally leave after they have
reached their native states. However, folding in the ER is often slow and inefficient and a
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substantial fraction of polypeptides fail to reach their native state. The cell must
continuously scrutinize these newly synthesized proteins and remove those that are
terminally misfolded (Smith et al., 2011). High fidelity of the ER quality control is
crucial for the functionally integrity of the cell. To illustrate this point, even subtle
mutations that would not dramatically affect protein function can lead to retention of the
mutant protein within the ER, thereby preventing its physiologically relevant activity.
This has been shown for a variety of proteins, including cystic fibrosis transmembrane
conductance regulator (CFTR), V2-vasopressin receptor, and many GPCRs, including the
MOR (Chaipatikul et al., 2003). However, retention and degradation of incorrectly
folded proteins may not be restricted to products of mutated genes. It has been proposed
that as much as 30% of newly synthesized proteins in various cell types never reach their
correct native structure (Schubert et al., 2000). In fact, it has been demonstrated that only
40% of newly synthesized delta opioid receptors are transported to the cell surface in
human embryonic kidney cells (Petaja-Repo et al., 2000). The number and activity of
opioid receptors on the cell surface are important factors in determining the capacity of
these receptors to modulate downstream signaling molecules (Law et al., 2000).
The involvement of the Siah proteins as potential E3 ligases involved in ERAD is
one explanation for the interaction between SIAH1 and 2 and MOR under non-stimulated
conditions. A number of ERAD ligases have been discovered in mammalian cells. Most
of them, like RNF5/RMA1, TEB4, RFP2, Kf-1, and TRC8 are poorly characterized with
only a few targets identified for each of them. Those that have been investigated in
greater detail, such as Hrd1 (synoviolin) and gp78 (autocrine motility factor, AMFR)
have numerous single and overlapping substrates (Mehnert et al., 2010). The majority of
135
these ERAD ligases have a transmembrane domain which tethers them to the ER
membrane. Analysis of the Siah proteins using PRED-TMR, a program designed to
predict transmembrane domains, did not detect any putative transmembrane domains
(Pasquier et al., 1999). In fewer cases, cytosolic ligases, such as Parkin, have been
demonstrated to promote turnover of selected proteins via ERAD (Imai et al., 2002;
Yoshida et al., 2002). Thus, the Siah proteins may be additional cytosolic E3 ligases that
participate in the ERAD pathway. Determining the precise location where the interaction
between the Siah proteins and the MOR occurs could help determine whether or not this
interaction may play a role in the ER-associated degradation of the MOR. This could be
tested using immunogold labeling of MOR and Siah proteins and electromicrography.
Currently, there is a lack of data on the expression profile of SIAH1 and SIAH2
and the contribution of these E3 ligases to ERAD. The criteria that drive individual
polypeptides into distinct ERAD pathways also remain unknown (Kostova et al., 2007;
Mehnert et al., 2010). However, one could hypothesize that the activity of individual
ligases or their cofactors may be controlled spatially, temporally, or in a tissue-specific
manner to allow cells to adapt to varying physiological conditions. In addition, the
amount of some of these ligases and cofactors may be regulated by the proteolysis of
other ERAD ligases (Mehnert et al., 2010). In fact, the ERAD ligase Hrd1 has been
shown to promote gp78 ubiquitination and degradation under certain conditions, thereby
affecting the degradation of gp78 target proteins (Shmueli et al., 2009). Thus, the
regulation of SIAH2 by SIAH1 may be important in preventing the targets of SIAH2
from ubiquitination under certain conditions, such as basal MOR turnover.
136
In the model of MOR degradation via ERAD, misfolded MOR would be
recognized by ER chaperones, which would bring it to an ER-resident E3 ligase for
ubiquitination (Figure 5.4A). Once ubiquitinated, the misfolded MOR would be
translocated through a channel protein to the cytosol. Cytosolic de-ubiquitinating
enzymes (DUBs) would then remove the ubiquitin from the MOR (Figure 5.4B).
Cytosolic chaperones would bind the misfolded MOR and SIAH2 would ubiquitinate the
MOR to signal for degradation (Figure 5.4C). The SIAH2-mediated ubiquitination of
MOR, would facilitate the shuttling of the misfolded protein to the proteasome by SIAH1
(Figure 5.4D). However, further evidence is needed to support or refute this model. One
method of determining the involvement of an E3 ligase in ERAD, is to induce ER stress
in cells. Sustained ER stress eventually leads to a stress response, unfolded protein
response (UPR). In an attempt to relieve this stress, cells will increase the synthesis of
ER-resident chaperones and other proteins involved in ER-associated protein
degradation. Therefore, if the Siah proteins are involved in ERAD, exposing cells to an
N-linked glycosylation inhibitor, such as tunicamycin, may result in increased expression
of the Siah proteins.
137
Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of
the MOR
(A) MOR (black line) is recognized by ER chaperones and is partially translocated
through a protein conducting channel complex/retrotanslocon (brown). MOR is
conjugated with chains of ubiquitin (gray circles) by an ER-resident ubiquitin ligase (E3)
and its cognate ubiquitin conjugating enzyme (E2) on the cytosolic face of the ER
membrane. (B) The ubiquitin chains on the MOR serve as an exit signal and MOR moves
out of the ER. This movement is facilitated by the removal of ubiquitin molecules by de-
ubiquitinating enzymes (DUB). (C) The dislocated MOR is ubiquitinated a second time
by SIAH2. (D) SIAH1 recognizes the ubiquitnated MOR and shuttles it to the
proteasome for degradation. Adapted from (Tsai and Weissman, 2011).
Taken together my results suggest that the roles for the Siah proteins in
ubiquitination of the opioid receptors are different. The majority of my results suggest
SIAH2 is a putative E3 ligase for all three of the opioid receptors. Testing the ability of
SIAH2 to conjugate ubiquitin to the individual opioid receptors in in vitro ubiquitination
assays would confirm or refute SIAH2 as an E3 ligase for the opioid receptors. In
contrast, SIAH1 does not appear to be a putative E3 ligase for the unstimulated MOR, but
instead may be a shuttle protein or chaperone that escorts the MOR. However, my
results do not eliminate SIAH1 as a putative E3 ligase for DOR and KOR.
138
The precise role that ubiquitination by SIAH1 or SIAH2 may be playing in the
life cycle of the opioid receptors is still unknown. For the MOR, ubiquitination by
SIAH2 appears to eventually lead to receptor degradation. In the future it will be
important to determine whether SIAH2- mediated ubiquitination results in lysosomal or
proteasomal degradation of the MOR. For the DOR and KOR, the role of SIAH1 and/or
SIAH2 ubiquitination is much less clear. Ubiquitination of DOR and KOR may also lead
to degradation, but it may also result in trafficking of these receptors. Tracking the
intracellular location of opioid receptors under both unstimulated and agonist-stimulated
conditions may shed some light on a more precise role for ubiquitination in the life cycle
of these receptors.
In summary, utilization of traditional and modified yeast two-hybrid (Y2H)
methods identified ten novel and two previously reported MORIPs. Directed Y2H and
GST-PD studies were used to confirm interaction and map the binding site for several
MORIPs to the second intracellular loop of MOR. Many MORIPs were demonstrated to
interact with full length MOR, as well as the DOR and KOR in the context of cultured
human cells. Additionally, chronic morphine treatment was shown to affect the protein
levels of WLS, SIAH1, and DOK4 in specific brain regions of mice. Functional
characterization of two of these novel MORIPs, SIAH1 and SIAH2, has suggested
distinct roles for these proteins in the basal (unstimulated) regulation of the MOR.
SIAH2 is a putative E3 ligase for the MOR, whereas SIAH1 appears to play a role in the
trafficking of the MOR to the site of degradation. The Siah proteins also may
ubiquitinate and degrade or traffic the DOR and KOR. Identification and
characterization of the full panorama of MORIPs represents a critical step in
139
understanding the normal regulation of the receptor life-cycle, as well as how receptor
signaling or trafficking may be hijacked in response to drugs of abuse. The MORIPs
identified in this and other studies may represent key players involved in other opioid-
mediated processes such as neural development, nociception, aversion, and synaptic
plasticity. As the list of MORIPs grows, and their contributions to receptor function
become better understood, it is possible that some of these proteins will also become
targets for new drug development to prevent and/or treat opioid addiction.
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VITA Stephanie Justice-Bitner
EDUCATIONAL BACKGROUND
Master of Science in Veterinary Science, 2004
University of Nebraska, Lincoln, NE
Non-thesis, emphasis in immunology
Bachelor of Science in Microbiology, 1997
Colorado State University, Fort Collins, CO
PROFESSIONAL EXPERIENCE
Visiting Assistant Professor of Biology, Department of Biology 8/11-present
King’s College, Wilkes-Barre, PA
Adjunct Professor of Biology, Department of Biology 8/09-5-09
Elizabethtown College, Elizabethtown, PA
Temporary Faculty- Biology, Kulpmont/Berwick 8/04-12/05
Luzerne County Community College, Nanticoke, PA
Research Tech III, Veterinary Diagnostic Center 8/03-1/04
University of Nebraska, Lincoln, NE
PUBLICATIONS
Justice-Bitner S, Petko, J, Jin J, Wong V, Kittanakom S, Ferraro T, Stagljar I, and
Levenson R, "MOR is Not Enough: Identification of Novel mu-Opioid Receptor
Interacting Proteins Using Traditional and Modified Membrane Yeast Two-
Hybrid Screens" (Submitted)
ABSTRACTS
“Discovery of Novel mu-Opioid Interacting Proteins Involved in the Ubiquitin
Pathway.”
Ubiquitin Drug Discovery and Diagnostics Conference, Hyatt at the Bellevue,
Philadelphia, PA, October 2009.