Development and Validation of a Novel Quantitative Assay for

Cell Surface expression of GPCRs using a Receptor β-lactamase

Fusion Protein and the Colourometric Substrate Nitrocefin

by

Vincent Lam

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Pharmacology and Toxicology

University of Toronto

© Copyright by Vincent Lam 2013

ii

Development and Validation of a Novel Quantitative Assay for Cell

Surface Expression of GPCRs using a Receptor β-lactamase Fusion

Protein and the Colourometric Substrate Nitrocefin

Vincent Lam

Master of Science

Department of Pharmacology and Toxicology

University of Toronto

2013

Abstract

Trafficking of GPCRs is a dynamic process that is tightly regulated and sometimes defective in

human diseases. Therefore it is important to develop new methods to allow simple and

quantitative measurement of surface expression of membrane proteins. Here we describe

the development and validation of a new assay for quantification of cell surface expression of

GPCRs using β-lactamase as a reporter. For this assay we N-terminally fused β-lactamase (βlac)

to the β2-adrenergic receptor (β2AR) and GABA b R1 (GBR1). The results obtained by the βlac

assay are quantitatively and qualitatively similar to well established ELISA when measuring

agonist induced internalization of β2AR. We also show that measurement of GBR1 surface

expression with GBR2 co-expression is quantitatively identical between the βlac and ELISA. In

conclusion, our results show that our newly developed βlac assay is quantitatively similar while

being less expensive, more robust and higher throughput compared to an ELISA.

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Acknowledgments

I would like to thank the following people who have made completion of the thesis possible.

First I would like to express my thanks and gratitude to Dr. Ali Salahpour for his

guidance, mentorship, support, and endless positive encouragement throughout the

completion of this thesis.

I would also like to thank Dr. Jane Mitchell (co-supervisor) for her advice and critical

analyses of my work.

I would also like to thank Dr. Amy Ramsey and Dr. David Riddick (Advisor) for their

support, guidance, and suggestions.

Furthermore I would like to thank the members of the Ramsey and Salahpour lab for their

support and intellectual discussion during my studies.

Lastly I would like to thank my friends and family for their endless encouragement and

support throughout my time in graduate studies, without them this thesis would not have

been possible.

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Table of Contents

Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv

List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii List of Appendices .......................................................................................................................... x

Chapter 1 Introduction .................................................................................................................... 1 Specific Aims and Working Hypothesis ......................................................................................... 1 1.0 GPCR Pharmacology .......................................................................................................... 2 1.1 GPCR Classification ........................................................................................................... 3

1.2 Structure and function ......................................................................................................... 4 1.3 GPCR Signalling ................................................................................................................. 5

1.3.1 Ligand binding to receptor ...................................................................................... 5 1.3.2 G-proteins ............................................................................................................... 7

1.3.3 Effectors .................................................................................................................. 8 1.4 GPCR Trafficking from the ER .......................................................................................... 9

1.4.1 Signal Sequences .................................................................................................. 10

1.4.2 Post translational modifications ........................................................................... 12 1.4.3 Molecular Chaperones .......................................................................................... 14 1.4.4 Pharmacological Chaperones and Diseases ......................................................... 16

1.5 GPCR Oligomerization ..................................................................................................... 17 1.6 GPCR Endocytic Trafficking ............................................................................................ 18

1.6.1 G-protein Coupled Receptor Kinases (GRK) ....................................................... 19 1.6.2 Arrestins ................................................................................................................ 20

1.6.3 Tonic/Constitutive Internalization ........................................................................ 22 1.6.4 Endocytic pathway ................................................................................................ 22

1.7 Summary of Assays for measuring surface expression of GPCRs ................................... 23 1.7.1 Fluorogen Activating Protein Biosensor ............................................................... 25 1.7.2 Internalization Assays ........................................................................................... 26

1.7.3 N-terminal GPCR Fusion Tags ............................................................................. 27 1.8 β-lactamase Assay ............................................................................................................. 28

Chapter 2 Materials and Methods ................................................................................................. 33 2.1 Reagents ............................................................................................................................ 33 2.2 Plasmid Construction ........................................................................................................ 33

2.3 Cell Culture ....................................................................................................................... 33 2.4 Generation of Stable Cell Lines and Transient Transfections .......................................... 34 2.5 Western Blotting ............................................................................................................... 34

2.6 βlac-β2AR Immunofluorescence ...................................................................................... 34 2.7 βlac-β2AR Functional Assay using BRET EPAC cAMP Biosensor ............................... 35 2.8 βlac Assay ......................................................................................................................... 35 2.9 ELISA ............................................................................................................................... 35 2.10 βlac-β2AR Agonist Studies............................................................................................... 36 2.11 βlac-β2AR Antagonist Studies .......................................................................................... 36

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2.12 βlac-β2AR Z’ Determination for Agonist Induced Internalization................................... 36

2.13 GBR1 Molecular Chaperoning Studies ............................................................................ 36 2.14 Data Analyses ................................................................................................................... 37

Chapter 3 Results .......................................................................................................................... 38 3.1 Generation of the βlac Plasmids and Stable Cell Lines Expressing βlac-GPCR Fusion

Constructs ................................................................................................................................. 38 3.2 Trafficking and Signalling of the βlac-β2AR ................................................................... 38 3.3 βlac-β2AR experiments .................................................................................................... 42

3.3.1 Comparison of Isoproterenol stimulated β2AR Internalization using the βlac

and the ELISA Assays ...................................................................................................... 42 3.3.2 Antagonist Blocking of Isoproterenol Induced Internalization of the β2AR ........ 46 3.3.3 Z’ Determination of the SS-HA-βlac-β2AR Internalization ................................. 46

3.3.4 Pharmacological Chaperoning using β2AR Antagonists ...................................... 48 3.4 Comparison of GBR1 Surface Expression using the βlac and the ELISA ....................... 50

Chapter 4 Discussion .................................................................................................................... 52

4.1 Summary of Key Findings ................................................................................................ 52 4.2 Nitrocefin Permeability ..................................................................................................... 52 4.3 Functional Experiments with the SS-HA-βlac-β2AR ....................................................... 52

4.4 SS-HA-βlac-β2AR Internalization .................................................................................... 55 4.5 Pharmacological chaperoning ........................................................................................... 57

4.6 Z’ Factor of βlac-β2AR internalization ............................................................................ 58 4.7 SS-HA- βlac-GBR1 Surface Expression by Co-expression with GBR2 .......................... 60 4.8 Cost Analyses .................................................................................................................... 60

Conclusion .................................................................................................................................... 61

References ..................................................................................................................................... 63 Appendices .................................................................................................................................... 83

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List of Tables

Table 1: Comparison of time and cost of ELISA and βlac assays ................................................ 62

vii

List of Figures

Figure 1.1: The conventional GPCR activation model of GPCR signaling ................................... 6

Figure 1.2: Schematic of the βlac assay ........................................................................................ 29

Figure 1.3: Nitrocefin as a chromogenic substrate for the βlac assay .......................................... 30

Figure 1.4: Mechanism of action for a class A β-lactamase ......................................................... 32

Figure 3.1: Western blot of stable clonal cell line expression levels of SS-HA-βlac-GBR1 and

SS-HA-βlac-β2AR ........................................................................................................................ 39

Figure 3.2: Immunofluorescence of HEK293 cells stably expressing SS-HA-βlac-β2AR .......... 41

Figure 3.3: β2AR functional assay using the BRET cAMP EPAC biosensor .............................. 43

Figure 3.4: Comparison of βlac and ELISA with the SS-HA-βlac-β2AR stable cell line ............ 44

Figure 3.5: Blocking of isoproterenol induced internalization with pre treatment of antagonists 45

Figure 3.6: Z’ of the βlac assay using the SS-HA-βlac-β2AR stimulated with isoproterenol ...... 47

Figure 3.7: Overnight incubation with alprenolol and propranolol increases surface expression of

SS-HA-βlac-β2AR ........................................................................................................................ 49

Figure 3.8: Comparison of βlac assay and ELISA Surface expression of GBR1 with GBR2 co-

expression ..................................................................................................................................... 51

Figure 4.1: Representative diagram for the BRET EPAC cAMP Biosensor ................................ 54

viii

List of Abbreviations

A1AR α1 adrenergic receptor

ANOVA Analysis of variance

AP2 Adaptor protein 2

β1AR β1 adrenergic receptor

β2AR β2 adrenergic receptor

β3AR β3 adrenergic receptor

βlac β-lactamase

cAMP Cyclic adenosine monophosphate

COPII Coat protein 2

CRLR Calcitonin receptor-like receptor

C-terminal Carboxyl-terminal

DAG Diacylglycerol

DOR δ-opioid receptor

DRIP 78 Dopamine receptor-interacting protein

EC50 Median effective concentration at 50%

ELISA Enzyme linked Immunosorbent Assay

EPAC Exchange proteins activated by cAMP

ER Endoplasmic Reticulum

FAP Fluorogen activating protein

GABA γ-aminobutyric acid

GBR1, GBR2 Metabotropic GABA bR1, bR2

GDP Guanosine diphosphate

GFP Green fluorescent protein

GRK G-protein coupled receptor kinase

GPCR G-protein coupled receptor

GTP Guanosine triphosphate

HA Hemaglutinin

HEK 293 Human embryonic kidney cells

HRP Horseradish peroxidase

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HTS High throughput screening

IC50 Median inhibitory concentration at 50%

IP3 Inositol 1,4,5-trisphosphate

mGluR Metabotropic glutamate receptor

MRAP Melanocortin-2 receptor accessory protein

NDI Nephrogenic diabetes insipidus

N-terminal Amino-terminal

PAR1 Protease activated receptor 1

PIP2 Phosphatidylinositol 4,5-bisphosphate

PKA, PKC Protein kinase A, C

PLCβ Phospholipase C β

RAMP Receptor activity modifying proteins

REEP Receptor expression enhancing protein

RTP Receptor transporting protein

RGS Regulators of G protein signalling

Rluc Renilla luciferase

TAAR1 Trace amine associated receptor 1

TM Transmembrane

TPβ Thromboxane A2 β receptor

V1R Vasopressin 1 receptor

V2R Vasopressin 2 receptor

WT Wild type

YFP Yellow fluorescent protein

x

List of Appendices

Appendix Figure 1: Cell permeability of the βlac substrate nitrocefin ......................................... 83

Appendix Figure 2: Dose response of isoproterenol mediated internalization quantified with flow

cytometry ...................................................................................................................................... 84

Appendix Figure 3: Time course of isoproterenol mediated internalization quantified with flow

cytometry ...................................................................................................................................... 85

Appendix Figure 4: Functionality of SS-HA-βlac-GBR1 using the BRET EPAC cAMP biosensor

....................................................................................................................................................... 86

1

Chapter 1 Introduction

Specific Aims and Working Hypothesis

Surface expression of G-protein coupled receptors (GPCR) is crucial for the correct

function of the receptor. Within the past 20 years proper trafficking of GPCRs and other

membrane proteins has seen an increase in interest within the scientific community. Specifically

the trafficking of GPCRs to the plasma membrane is of interest. Indeed there are an increasing

number of GPCRs that require the use of specific molecular chaperones to aid in their proper

trafficking to the plasma membrane (see section 1.4.3). In addition there are an increasing

number of diseases that are linked to alterations in normal trafficking of GPCRs and other

membrane proteins (see section 1.4.4). Therefore there is a need for fast and robust assays that

can quantify surface expression and the effects of small molecules or proteins on the trafficking

of GPCRs. Unfortunately the current assays that are commonly used for measuring surface

expression of GPCRs, while being robust and quantitative, are time consuming and relatively

low throughput. In this context, an assay that is capable of high throughput screening would

provide a powerful tool in the search for specific ligands or proteins that can affect the surface

expression of membrane proteins. Indeed, within the last 5 years, there has been several novel

assays created that address the current limitations as discussed above (see section 1.8). Here we

describe a new assay for the quantification of GPCRs on the plasma membrane using an N-

terminal β-lactamase-GPCR fusion protein (βlac-GPCR). We hypothesize that this new assay has

the ability to quantify receptor surface expression in an equivalent manner to current standard

assays such as the enzyme linked immunosorbent assay (ELISA). In addition we also aim to

optimize and miniaturize this assay for use in a high throughput manner. We validated this assay

according to the following aims with well-established trafficking profiles of two separate

GPCRs.

Aim 1: β2-adrenergic receptor (β2AR) internalization

The β2-adrenergic receptor (β2AR) is a prototypical GPCR that has been used to validate

multiple surface expression assays (Hammer et al., 2007; Fisher et al., 2010; Yano et al., 2012).

In our first aim, we created a stable cell line expressing a βlac-β2AR construct which was used to

2

validate our assay. The internalization of β2AR is well characterized with reproducible EC50 and

half-life values for internalization upon stimulation with isoproterenol, a β2AR full agonist.

Taking advantage of this internalization profile, we compared the βlac assay in parallel with an

ELISA. In addition, we looked at the ability of antagonists to block ligand induced

internalization of β2AR using the βlac assay. Lastly it has been shown that the β2AR/β1AR

selective antagonists alprenolol and propranolol can act as pharmacological chaperones

increasing the surface expression of the β1AR and potentially β2AR (Kobayashi et al., 2009).

We used the βlac assay in order to assess whether these antagonists can increase surface

expression of the β2AR. In summary we compared the ability of our new βlac assay with an

ELISA for the general internalization profile of β2AR. This profile includes the time course and

dose response of isoproterenol mediated internalization as well as antagonist blockade of

isoproterenol induced internalization. Lastly, pharmacological chaperoning effects of alprenolol

and propranolol were assessed by overnight treatment of βlac-β2AR cells.

Aim 2: GΑBA b R1 (GBR1) surface expression

In the second aim we used the metabotropic GΑBA b R1 (GBR1) receptor. The GBR1

receptor has a well characterized surface expression profile where the receptor is retained in the

endoplasmic reticulum (ER) unless co-expressed with its molecular chaperone GΑBA bR2

(GBR2). We compared the ability of the βlac assay and ELISA to measure surface expression of

GBR1 by transiently transfecting GBR2 into stable cell lines expressing a βlac-GBR1.

Overall, we validated the βlac assay as being quantitatively equivalent to an ELISA while being

less expensive, more robust and higher throughput than an ELISA.

1.0 GPCR Pharmacology

With over 800 known members, GPCRs are the largest family of receptors in the human

genome (Audet and Bouvier, 2012) where these receptors are currently the target of

approximately 40% of the prescribed drugs on the market (Wise et al., 2002). These receptors

play pivotal roles in a wide range of biological functions that include but are not limited to:

olfaction, cardiovascular function, and neurotransmission (Ferguson et al., 1998). In the

introduction, we will present a general overview on the current state of GPCR knowledge with

specific emphasis on GPCR trafficking and assays for measuring surface expression.

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1.1 GPCR Classification

With over 800 known receptors, GPCRs represent the largest class of receptors in the

human genome (Foord, 2002). Although GPCRs all share similar signalling mechanisms and

receptor topology, there is very little sequence homology shared between receptors. Therefore

GPCRs have been divided into five or six (depending on classification criteria) major families of

receptors based primarily on sequence similarity (Foord et al., 2005). The vast majority of

GPCRs are classified into the three following classes: Class A (rhodopsin-like), B (secretin-like),

and C (metabotropic glutamate-like) (Foord et al., 2005). The details regarding the classification

criteria for GPCRs are beyond the scope of this study, for a detailed description please see the

following review from the International Union of Pharmacology (Foord, 2002).

Class A GPCRs (rhodopsin-like) comprise the largest family of GPCRs with roughly 700

genes found in the human genome (Fredriksson et al., 2003). It is important to note that olfactory

receptors make up the bulk of the class A family of receptors consisting of over 65% of the

receptors within this class. Class A receptors in general have two conserved motifs. The first

motif is the DRY motif found in the third transmembrane domain; where this motif has been

shown to be involved in the activation of various members of the class A family through an

‘ionic lock’ with the sixth transmembrane domain (Scheer et al., 1996; Rasmussen et al., 1999;

Ballesteros et al., 2001; Rovati et al., 2007). In addition the NPXXY motif is also a highly

conserved motif within this family of receptors and is involved in signalling as well as

internalization of GPCRs (Bouley et al., 2003; Fredriksson et al., 2003).

Class B GPCRs (secretin-like) are characterized by their large (~100 amino acid) cysteine

rich N-terminal domain responsible for ligand binding. In contrast to class A receptors, class B

receptors bind peptides as their endogenous ligands (Harmar, 2001). Further distinction between

Class B and Class A receptors are the absence of the DRY and NPXXY motifs in class B

receptors. However it has been proposed that the motifs REY and VAVLY act as equivalent

motifs to the DRY and NPXXY motifs respectively for class B receptors (Frimurer and Bywater,

1999; Vohra et al., 2013).

Class C receptors (metabotropic-glutamate like) are characterized by their large 500-600

amino acid N-terminal domains. The large N-terminal domain binds the ligands and consists of a

two lobed structure termed the ‘venus fly trap’ domain for the mechanism by which it binds

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ligands (Bräuner-Osborne et al., 2007). However, the mechanisms by which the binding of a

ligand to the venus fly trap domain translates into GPCR signalling remains elusive (El

Moustaine et al., 2012). In addition, a unique distinction for class C receptors is the finding that

all these receptors function as dimers (Bräuner-Osborne et al., 2007; El Moustaine et al., 2012).

Interestingly a subset of class C receptors contain the DRY motif found in class A receptors (Pin

et al., 2003). Lastly class C receptors contain a well conserved xPKxY domain that has been

proposed to function similarly to the NPXXY domain of class A receptors (Pin et al., 2003).

1.2 Structure and function

GPCRs are 7-transmembrane integral proteins localized predominately at the plasma

membrane with evidence of some receptors localized to intracellular membranes as well

(Tadevosyan et al., 2012). All GPCRs share the same general topography: a plasma membrane

spanning 7-transmembrane hydrophobic core, three intracellular loops, three extracellular loops,

an extracellular N-terminal domain, and an intracellular C-terminal domain (Baldwin, 1993;

Bockaert and Pin, 1999; Duvernay et al., 2005). Although this topology and organization of

GPCRs has been proposed for many years, this layout has only been recently confirmed with the

crystallization of mammalian GPCRs starting with bovine rhodopsin (Palczewski et al., 2000).

Due to a methodological breakthrough there has recently been a large increase of solved crystal

structures starting with the 2AR (Rasmussen et al., 2007). Indeed at the time of writing of this

section, there are 57 structures solved for 13 distinct GPCRs all within the class A family of

GPCRs (see section 1.1). These crystal structures have confirmed the structure of GPCRs

illustrating the high diversity of orthosteric binding sites, as well as revealing the crucial link

between ligand binding and receptor activation. Even though early structures were crystallized

with agonists or antagonists bound, a surprising observation has been the similarity of the

tertiary structure for these ligand bound GPCRs (see review Audet and Bouvier, 2012). Indeed

the apparent similarity between antagonist and agonist bound structures can be attributed to the

lack of co-crystallization of the G-proteins, validating the model that GPCR activation requires

both the binding of ligand and the presence of the G-protein.

Fortunately, recent innovation in the crystallization methods have also yielded receptors

co crystallized with G-proteins where further insight into GPCR activation has been achieved

through the analysis of three separate crystal structures. The first was the crystallization of the

5

ligand free form of opsin (active-like state of rhodopsin) with co-crystallization of the C-terminal

domain of transducin, the Gα subunit for rhodopsin (Scheerer et al., 2008). This structure when

compared to the inactive rhodopsin structure, showed an outward movement of TM VI which

forms the binding site for transducin. Subsequently two additional structures added to this

observation and confirmed the movement of TM VI as a function of G-protein binding and

activation. These structures were the co-crystallization of the agonist bound β2AR and the

heterotrimeric G-protein (Rasmussen et al., 2011a) and the agonist bound β2AR with a nanobody

mimicking Gαs (Rasmussen et al., 2011b). Additionally, these structures provided a mechanism

for how GPCRs facilitate nucleotide exchange upon the activation of the receptor. In the

activated agonist bound GPCR, the outward movement of the TM VI domain causes a 130°

rotation of the Gα subunit. It is proposed that this rotation in the Gα subunit allows for the

exchange of GDP for GTP (Rasmussen et al., 2011b). Although these structures have provided

invaluable information for how GPCRs are activated, there are still some questions that remain.

For instance, this model shows one receptor activating one G-protein complex, however it is well

established that some functional receptor complexes require GPCR dimers (see section 1.5).

Therefore it is still unclear how these dimers affect signalling or binding to G-proteins.

1.3 GPCR Signalling

The simplest model of GPCR signalling involves three steps. First, ligand binding to the

receptor, where each family of GPCR, in general, has specific endogenous ligands that bind the

receptors with high affinity. Once an agonist is bound and the receptor is activated the second

step of GPCR signalling occurs, whereby the receptor acts to catalyze the exchange of GDP for

GTP on the Gα subunit of the heterotrimeric Gαβγ G-protein complex. The third step involves

the Gα subunit and Gβγ dimer interacting with membrane bound/associated effector proteins or

ion channels leading to an increase/decrease in cytosolic secondary messengers (see Figure 1.1

for more detail of this model) (Luttrell, 2006).

1.3.1 Ligand binding to receptor

The first step of GPCR signalling involves the binding of the extracellular ligand to the GPCR.

GPCR ligands are separated into two separate classes: agonists and antagonists. Agonists are

ligands that promote receptor activation while antagonists are ligands that block activation of the

receptor. Antagonists can be further separated into two subclasses: inverse-agonists, ligands

6

Figure 1.1: The conventional GPCR activation model of GPCR signaling. There are three

main components to this model of signaling. First an extracellular hormone/ligand (H) binds to

the 7-TM protein that is coupled to a heterotrimeric G-protein. Upon hormone binding, the

receptor is activated and catalyzes the exchange of GDP to GTP for the Gα subunit of the

heterotrimeric G-protein complex. The binding of GTP causes the trimer to dissociate into the

GTP-Gα subunit and the Gβy heterodimer. These dissociated G-proteins activate effector

proteins located on the plasma membrane which activate or inhibit secondary messenger

production. Typical effector proteins include enzymatic effectors (ie adenylyl cyclase and

phospholipases) and ion channels. Signaling is halted once the intrinsic GTPase of the Gα

subunit hydrolyzes the GTP to GDP (Luttrell, 2006).

7

that block intrinsic activity of a GPCR, and neutral antagonists that have no effect on the

activation state of the receptor (Audet and Bouvier, 2012). In order to predict and correctly

model GPCR signalling using a mathematical model, several factors have to be considered. First

and most importantly, the binding of a ligand to the receptor can be affected by the presence or

absence of G-proteins. This was first observed in the 2AR where it was shown that the receptor

exists in high and low affinity receptor states for the binding of ligands to the receptor. To

account for this observation it was discovered that G-protein binding to the receptor acts as an

allosteric modulator that can alter the affinity state of the receptor for ligands. Therefore to

model this effect, the ternary complex model (De Lean et al., 1980) was created to predict

agonist binding and the effects of an allosteric modulator, in this case G-proteins. Furthermore

the discovery of GPCR constitutive activity (receptor activity in the absence of agonist binding)

led to the addition of active (R*) and inactive (R) states of the receptor in the extended ternary

complex (Samama et al., 1993). Although the extended ternary complex model is the most

accepted model for GPCR signalling through G-proteins, other models have been generated to

take into account the effects of biased signalling (Whistler and von Zastrow, 1998; Holloway et

al., 2002; Kohout et al., 2004; Swaminath et al., 2004) and arrestin binding (Gurevich et al.,

1997) on effects of ligand binding to the GPCR. For a detailed description of current state of

receptor ligand binding and activation, refer to the following reviews (Kenakin, 2002, 2003,

2011).

1.3.2 G-proteins

The second component for GPCR signalling is the activation and dissociation G-proteins

from the receptor. The heterotrimeric G-protein complex is composed of three subunits: the Gα

GTPase, Gβ, and Gγ. The complex is associated together when the Gα subunit is in its inactive

GDP bound form. In its inactive form, the heterotrimeric G-protein is associated with the

receptor complex. As described in figure 1.1, GPCR activation induces the exchange of GDP to

GTP in the Gα subunit causing the heterotrimer to dissociate from the receptor into membrane

tethered Gα and Gβγ subunits. Once dissociated, the Gα and Gβγ subunits interact with

membrane bound effector proteins. The subsequent response is determined by the structural

composition of the G-protein subunits. There are 16 genes encoding for Gα subunits yielding a

total of 20 expressed Gα subunits through alternative splice variants. The Gα subunits can be

separated into four families based upon sequence homology: Gαs, Gαi, Gαq, and Gα12. The Gαs

8

family (Gαs and Gαolf) of G-proteins are stimulatory toward adenylyl cyclase causing an

increase in intracellular cAMP. Conversely the Gαi family (Gαi1-3, Gαt1-2, Gαo1-2, Gαz and

Gαgust) are in general inhibitory to adenylyl cyclase causing a decrease in intracellular cAMP,

although Gαt is an activator for the cGMP phosphodiesterase 6 (Hingorani and Ho, 1987). The

Gαq family (Gαq, Gα11, Gα14, and Gα15) activates membrane associated phospholipase C β

(PLCβ) forming inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from

phosphatidylinositol 4,5-bisphosphate (PIP2). Lastly the Gα12 family (Gα12 and Gα13)

activates Rho-specific guanine nucleotide exchange factors (Tanabe et al., 2004; Luttrell, 2006).

In addition to the Gα subunits the Gβγ heterodimers also have important functions in GPCR

signalling. Currently, there are 5 and 12 known Gβ and Gγ subunits respectively. Although there

are almost 1000 possible combinations of Gβγ heterodimers with Gα subunits, whether all these

combinations exist in vivo is currently unknown (see review Hildebrandt, 1997). Nevertheless

there are common combinations of the Gβγ heterodimer found expressed in specific tissues. For

example the Gβ1γ1 is the predominant heterodimer found associated with the Gαt in the retina

(Ford et al., 1998). Although it was not initially recognized, it is now generally accepted that Gβγ

subunits can mediate as many functions as Gα subunits (Milligan and Kostenis, 2006) and

activate or inhibit effectors on their own such as the PLC-β isoforms, adenylyl cyclase, ion

channels, and GRKs (Ford et al., 1998).

1.3.3 Effectors

The last component of GPCR signalling are the effectors that are modulated by the

binding of the G-protein subunits (Clapham and Neer, 1993). The most well studied effectors are

the family of adenylyl cyclases that convert intracellular ATP to the secondary messenger cyclic

adenosine monophosphate (cAMP). The 10 members of the 12-transmembrane adenylyl cyclase

family of proteins can all be activated by Gαs where most adenylyl cyclases are inhibited by Gαi

or Gβγ G-proteins (Sunahara et al., 1996). Lastly it has been shown that Gβγ can also interact

and activate or inhibit adenylyl cyclase (Rebois et al., 2006; Wang and Burns, 2006; Boran et al.,

2011). Once the adenylyl cyclase has been activated the secondary messenger, cAMP, in turn

activates further downstream signalling cascades. Some important proteins that are directly

activated by cAMP include, but are not limited to, the cAMP-dependent protein kinases (PKA)

and cAMP-regulated guanine nucleotide exchange factors (EPAC) (Zwartkruis and Bos, 1999).

9

In addition to adenylyl cyclases, the second major class of effector proteins are the PLCβ

family (Isoforms 1-4). These lipases hydrolyze membrane bound PIP2 to yield the intracellular

second messenger IP3 and the membrane bound DAG. The cytosolic IP3 binds to ligand gated

calcium channels found on the ER releasing calcium into the cytoplasm. Meanwhile DAG is the

primary activator of several isoforms of the membrane bound protein kinase C (PKC) (Morris

and Scarlata, 1997).

Lastly ion channels can be inhibited or activated by G-protein subunits. For example, the

‘N-type’ calcium channels are inhibited by Gαo and βγ (Albert and Robillard, 2002), the ‘L-

type’ calcium channels are activated by Gαs, and the inward rectifying muscarinic gated

potassium channels are regulated by the Gβγ subunits (Wickman and Clapham, 1995; Rebois et

al., 2006).

The main mechanism of stopping GPCR signalling is through the hydrolysis of the GTP

to GDP by the Gα subunit. This GTPase activity of the Gα subunit can be enhanced by a family

of regulators of G protein signalling (RGS) proteins. For details on the role of RGS see the

following review (Kach et al., 2012). Further desensitization occurs at the receptor level and is

explained in section 1.6.

Although GPCR signalling through G-proteins has been well established as the canonical

signalling pathway, over the last decade, extensive studies have shown that GPCRs can also

signal in non-canonical pathways that are G-protein independent (see section 1.6.2).

1.4 GPCR Trafficking from the ER

Receptor trafficking is a dynamic process that contributes to the regulation and proper

function of GPCRs. While the endocytic pathway has been studied extensively for GPCRs, the

molecular mechanisms for GPCR trafficking from the ER to the plasma membrane is less

understood (Duvernay et al., 2005; Dong et al., 2007). The general mechanism for ER export of

GPCRs first involves translation and folding of the receptor in the ER, where the receptors are

subsequently packaged into transport vesicles and targeted to the ER-Golgi intermediate

complex. Incomplete or misfolded receptors in the ER are ubiquitinated and degraded in a

process known as ER-associated degradation (Meusser et al., 2005). During receptor maturation

various post translational modifications (see section 1.4.2) occur while the receptor is transported

10

from the ER to the ER-Golgi intermediate complex and finally to the trans-Golgi network before

being delivered to the plasma membrane (see review Duvernay et al., 2005). For GPCRs,

trafficking and transport from the ER to the plasma membrane is generally considered the rate

limiting step in their biogenesis (Petaja-Repo et al., 2000). The following section will give a brief

overview of the different factors that can affect the trafficking of GPCRs from the ER to the

plasma membrane.

1.4.1 Signal Sequences

The first determinant for cell surface targeting is found in the primary amino acid

sequence in the form of distinct and conserved motifs. It is well established that specific motifs

aid in the export of membrane proteins from the ER. In general, there are two classes of ER

export motifs for non-GPCRs. The first is a diacidic DXE motif (Nishimura and Balch, 1997)

that is required for the proper surface expression of transmembrane proteins such as CFTR

(Wang et al., 2004) and the Kir2.1 potassium channel (Ma et al., 2001). The second class is the

dihydrophobic (FF) motif that is required for the trafficking of the following membrane proteins:

ERGIC-53 (Nufer et al., 2003), Erv41-46 (Otte and Barlowe, 2002), and p24 family of

proteins(Dominguez et al., 1998).

In contrast, GPCRs have no consensus export motifs that are found between different

classes of receptors. Nevertheless there are receptor specific motifs located on the membrane

proximal C-terminal domain of GPCRs that are essential for the proper ER export. To date four

distinct C-terminal motifs required for ER export of specific GPCRs have been identified. These

motifs include the following: E(X)3LL on the vasopressin 2 receptor (V2R) (Schülein et al.,

1998), F(X)3F(X)3F on the Dopamine D1 receptor (Bermak et al., 2001), FN(X)2LL(X)3L on the

vasopressin 1B and vasopressin 3 receptors (Robert et al., 2005), and F(X)6LL on the α2

adrenergic receptor (α2AR) and angiotensin II type 1 receptor (Duvernay et al., 2004).

Interestingly although these motifs are distinct they all share the common feature of having

hydrophobic residues spaced apart in such a way as to reside on one side of an alpha helix.

Despite sharing similar chemical features, it is important to note that these motifs are receptor

specific. For instance mutating the FN(X)2LL(X)3L motif of the vasopressin 1B receptor to

F(X)6LL found on the α2AR, caused the receptors to be retained in the ER rather than being

11

targeted to the plasma membrane indicating that the FN(X)2LL(X)3L motif is necessary for

proper surface expression of the vasopressin 1B receptor (Robert et al., 2005).

In addition to the motifs located on the C-terminus, the intracellular loops and even the

N-terminus of GPCRs can also regulate export from the ER to the plasma membrane. For

example, in the α2bAR a triple arginine motif in the third intracellular loop is required for proper

binding of COPII transport vesicles that mediate receptor export from the ER (Dong et al., 2012).

In numerous other class A GPCRs, a single conserved leucine in the intracellular loop 1 is

required for the proper export of the receptors from the ER (Duvernay et al., 2009). Lastly the

motif ALAAALAAAAA in the α2cAR is found on the extracellular N-terminus and functions to

aid the trafficking of the receptor to the plasma membrane (Angelotti et al., 2010).

In general the interaction between the coat protein 2 (COPII) in transport vesicles and the

non-GPCR-ER export motifs is the main mechanism of membrane protein export from the ER. It

is well established that the DXE and FF motifs, present in non-GPCRs, interact with components

of the COPII complex to mediate ER export (Miller et al., 2003). However for GPCRs, the

mechanisms by which the above mentioned ER export motifs mediate surface expression is

currently unknown. It is hypothesized that these motifs traffic GPCRs to the plasma membrane

by one of the following mechanisms. Firstly, like the non-GPCR export motifs, the interaction of

these motifs with proteins in the COPII complex has been suggested. The only evidence for

direct binding of GPCRs to a COPII complex are from studies on a triple arginine motif present

in the third intracellular loop of several GPCRs (Dong et al., 2012). The second potential

mechanism is through the direct binding of GPCR with specific molecular chaperones. Indeed

this mechanism has been shown for the F(X)3F(X)3F motif of the dopamine D1 receptor which

interacts with the ER chaperone dopamine receptor-interacting protein 78 (DRIP 78) (Bermak et

al., 2001). The third mechanism involves the participation of these motifs in the dimerization

with other receptors. However there is no evidence to support this mechanism. For example

mutation of the F(X)6LL motif in the α2bAR retains the receptors ability to form dimers (Zhou et

al., 2006). Lastly it is hypothesized that the motifs themselves aid in the folding and trafficking

of the receptor in a yet unknown mechanism (Duvernay et al., 2005). In the end it can be seen

that ER export motifs are important for certain receptors, however the specific mechanism of

how these motifs aid in ER export appears to be different for each receptor.

12

In contrast to ER export motifs, some receptors contain ER retention motifs on the C-

terminus. As with ER export motifs, there are three conserved ER retention motifs (KDEL,

KKXX, and RXR (Dong et al., 2007)) found for membrane proteins. However, only one of the

motifs mentioned above has been found to be present in GPCRs (the RXR motif). The RSRR

motif of the GBR1 acts to retain the receptor in the ER and only upon binding with GBR1’s

molecular chaperone, GΒR2, does the retention motif become masked (Margeta-Mitrovic et al.,

2000a). In addition, the metabotropic glutamate receptor splice variant 1b is retained in the ER

due to the presence of the RRKK motif in the C-terminus (Chan et al., 2001). Unlike the GBR1

the mechanism by which mGluR1b is trafficked to the plasma membrane is unknown. Although

ER export motifs can be present in both the C-terminus and intracellular loops, it is important to

note that the ER retention motifs only retain receptors if present on the C-terminus. For example

the V2R contains two RXR motifs in the third intracellular loop, however only truncation

mutants of this receptor, where the motifs are now present at the C-terminus, show a phenotype

of ER retention (Hermosilla and Schülein, 2001).

As noted above, the presence of specific motifs on the intracellular portion of the GPCR

is important for either the export or retention in the ER. However unlike other membrane

proteins, there is a lack of consensus motifs that are present in more than one receptor indicating

that the vast majority of receptors are targeted to the plasma membrane via a mechanism that is

currently not known.

1.4.2 Post translational modifications

GPCRs go through a number of post translational modifications that can affect function

and expression of the receptors. In general, most receptors undergo N-linked glycosylation

(Renthal et al., 1973) as well as O-linked glycosylation (Sadeghi and Birnbaumer, 1999; Petaja-

Repo et al., 2000; Nakagawa et al., 2001). N-linked glycosylation is the most common post

translational modification and occurs at the consensus sequence NXS/T (Nita-Lazar et al., 2005).

N-linked glycosylation is reported to affect the targeting of GPCRs to the cell surface although

this effect is receptor specific. For example the mutation of two N-terminal glycosylation sites on

the β2AR show a marked decrease in the surface expression of the receptor but with no change

in function for the receptor that is on the plasma membrane (Rands et al., 1990). Furthermore

when the same two N-terminal glycosylation sites of β2AR are added to the trace amine

13

associated receptor 1 (TAAR1) there is an increase in the surface expression of this receptor

which otherwise is normally retained in the ER (Barak et al., 2008). However when the

glycosylation sites of the AT1R (Deslauriers et al., 1999) and FSH (Davis et al., 1995) receptors

are removed, the receptors are retained in the ER. In contrast, the surface expression of the

muscarinic M2 (van Koppen and Nathanson, 1990), histamine H2 (Fukushima et al., 1995) and

α1AR (Sawutz et al., 1987) are not affected when the N-glycosylation sites are removed.

Although N-linked glycosylation is the most common glycosylation, receptors can also

undergo O-linked glycosylation. However, the precise role and function of O-linked

glycosylation for GPCRs is not well understood. While there is no consensus sequence for O-

linked glycosylation, it is known to occur within the golgi apparatus where serine and threonine

residues are glycosylated (Duvernay et al., 2005). Much as for N-glycosylation, effects of O-

glycosylation on receptor function are not universal. For example, the removal of O-linked

glycosylation sites in the V2R receptor does not alter the surface expression of that receptor

(Sadeghi and Birnbaumer, 1999). In contrast, O-glycosylation is essential for the maturation and

surface expression of the δ-opioid receptor (DOR) (Petaja-Repo et al., 2000). Therefore it can be

seen that glycosylation is an important post translational modification for most receptors.

However much like the ER export and retention motifs, the effects on receptor surface

expression are receptor specific and not universal to all GPCRs.

The third most common post translational modification to GPCRs involves

palmitoylation. GPCR palmitoylation is a dynamic and reversible process of the addition or

removal of palmitic acid to mostly cysteine residues via a thioester bond (Qanbar and Bouvier,

2003). Palmitoylation has been shown to occur early in the maturation process of GPCRs in the

ER where palmitoylation is a requirement for proper GPCR targeting to the plasma membrane of

several GPCRs (Karnik et al., 1993; Zhu et al., 1995; Schülein et al., 1996; Fukushima et al.,

2001; Percherancier et al., 2001). Apart from palmitoylation’s role in surface expression,

palmitoylation can act to orientate the C-terminal tail in a position to increase the affinity for

arrestin binding and internalization of the receptor. This is clearly seen with the V2R receptor

where palmitoylation deficient mutants are internalized much less efficiently upon stimulation of

agonists even though their phosphorylation states are similar to the WT receptor (Charest and

Bouvier, 2003).

14

In sum, glycosylation and palmitoylation have large impacts on not only receptor

trafficking to the plasma membrane but on the signalling of receptors as well. The effects of

these post translational modifications are elegantly reviewed in the following references (Qanbar

and Bouvier, 2003; Duvernay et al., 2005)

1.4.3 Molecular Chaperones

As explained in the previous section, some GPCRs require post-translational

modifications for proper trafficking to the plasma membrane, such as presence of ER export

motifs, or the masking of ER retention motifs. There are a multitude of different GPCR

chaperone proteins that can be classed into proteins that bind the C-terminus, intracellular loops,

and the N-terminus. For a review of different interactor proteins that promote and increase

surface expression see the following reviews (Duvernay et al., 2004; Dong et al., 2007; Dunham

and Hall, 2009). For the purposes of this thesis, we will focus on the interactors that are required

for the surface expression of GPCRs which we term here as molecular chaperones.

Some receptors require the binding of specific chaperones for proper trafficking to the

plasma membrane. The existence of molecular chaperones was postulated based on the

observation that certain receptors expressed little to no functional protein when expressed in

heterologous systems (McClintock et al., 1997; Couve et al., 1998; Borowsky et al., 2001;

Bunzow et al., 2001).

The first class of chaperones are a general class of ER localized chaperones that bind and

aid the folding of receptors, these include calnexin, calreticulin and ER heatshock proteins such

as Grp78 and Grp94 (Siffroi-Fernandez et al., 2002; Mizrachi and Segaloff, 2004). The function

of these chaperones is to promote the correct folding of GPCRs as well as aiding the trafficking

of correctly folded receptors to the plasma membrane. For instance calnexin and calreticulin

recognize and bind to early glycosylated receptors and promote the N-linked glycosylation of

certain receptors (Williams, 2006). On the other hand, heatshock proteins such as Grp78 and

Grp94 aid the proper folding of receptors by binding the exposed hydrophobic patches of newly

synthesized proteins in the ER (Hamman et al., 1998).

The second class of chaperones are specific for GPCRs and are known as molecular

chaperones. The first example of such molecular chaperones is other GPCRs themselves. As

15

described in section 1.4.3 the GBR1 receptor contains an ER retention motif on its C-terminus

(Couve et al., 1998). It is upon dimerization with its molecular chaperone, GΒR2, that proper

surface expression of the GBR1 receptor is achieved (White et al., 1998), whereby the

dimerization of GΒR2 masks the retention signal of GBR1 through a coiled coil interaction of

the two C-terminal domains (Margeta-Mitrovic et al., 2000b; Villemure et al., 2005). Similarly

the dimerization of α1dAR with α1bAR or α2bAR strongly promotes the surface expression of

the α1dAR (Uberti et al., 2003, 2005; Hague et al., 2004). Conversely dimerization can retain

receptors in the ER rather than promote surface expression. The clearest examples are the

following dominant negative interactions of receptors. For example the heterodimerization of the

common DOR Cys-27 with the wild type DOR Phe-27 caused a decrease in surface expression

of the WT DOR Phe-27 (Leskelä et al., 2012). This is also seen when a mutated β2AR, that

harbours an ER retention motif, is able to significantly decrease the surface expression of a co-

expressed WT β2AR (Salahpour et al., 2004).

In addition to GPCRs, there are an increasing number of single transmembrane proteins

that act as molecular chaperones for specific receptors. These small proteins act as molecular

chaperones but have also been shown to modify receptor signalling as well. The first of these

small membrane proteins discovered are the receptor activity modifying proteins 1-3 (RAMPs).

RAMPs are single transmembrane proteins that are key regulators of receptor trafficking and

signalling. RAMPs act as molecular chaperones for the calcitonin receptor-like receptor

(CRLR) where binding with RAMPs enables proper surface expression of the receptor

(McLatchie et al., 1998). In addition to the CRLR it has been shown that RAMPs interact and

promote the surface expression of several other class B and C GPCRs (reviewed by Hay et al.,

2006).

The family of receptor transporting proteins (RTP 1,1s,2-4) are also single

transmembrane proteins that act as molecular chaperons for odorant receptors (Saito et al., 2004).

REEP1 (receptor expression enhancing protein 1), which is also a single transmembrane protein,

also increases the surface expression of odorant receptors but to a lesser extent then RTP1 and

RTP2 (Saito et al., 2004). Lastly the melanocortin-2-receptor accessory protein (MRAP) family

of single transmembrane proteins bind, traffic, and modify the signalling of all 5 members of the

melanocortin receptors (Chan et al., 2009). Based on structure function studies made with

RTP1s, it has been proposed that the N-terminus is crucial for the exit of the receptor complex

16

from the ER, their transmembrane domain is important for the trafficking out of the Golgi, and

their C-terminus required for interaction with the receptor itself (Wu et al., 2012a).

In summary molecular chaperones are an important aspect of GPCR surface expression

where they are required for the surface expression of specific receptors or receptor classes. While

a number of specific molecular chaperones have been identified, there remains a list of receptors

that do not traffic to the plasma membrane in heterologous systems, potentially requiring yet

undiscovered molecular chaperones (ie TAAR1).

1.4.4 Pharmacological Chaperones and Diseases

Pharmacological chaperones (also referred to as pharmacochaperones and

pharmacoperones) are compounds that bind and increase the efficiency of receptor folding

leading to an increase in total and surface expression. The discovery of pharmacological

chaperones came from the study of human diseases that are the result of mutations that result in

ER retention and lack of surface expression of the mutant receptor. One of the first diseases to

be described is nephrogenic diabetes insipidus (NDI) that results from a variety of mutations on

the V2R (Bernier et al., 2004). The mutant receptors are, for the most part, retained in the ER

and do not traffic to the plasma membrane. The lack of resulting signalling from these receptors

does not allow the kidneys to concentrate urine correctly, resulting in chronic dehydration. It was

found that the addition of lipophilic antagonists to heterologous cell lines expressing these V2R

mutants rescued their surface expression and allowed for proper V2R based signalling (Morello

et al., 2000).

Autosomal dominant retinitis pigmentosa can be caused by one of thirteen point

mutations in rhodopsin. These mutations cause the retention of rhodopsin in the ER with no 11-

cis-retinal binding (Dryja et al., 1990; Sung et al., 1991; Dejneka and Bennett, 2001). Surface

expression of these mutants can be rescued with 11-cis-ring-retinal analog of 11-cis-retinal

rescuing the surface expression of these mutant rhodopsins (Noorwez et al., 2003).

Specific mutations in the gonadotropin releasing hormone receptor result in receptor

retention in the ER and can result in hypogonadotropic hypogonadism. Interestingly this study

was the first to do a screen of compounds to discover novel pharmacological chaperones using

this mutant receptor. The result of this screen found a wide range of compounds that act as

17

pharmacological chaperones with differing efficacy in the rescue of surface expression of the

receptor (Janovick et al., 2003).

Lastly, heterozygous null mutations in the MC4R receptor cause an imbalance in

metabolic homeostasis leading to the early onset of obesity in humans (Martinelli et al., 2011).

Treatment with a novel MC4R antagonist causes an increase in the folding and surface

expression of the receptor, consistent with pharmacological chaperoning (René et al., 2010).

Although the study of pharmacological chaperones is relatively new, it has been proposed

that pharmacological chaperones exist for almost all GPCRs (see Maya-Núñez et al., 2012).

Indeed clinical interest in the therapeutic benefits of pharmacological chaperones is on the rise.

One interesting example is the use of β2AR antagonists as a long term treatment for asthma

(Walker et al., 2011). The rationale behind this treatment is paradoxical where current treatment

for asthma involves the acute administration of β2AR agonists that act to relax the muscles of the

airway. However the authors propose that adding low doses of the β2AR antagonist nadolol

increases the surface expression of the endogenous receptors (presumably through

pharmacological chaperones or stabilization of the receptors to the plasma membrane), allowing

for long term alleviation of asthma symptoms without the need for β2AR agonists which are

known to have adverse effects when used for long term treatment of asthma.

1.5 GPCR Oligomerization

It is well established that many non-GPCR receptors, like receptor tyrosine kinases, exist

and function as dimers (Heldin, 1995; Bain et al., 2007). Although early studies with purified

rhodopsin showed this receptor existing in ordered oligomers, these were mainly attributed to

experimental artifacts and thought to have no physiological function (see reviews Hébert and

Bouvier, 1998; Salahpour et al., 2000). However many recent studies have shown the existence

of GPCR oligomerization and its importance in vivo and in mediating physiological responses

(Angers et al., 2002).

The assembly of dimers can occur as early as in the ER, as part of the maturation of the

receptor. Specifically this is the case with the GABA b receptors and the V2R (See section

1.4.3). Indeed these receptors form stable dimers in the ER and are trafficked to the plasma

18

membrane as dimers. However other receptors form oligomers quite transiently while on the

plasma membrane via yet unknown mechanisms (please see review Lohse, 2010).

There are several important functional consequences for oligomerization. As described

above, the first role of dimerization is the regulation or requirement for the export of GPCRs

from the ER (see section 1.5). The second function of oligomerization is the alteration of

pharmacological properties of receptors. One such example is the δκ-opioid heterodimer losing

the ability to bind to the selective ligands for either of the monomers (Gomes et al., 2000). This

is also seen with the co-expression of the μδ-opioid receptors (George et al., 2000). In addition

dimerization has also been shown to alter G-protein coupling whereby the dimerization of the D1

and D2 dopamine receptors leads to coupling of the heterodimer to Gαq where individually the

D1 and D2 couple to Gαs and Gαi respectively (Lee et al., 2004; Rashid et al., 2007).

Lastly, it has been shown that the GPCR oligomer is the functional receptor unit that

binds and signals upon agonist activation. For example the heterodimerization of the GBR1 and

GBR2 receptor is necessary for a fully functioning GABAb receptor. By creating a chimeric

GBR1/2 receptor, it was shown that the intracellular helical domain of GBR2 is required for G-

protein binding and the extracellular domain of GBR1 is required for GABA binding (Galvez et

al., 2001). This cooperative binding and signalling seen in the GBR1/GBR2 heteromer has been

termed asymmetrical activation where activation of one GPCR affects the other receptor within

the dimer complex (Damian et al., 2006; Hugo et al., 2006; Albizu et al., 2010). Further evidence

for a functional receptor as an oligomer includes the homodimer of the metabotropic glutamate

receptor 2 (mGluR2) being required for the proper binding and signalling of the receptor. While

the monomeric mGluR2 is sufficient for G-protein coupling, only dimerization allows the

receptor to signal when bound to its endogenous ligand, glutamate (El Moustaine et al., 2012).

Although oligomerization of GPCRs is a relatively new development, it is quite clear that

GPCR oligomerization is involved in GPCR regulation and signalling.

1.6 GPCR Endocytic Trafficking

The other important trafficking aspect of GPCRs involves the internalization and

desensitization of the receptors. The first step of internalization relies on the uncoupling of the

19

G-protein upon receptor activation. Once the GPCR is de-coupled, the receptor can undergo

phosphorylation in the intracellular loops and C-terminus via GRKs or PKA (Lefkowitz, 1993).

The phosphorylation of the receptor promotes the binding of arrestins allowing for the receptor

to enter the endocytic pathway for internalization. Once internalized the fate of the GPCR can be

one of the following: recycling of the receptor back to the plasma membrane, proteolytic

degradation, or mediating alternative signalling pathways. In general receptor desensitization can

be characterized into homologous and heterologous desensitization. Heterologous desensitization

refers to inactive receptors being phosphorylated and internalized through an agonist

independent manner. This phosphorylation is done by the activation of secondary messenger

dependent protein kinases (ie PKC) (Lefkowitz, 1993). The term homologous desensitization

refers to the requirement of agonist occupation of a receptor for desensitization and will be

described below.

1.6.1 G-protein Coupled Receptor Kinases (GRK)

The first stage of GPCR desensitization is through the phosphorylation of specific serine

and threonine residues on the third intracellular loop and the C-terminal domain of the GPCR

(Premont et al., 1995; Ferguson et al., 1996). This phosphorylation is mediated by the G-protein

coupled receptor kinases (GRK) which is the initial step of GPCR desensitization and

propagation to the following three pathways. 1) The uncoupling of G-proteins from the receptor

through this phosphorylation and subsequent binding of arrestins, 2) endocytosis of the receptor

into endosomes and 3) GPCR signaling through G-protein independent mechanisms. The

mammalian family of GRKs consists of 7 GRKs where GRK 2, 3, 5, and 6 are ubiquitously

expressed while GRK 1 and 7 are expressed selectively in the visual system. Structurally, all

GRKs contain three functional domains: N-terminal regulator of G-protein signalling (RGS)

homology domain, central catalytic domain, and a C-terminal targeting domain. (for a detailed

review of specific GRK domains and their function see (Marchese et al., 2008)). Although

phosphorylation is the first step of desensitization, it is not sufficient to completely uncouple the

GPCR from the G-protein. This was first reported with the discovery of GRK1 where it was

observed that phosphorylated rhodopsin could still bind and signal through transducin

(McDowell and Kühn, 1977). Therefore the role of GRKs is necessary to initiate desensitization

and internalization of GPCRs but is not sufficient to mediate complete desensitization.

20

1.6.2 Arrestins

It is the binding of arrestins to the phosphorylated receptor that completes the

desensitization of the GPCR by blocking the G-protein binding site as well as mediating receptor

endocytosis. This was first reported with the discovery of the visual arrestin (arrestin 1) whereby

complete decoupling of rhodopsin from transducin was mediated through the binding of arrestin

1 (Bennett and Sitaramayya, 1988). In general arrestins are intracellular proteins that bind

phosphorylated GPCRs and mediate their endocytosis through a clathrin dependent pathway

(Ferguson et al., 1996; Goodman et al., 1996). Four arrestin isoforms are expressed in mammals:

arrestin 1 -4. It is important to note that arrestin 2 and 3 are also named β-arrestin1 and β-

arrestin2 respectively and this will be the notation used from this point forward. Conversely

arrestin 1 and 4 will be termed visual arrestins. The key difference between the visual arrestins

and β-arrestins lies in the C-terminal tail of the protein. β-arrestins contain two motifs that link

the GPCR to the clathrin dependent endocytic machinery. It has been shown that β-arrestins bind

with high affinity with clathrin in vitro while visual arrestins do not (Goodman et al., 1996).

There exists two classes of receptors based on the β-arrestin binding profile: Class A and B.

Class A receptors such as the β2AR preferentially bind β-arrestin2 over β-arrestin1 while Class

B receptors such as the V2R bind β-arrestin1 and β-arrestin2 with similar affinities as well as

having the ability to interact with visual arrestins (Oakley et al., 2001). Furthermore class A

receptors bind β-arrestin more transiently where β-arrestin2 is dissociated from the receptor

during endocytosis(see reviews Ferguson, 2001; Luttrell, 2008). Class B receptors on the other

hand form a more stable association with the arrestins where the arrestins do not dissociate upon

receptor endocytosis (Zhang et al., 1999a). In the case of class A receptors, transient interaction

with β-arrestin2 allows for the dephosphorylation and recycling of the receptors to the plasma

membrane. Conversely, class B receptors recycle less efficiently since they have stable

interactions with arrestins (see section 1.6.2). The structural determinant for the stability of

arrestin interaction is found within the serine and threonine clusters of the C-terminal tail. Indeed

switching the C-terminus of the β2AR with that of the V2R diminishes the recycling of β2AR.

Conversely the V2R receptor recycling is increased with the C-terminal tail of β2AR, a typical

class A response (Oakley et al., 1999, 2001). The stable or transient β-arrestin association with

GPCRs has been shown to be mediated through different phosphorylation patterns by different

GRK isoforms. For instance while the β2AR is traditionally a class A receptor, over expression

21

of GRK5 and 6 leads to a stable β2AR -βarrestin complex during internalization, a typical class

B response (Shenoy et al., 2006).

In addition to arrestin’s role in mediating endocytosis of GPCRs, it is now well

established that internalized GPCRs can still signal through arrestin mediated pathways in a G-

protein independent manner. The GPCR – arrestin signalling pathway has now been shown to

bind a wide variety of proteins including, kinases, small GTPases, guanine nucleotide exchange

factors, E3 ubiquitin ligases, phosphodiesterases, and transcription factors (Reiter and Lefkowitz,

2006; Gesty-Palmer and Luttrell, 2008; Luttrell, 2008; Rajagopal et al., 2010). Indeed the

evidence for the biological significance of arrestin signalling has been increasing. For instance it

has been postulated that the anti-psychotic effects of lithium are primarily due to its ability to

inhibit glycogen synthase kinase 3, a downstream effector protein of the dopamine D2-β-arrestin

signalling pathway (Beaulieu et al., 2008). Furthermore what is arguably more interesting is the

observation that ligands for GPCRs can bias the signalling of GPCRs through either the G-

protein dependent pathway or the β-arrestin pathway. This type of signalling is termed,

functional selectivity or biased signaling (Gesty-Palmer and Luttrell, 2008; Luttrell and Kenakin,

2011).

Although arrestin mediated endocytosis in clathrin coated pits is the most common form

of receptor endocytosis, some GPCRs can also internalize through an arrestin independent

manner. These receptors internalize using a C-terminal motif that interacts with adaptor protein 2

(AP2) found in clathrin coated pits, resulting in clathrin dependent endocytosis (Blanpied et al.,

2002; Santini et al., 2002; Scott et al., 2002). The motifs includes two tyrosine based motifs

YXXϕ and YXXXϕ (where ϕ represents a bulky hydrophobic amino acid), and the more

common dileucine motif. For example the protease activated receptor 1 (PAR1) is a receptor that

internalizes through this mechanism. Mutation of the YXXϕ domain to a aXXa domain in this

receptor completely impairs its internalization upon agonist stimulation (Paing et al., 2004). The

YXXXϕ motif is found in the thromboxane A2 β (TPβ) receptor and is responsible for the tonic

internalization of the receptor (see section below) (Parent et al., 2001). The dileucine motifs are

present in multiple GPCRs such as the β2AR (Moore et al., 2007). Although the β2AR

undergoes arrestin mediated endocytosis, the mutation of the dileucine motif decreases the

amount of receptors internalized upon agonist stimulation indicating that two mechanisms of

internalization are occurring (Gabilondo et al., 1997). While these mutations reduced ligand

22

induced internalization, the signalling through the receptor was otherwise not affected

(Gabilondo et al., 1997; Kohout et al., 2001). Although the dileucine motif is important for

internalization of certain receptors, it might also play a role as an ER export signal (see section

1.4.1). It is clear that internalization is a complex process where dynamics between arrestin

independent and dependent internalization are currently unknown (Magalhaes et al., 2012).

1.6.3 Tonic/Constitutive Internalization

Receptors can also undergo tonic internalization in the absence of any ligand stimulation

following a distinct mechanism. For example the TPβ internalizes through the arrestin pathway

when stimulated with an agonist while tonic internalization is mediated through the YXXXϕ

domain mentioned above (Parent et al., 2001). Beyond the regulation of receptor homeostasis, it

is not yet known what role tonic endocytosis plays in the signalling pathways mediated by

GPCRs.

1.6.4 Endocytic pathway

Once the receptors are internalized, they are either recycled back to the plasma membrane

or targeted for degradation. The stability of β-arrestin interaction with the receptor acts as the

major contributor as to whether or not the receptor is recycled back to the plasma membrane. For

example the class A receptor β2AR, has a rapid dissociation with β-arrestin upon internalization

through clathrin coated pits, allowing for the receptor to enter a more acidic endosomal

compartment that leads to dephosphorylation of the receptor and its recycling back to the plasma

membrane (Pitcher et al., 1995). In contrast, class B receptors like the V2R internalize in

complex with β-arrestins slowing the dephosphorylation of the receptor which leads to

subsequent proteolytic degradation of the receptor (Oakley et al., 1999). Therefore it appears that

the stability of receptor-β-arrestin interaction determines whether the receptor is recycled to the

plasma membrane or degraded (Luttrell and Lefkowitz, 2002). Further regulation of GPCRs in

the endosome has been found with the NPXXY motif on the cytoplasmic end of the 7th

transmembrane domain which contributes to the endocytosis of many GPCRs (Gripentrog et al.,

2000; Bouley et al., 2003; Kalatskaya et al., 2004). Although less understood, cytoplasmic C-

terminal motifs such as the PDZ (PSD-95 protein), DLG (Drosophila discs large protein) and

ZO-1 (zonula occludens-1 protein) motifs regulate endocytic sorting and are present in a large

number of GPCRs (see review by Marchese et al., 2008).

23

In addition to the motifs present on the receptors, post translational modification of

arrestins can also be important for internalization of receptors. For instance ubiquitination of β-

arrestin 2 by the E3 ubiquitin ligase MDM2 is essential for the internalization of β2AR (Shenoy

et al., 2001).

Lastly if the receptor is not recycled to the plasma membrane, the receptor-arrestin

complex is ubiquitinated and subsequently targeted to the lysosome for degradation. In most

cases, targeting of the receptor-arresting complex to the lysosome occurs most commonly

through ubiquitination however ubiquitination independent targeting has also been reported (see

review Marchese and Trejo, 2013).

In summary, the endocytic pathway is important in the long term regulation of GPCR

signalling. Once internalized, the receptor is sorted through the recycling or degradation

pathways. Recycling of the receptor is thought to be the main mechanism for receptor

resensitization while receptor degradation is the main mechanism for down regulation of the

receptors (Hanyaloglu and von Zastrow, 2008). Furthermore internalized receptors can initiate

other signalling pathways in a G-protein independent manner.

1.7 Summary of Assays for measuring surface expression of GPCRs

The concept for quantification of surface receptors is a relatively simple idea, whereby

only receptors on the plasma membrane of the cell are detected. The most widely adopted assays

for quantification of surface expression use either hydrophilic ligands or antibodies in order to

quantify cell surface expression of GPCRs. However, as will be discussed later in this section,

there are several newly developed assays for quantification of cell surface receptors where the

primary purpose of these assays has been miniaturization and their adaptation and use for high

throughput approaches.

The first assays used to quantify surface expression of GPCRs utilized hydrophilic

radioligands that do not cross the plasma membrane (Lohse et al., 1990; Green and Liggett,

1994; Mialet-Perez et al., 2004). In the absence of radiolabelled hydrophilic ligands, cell

fractionation was used to isolate the plasma membrane from whole cell lysates (Lohse et al.,

1990). Once isolated, surface receptors were quantified using radioligands and autoradiography

(Jockers et al., 1996, 1999) or immunoblotting (Jockers et al., 1999; Lee et al., 2000). Given the

24

limited availability of radioactive hydrophilic ligands as well as the time consuming process of

cell fractionation, there has been a transition to simpler and more efficient assays over the last 20

years.

Currently, the gold standard for the quantification of receptors at the plasma membrane is

through antibody mediated assays (flow cytometry and ELISA) or biotinylation. These methods

are well established and have been used in multiple publications looking at surface expression of

GPCRs. The following are examples of studies using flow cytometry (Ramprasad et al., 1996;

Jockers et al., 1999; Morello et al., 2000; Compton et al., 2002), ELISA (Salahpour et al., 2004;

Rochdi et al., 2010; Lan et al., 2011), and biotinylation (Ramprasad et al., 1996; Ray et al.,

1998; Petaja-Repo et al., 2000; Wüller et al., 2004). ELISA and flow cytometry are

accomplished using live whole cell preparations expressing recombinant GPCRs with the

addition of N-terminal epitopes. Exogenous epitopes are used due to the availability of selective

high affinity primary antibodies towards them. The basic steps for both flow cytometry and

ELISA include the following: Blocking, probing with 1˚ antibody, fixing the cells, and adding 2˚

antibody. In the case of an ELISA the 2˚ antibody is a horseradish peroxidase (HRP) conjugated

antibody where HRP enzymatically converts a colourometric compound (o-Phenylenediamine

dihydrochloride) that can be quantified using a spectrophotometer (for procedure reference

please see Salahpour et al., 2004). For flow cytometry the secondary antibody is a fluorophore

conjugated antibody that can be detected and quantified by a flow cytometer (Jockers et al.,

1999). Both of these assays can be done within a day and have been shown to produce reliable

and reproducible results. In addition, once optimized, these assays can be miniaturized to at least

96-well plate format, therefore increasing their throughput. Although there have been no

attempts to optimize these assays for high throughput screening of surface expression, ELISA

and flow cytometry have been optimized for other systems to the point where they are suitable

for high throughput screening. Examples include high throughput discovery of protein

biomarkers for ELISA and identification of small molecule inhibitors for proteins using flow

cytometry. For reviews of this topic please see (Jin and Zangar, 2010) and (Sklar et al., 2007) for

ELISA and flow cytometry respectively. The main advantages of flow cytometry and ELISA

stem from the increase in robustness and throughput from previous generation surface expression

assays (see above) where these assays can be completed in a 96 well plate compared to cell

fractionation and radioligand binding. Furthermore flow cytometry can be used to measure

25

multiple labeled receptors versus just one for the ELISA. The disadvantage of flow cytometry

and ELISA are the relative low throughput of these assays when compared to new surface

expression assays as described below (section 1.7.1-1.7.3).

The third standard for measuring surface expression is the use of cell surface

biotinylation. Biotinylation allows for the purification of surface proteins by taking advantage of

the high affinity binding of biotin to streptavidin. The method requires the biotinylation of all

surface proteins of a live cell. Once biotinylated, the cells are lysed and run through a

streptavidin column, binding all biotin labelled cell surface proteins. After several washes, the

column is eluted with the addition of free biotin. The eluted fractions are then run on an SDS-

PAGE gel where immuno blotting is subsequently used to quantify the protein of interest. Unlike

an ELISA or flow cytometry, biotinylation can be used for native proteins if a suitable antibody

is available. However, biotinylation cannot currently be done in a high throughput manner

because of the affinity column and the long wash and incubation steps required.

Within the past 5 years several other assays have been introduced that significantly

increase the efficiency, cost, and robustness of surface expression measurements. The first

method is the conjugation of the primary antibody with a quantum dot (Fichter et al., 2010).

Quantum dots are inorganic semiconductors that can undo go fluorescence, like organic

fluorophores, but have distinct advantages over traditional organic fluorophores. They can be

engineered, based on the size of the quantum dot, to have specific excitation and emission

spectra as well as a wide band gap increasing the signal to noise ratio. In addition, quantum dots

are immune to photobleaching and are 10-20 times brighter than organic fluorophores (For a

review of quantum dots for cellular imaging please see Alivisatos et al., 2005). By using

quantum dots, the signal to noise of the assay when using flow cytometry is greatly improved.

1.7.1 Fluorogen Activating Protein Biosensor

The second assay that improves upon traditional flow cytometry uses a fluorogen

activating protein (FAP) based biosensor. FAP is a relatively small 200 amino acid protein that

upon binding of a fluorogen increases the fluorescence of the fluorogen dramatically as part of a

non-covalent reaction (Szent-Gyorgyi et al., 2008). Although this FAP biosensor can be

conjugated with antibodies for cell surface labeling (Szent-Gyorgyi et al., 2008), the FAP

biosensor can actually be fused on the N-terminus of GPCRs (Fisher et al., 2010). This method

26

requires the use of cell impermeable fluorogens that selectively label cell surface receptors. This

assay improves upon traditional flow cytometry by removing the need for antibodies thereby

improving the signal to noise and throughput of the assay. Recently, the FAP assay was

optimized for high throughput screening of ligands that promote internalization of the β2AR (Wu

et al., 2012b).

1.7.2 Internalization Assays

In addition to the approaches developed for measuring surface expression, assays have

also been designed to specifically monitor receptor internalization rather than total surface

expression. These assays can be split into two separate types. The first is the binding of β-

arrestin to the GPCR, while the second assay is the recruitment of receptors to endosomes. For

binding of β-arrestins to GPCRs, two distinct techniques have been developed. The first is the

use of bioluminescence resonance energy transfer (BRET) whereby the β-arrestin is tagged with

either the donor or acceptor BRET partner (or both acceptor and donor Charest et al., 2005). The

assay relies on measuring the energy transfer between β-arrestin and the GPCR upon agonist

activation (for a review see Salahpour et al., 2012). The second assay utilizing β-arrestin is a

complementation assay whereby fragments of β-galactosidase are found on the C-terminus of the

GPCR and on the β-arrestin (Hammer et al., 2007). The binding of β-arrestin completes the β-

galactosidase enzyme whereby a β-galactosidase substrate can then be added allowing for a

quantifiable signal. The advantages of these β-arrestin assays are their simplicity and ability to

be used in high throughput screening (Hamdan et al., 2005; Hammer et al., 2007).

The second technique looking at receptor localization to endosomes relies on pH

sensitive fluorescent proteins (phlourins) that will have altered fluorescent properties due to the

lower pH of endosomes (Geisow and Evans, 1984). The first assay using phlourins has

traditionally been used for imaging GPCRs in endosomes rather than quantification of receptor

internalization (Miesenböck et al., 1998; Mahon, 2011; Yudowski and von Zastrow, 2011).

However this technique has evolved to allow for quantification of internalization using a N-

terminal coil-coiled tag probes (Yano et al., 2012). These probes are different than typical

phlourins due to the decrease in protein size on the N-terminus of the GPCR as well as allowing

for a wide range of conjugated pH sensitive fluorophores to be used (Takeda et al., 2012). Lastly

by tagging a β-Galactosidase fragment with the FYVE domain, of the endosomal protein

27

endofin, receptor localization within endosomes can be quantified in a similar fashion to the β-

arrestin complementation assay (Hammer et al., 2007). The advantage of these assays compared

to the β-arrestin assays, is the ability to quantify the amount of receptors internalized to

endosomes.

1.7.3 N-terminal GPCR Fusion Tags

Next, a multitude of fusion proteins have been developed to observe specific

compartments in cellular imaging. These fusion proteins, or tags, rely on small fluorescent

organic molecules covalently linked to the tags through enzymatic ligation (Sun et al., 2011).

Having compounds that are sensitive to their chemical environment (eg. cell permeable

compounds) can allow for the specific labelling of fusion proteins in different cellular

compartments. Although various examples for each tag exist, only the SNAP and CLIP tags have

been developed and optimized to measure surface expression (Doumazane et al., 2011). The

SNAP tag is derived from the 20kDa DNA repair protein O6-alkylguanine-DNA alkyltransferase

(AGT) that covalently binds O6-benzylguanine derivatives that are fluorescently labeled

(Juillerat et al., 2003). The CLIP tag is a derivative of AGT that selectively binds O6-

propylguanine analogs (Gautier et al., 2008). Using different combinations of CLIP and SNAP

tags in addition to cell impermeable substrates, surface expression can be quantified using this

technique (Maurel et al., 2008; Zwier et al., 2010; Doumazane et al., 2011; Ward et al., 2011). In

addition the substrates for both the SNAP and CLIP tags can be modified to contain two

fluorophores allowing for FRET (Maurel et al., 2008). By utilizing FRET, the signal to noise of

this assay is dramatically increased to such a point that SNAP-tagged receptors can be used for

high throughput screening (Haruki et al., 2012). Although assays using SNAP-tagged fusion

proteins have yielded a Z’ > 0.7 (see section 4.6 for definition of Z’), quantification of surface

expression of SNAP tagged GPCRs in a high throughput manner has not yet been optimized.

In this thesis, we describe a new assay to quantify surface expression of GPCRs. We

utilize β-lactamase (βlac) fused to the N-terminus of GPCRs, as our reporter for this assay

(Figure 1.2). In addition we utilize the chromogenic β-lactamase substrate nitrocefin in our assay

for quantification (Figure 1.3). Nitrocefin is a well validated substrate for β-lactamases and is the

main substrate used when screening for β-lactamase activity. Nitrocefin on its own is a coloured

substrate that has a max absorption at 390 nm (yellow). Upon the cleavage of the β-lactam ring

28

by β-lactamase, there is a shift in the peak absorption to 486 nm (red) (O’Callaghan et al., 1972)

(Figure 1.2).

1.8 β-lactamase Assay

Although the use of β-lactamases as reporters for proteins is not a new concept (Moore et al.,

1997; Watanabe et al., 2011), we describe a new assay that utilizes βlac-GPCR fusion receptors

to quantify the surface expression levels of GPCRs (Figure 1.2). While others have used β-

lactamase to differentiate the localization of specific proteins in the cell (Watanabe et al., 2011),

we are the first to utilize N-terminal βlac on GPCRs to quantify the surface expression on the

plasma membrane in a robust manner. Since βlac is a relatively large protein (~30 kDa), it is

possible that the function of the βlac tagged receptor could potentially be affected. However it

has been shown in the literature that other bulky N-terminal tags (ie SNAP-tag) do not affect the

expression or function of multiple GPCRs (Maurel et al., 2008) and our own results indicate that

for the GPCRs tested, βlac fusion does not affect receptor activity.

β-lactamases are a family of enzymes that are expressed in prokaryotes and confer a form

of antibiotic resistance (review of the last 30 years of β-lactamase inhibitors see Drawz and

Bonomo, 2010). β-lactam based antibiotics act on the penicillin binding proteins (PBP) that

inhibit the transpeptidase action of PBPs which are necessary for the cell wall synthesis of

prokaryotes (Zapun et al., 2008). Therefore β-lactamases act as enzyme mimics of PBPs and are

serine hydrolases that acylate the β-lactam ring of antibiotics (Minasov et al., 2002). The

activated serine of the β-lactamase enzyme acylates the β-lactam containing compound, whereby

the active site of the enzyme is regenerated following the deacylation of the enzyme with

activated water (figure 1.4). We chose to use β-lactamase as our reporter due to a lack of an

eukaryotic homolog resulting in no background hydrolysis of nitrocefin (Moore et al., 1997). We

also chose to use nitrocefin as the substrate for our assay due to its commercial availability and

its thorough characterization in the literature as a suitable substrate for β-lactamase (Jones et al.,

1982; Zygmunt et al., 1992; Moore et al., 1997; Bouillenne et al., 2000; Tan et al., 2003). Apart

from nitrocefin, there are other compounds that can act as quantifiable substrates for β-

lactamase. For example alternative chromogenic β-lactamase substrates have been described in

the literature. The compounds PADAC (Jones et al., 1982) and CENTA (Bebrone et al., 2001)

have been shown to have similar kinetics to nitrocefin and would also be suitable ligands for our

29

Figure 1.2: Schematic of the βlac assay. The βlac assay uses an N-terminal βlac-GPCR fusion

protein that is transfected into cells. If there is no surface expression of this construct (left panel)

then the addition of the cell impermeable nitrocefin will cause no colour change. If the construct

is expressed on the plasma membrane, the extracellular βlac can now cleave nitrocefin causing a

shift in absorbance from 390nm (yellow) to 486nm (red).

30

Figure 1.3: Nitrocefin as a chromogenic substrate for the βlac assay. Nitrocefin is a β-lactam

compound that absorbs light at λ=390nm natively. Upon hydrolysis of the β-lactam ring by β-

lactamase the absorbance shifts at λ=486 nm.

31

assay. Unfortunately none of these two alternatives is commercially available. In addition it has

been shown that ampicillin and its esterified analog bacampicillin; can be chemically conjugated

with cationic fluorophores to become suitable fluorescent β-lactamase substrates (Watanabe et

al., 2011). By using ampicillin and its pro-drug variant, cell permeant and cell impermeant

fluorophore conjugated β-lactam analogs were created. However these β-lactam analogs are not

well characterized in the literature and are only available through chemical synthesis. Lastly

there exists a commercially available FRET based cephalosporin analog substrate for βlac

(Zlokarnik et al., 1998; Rukavishnikov et al., 2011). However this substrate is cell permeable and

therefore not suited for quantifying surface expression of GPCR. The kinetics of nitrocefin on

our Class A β-lactamase (TEM-1) has been reported with a Km and Kcat of 52μM and 930s-1

respectively (Bouillenne et al., 2000). In addition, nitrocefin is impermeable to polymer based

liposomes (Städler et al., 2009), however plasma membrane permeability has not yet been

experimentally evaluated.

In summary we have developed a novel assay for GPCR surface expression

quantification. This assay is sufficiently different from current assays on the market by using a

chromogenic substrate that is commercially available. In this thesis, we aimed at validating this

novel assay against ELISAs looking at various conditions that affect surface expression of

prototypical GPCRs.

32

Figure 1.4: Mechanism of action for a class A β-lactamase. (1) The β-lactam carbonyl group

undergoes nucleophilic attack by the activated ser70, resulting in an acylation intermediate (2).

(3) Protonation of the β-lactam nitrogen leads to cleavage of the C-N bond and formation of the

covalent acyl-enzyme (4). (5) Attack by a catalytic water leads to a high-energy deacylation

intermediate, with subsequent hydrolysis of the bond between the β-lactam carbonyl and the

oxygen of Ser70. Deacylation regenerates the active enzyme and releases the inactive β-lactam

(Drawz and Bonomo, 2010)

33

Chapter 2

Materials and Methods

2.1 Reagents

Nitrocefin (BD Biosciences) was dissolved in DMSO at a concentration of 10mM. Isoproterenol

(Sigma) was dissolved in PBS containing 170μM ascorbic acid. Alprenolol and propranolol

(Sigma) were dissolved in PBS. Mouse anti-HA primary antibody (12CA5 hybridoma), anti-

mouse HRP conjugated (Cell Signaling Technology), and alexfluor-680 anti-mouse conjugated

(Invitrogen) secondary antibodies were diluted in PBS containing 1% BSA.

2.2 Plasmid Construction

The cDNA expression vectors for human GBR1 and β2AR were provided by Dr. Michel Bouvier

(Salahpour et al., 2004; Villemure et al., 2005). The cDNA expression vector for human GBR2

was obtained from Missouri S&T cDNA.

The β-lactamase sequence was cloned from the ampicillin resistance gene within the pcDNA3.1

plasmid, with the restriction sites HindIII and AscI at 5’ and 3’ of the βlac respectively. The βlac

was further modified by the additions of a chicken α7 nicotinic receptor signal sequence (SS) [9],

and an HA epitope at the N-terminus, yielding the following cDNA: SS-HA-βlac. The βlac

construct was subsequently cloned into the multiple cloning site of the pcDNA3.1 plasmid. A 5’

Asc I and 3’ Not I restriction site were added to the GBR1 and β2AR. These cDNA constructs

were then ligated into the plasmid in frame with the βlac creating SS-HA-βlac-β2AR and SS-

HA-βlac-GBR1.

2.3 Cell Culture

HEK 293 and HEK 293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM,

Wisent) and supplemented with 10% FBS (Wisent), 100 U/ml penicillin and 100µg/ml

streptomycin. HEK 293 cells stably expressing SS-HA-βlac-β2AR or SS-HA-βlac-GBR1 were

further supplemented with 1µg/ml puromycin. All cells were kept at 37˚C and 5% atmospheric

CO2.

34

2.4 Generation of Stable Cell Lines and Transient Transfections

Cells (2x106 cells) were seeded into 10cm tissue culture plates. The following day, cells were

transfected with 3 µL of polyethylenimine (1mg/mL) (Polyscience Inc) per µg of plasmid DNA.

For transient transfections, cells (HEK 293T) were seeded 24h post-transfection for experiments.

For stable cell line creation (HEK 293), media was replaced with the proper selection antibiotic

24 hours post transfection. Clonal cell lines were generated by picking individual colonies and

expression was confirmed by western blot. For ELISA and βlac experiments, 1x105 cells were

plated into individual wells of a poly-D-lysine coated 48 well plate.

2.5 Western Blotting

Clonal cell lines for the SS-HA-βlac-β2AR and SS-HA-βlac-GBR1 were lysed with the addition

of RIPA buffer (25mM Tris-Hcl, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%SDS.

1.5ug/ml aprotinin, 10ug/ml pepstatin A, 10ug/ml leupeptin, 0.25mM PMSF) on ice. The

supernatant was collected from the lysates through centrifugation at 15 000 RPM at 4°C. Protein

concentration was quantified using the BCA protein assay (Pierce). Samples were heated to 95°C

in the presence of β-mercaptoethanol. 30ug of protein was resolved on a 7.5% SDS-

polyacrylamide gel and subsequently transferred to a polyvinyl fluoride membrane. Protein

expression levels were determined by immunoblotting using the monoclonal anti-HA antibody.

Immunoreactivity was detected using the LiCor Odyssey infrared imaging system.

2.6 βlac-β2AR Immunofluorescence

SS-HA-βlac-β2AR stable cell lines were plated at 1x106 cells per well in a 6-well plate

containing glass microscope cover slips. 24 hours after the cells were plated, the wells were

washed with PBS and blocked with PBS containing 1% BSA and kept on ice. Primary antibody

(1:1000) was then incubated for one hour on ice. The primary antibody was removed and warm

cell media was added. Cells were then treated for 30 minutes with vehicle or 10μM

isoproterenol. The media was then aspirated and the plate washed with PBS. The cells were then

fixed with 4% PFA for 15 minutes and subsequently blocked at room temperature with PBS with

1% BSA for 30 minutes. A fluorescent secondary antibody (1:5000) was subsequently added and

incubated for 30 minutes. The cells were then washed 3 times with PBS and 1% BSA with a

35

final wash of PBS. The cover slips were then removed and mounted onto microscope slides

using Vectashield mounting media (Vector Laboratories). Images were acquired using the

eclipse 80i fluorescent microscope (Nikon).

2.7 βlac-β2AR Functional Assay using BRET EPAC cAMP Biosensor

HEK293 cells stably expressing the BRET EPAC cAMP biosensor (Barak et al., 2008) were

transfected with 50ng of SS-HA-βlac-β2AR or HA- β2AR and seeded into 96-well plates (1x105

cells) 24 hours post transfection. After 24 hours incubation, cells were washed once with PBS.

Coelenterazine H (5 μM, final conc) was added and the plate was incubated for 5 minutes in the

dark, after which isoproterenol (1x10-14

– 1x10-4

M) was added. Each well was read once every 5

minutes in the Mithras luminometer (Berthold Technologies).

2.8 βlac Assay

Nitrocefin was first diluted to a final concentration of 100 μM in PBS. After drug treatment the

cells were washed once with PBS and, after removal of the PBS wash, 200 μL of the nitrocefin

solution was added to each well. Immediately after the addition of the nitrocefin solution,

absorbance for each well was read kinetically once every minute for 30 minutes at 486 nm using

the EPOCH microplate spectrophotometer (Biotek). The rate of reaction (slope of the curve in

the linear range) was taken as the readout for this assay

2.9 ELISA

All ELISAs were performed as described previously (Lavoie et al., 2002). Briefly, 24 hours after

plating the cells and after drug treatment, plates were washed with PBS and blocked with PBS

containing 1%BSA and kept on ice. Primary antibody (1:1000) was then incubated for one hour

on ice. The cells were then fixed with 4% PFA for 15 minutes and subsequently blocked at room

temperature with PBS with 1% BSA for 30 minutes. Secondary antibody (1:1000) was added

and incubated for 30 minutes. The cells were washed 3 times with PBS and 1% BSA with a final

wash of PBS where the HRP substrate Sigmafast OPD (Sigma) was subsequently added. After

30-45 minutes of substrate incubation, the reaction was stopped with the addition of 3M HCl.

The supernatant was then transferred to a 96 well plate and absorbance was read using the

EPOCH microplate spectrophotometer (Biotek) at 492 nm.

36

2.10 βlac-β2AR Agonist Studies

SS-HA-βlac-β2AR cells were seeded at 1x104 cells (βlac assay) or 1x10

5 cells (ELISA) per well

in poly-D-lysine coated 48 well plates 24 hours prior to experimentation. Isoproterenol was

weighed out the day of the experimented and dissolved in PBS containing 170μM ascorbic acid

where the doses of isoproterenol were diluted to 100x solutions. To determine the time course of

internalization a dose of 10μM of isoproterenol every 5 minutes in triplicate. The dose response

of internalization was done by the addition of individual doses (triplicates) of isoproterenol

(1x10-11

- 1x10-4

M) followed by incubation at 37°C for 30 minutes. Both the βlac and ELISA

were done as stated in section 2.8 and 2.9 for the time course and dose response for SS-HA-βlac-

β2AR internalization.

2.11 βlac-β2AR Antagonist Studies

SS-HA-βlac-β2AR cells were seeded at 1x104 cells per well in poly-D-lysine coated 48 well

plates 24 hours prior to experimentation. Internalization blocking assays were done by treatment

with 30 minute treatment with individual doses (triplicates) of alprenolol or propranolol (1x10-10

- 1x10-4

M) followed by 30 minute incubation with 10μM isoproterenol. Pharmacological

chaperoning experiments were done with overnight incubation of individual doses of either

alprenolol or propranolol (1x10-10

- 1x10-4

M). The βlac assay was done as stated in section 2.8

for both the blocking and pharmacological chaperoning experiment.

2.12 βlac-β2AR Z’ Determination for Agonist Induced Internalization

SS-HA-βlac-β2AR cells were seeded at 5x103 per well in a 96 well plate 24 hours prior to

experimentation. Isoproterenol was weighed out the day of the experiment and dissolved in PBS

containing 170μM ascorbic at a 10X concentration of 100μM. Half the plate was stimulated by

isoproterenol (10μM final) as the positive control and vehicle was added to the other half of the

plate as the negative control. The βlac assay was done as stated in section 2.8 for Z’

determination.

2.13 GBR1 Molecular Chaperoning Studies

SS-HA-βlac-GBR1 stable cell lines were transfected with empty vector or an increasing amount

of GBR2 and subsequently plated into a 48 well plate at a density of 1x105 cells per well 24

37

hours post transfection. A βlac assay or ELISA was done 48 hours post transfection as stated in

sections 2.8 and 2.9.

2.14 Data Analyses

Data analyses were performed with Graphpad Prism 5.01 (Graphpadsoftware inc). Linear

regression was performed on βlac data to determine the slope of the curve for comparisons.

The Z’ factor is a measure of the quality of an assay that takes into account both the signal

dynamic range and variation of data. The Z’ was determined using the following

equation(Zhang, 1999):

(1) Z’=1-(3σc+ + 3σc-) / (|μc+ - μc-|)

Where μc represents the mean and σc the standard deviations of the controls, where the (+) and

(-) denote positive (isoproterenol) and negative (vehicle treated) controls respectively.

Dose response curves were fitted with the following equation:

(2) Y = Ymin + (Ymax – Ymin) / (1 + 10[(pEC50 – logX ) * Hill Slope])

Y represents %response with Ymin and Ymax being the minimum and maximum response

respectively. pEC50 is the negative log of the molar concentration to yield 50% maximal

response and logX is the molar concentration for response Y. Hill slope represents the degree of

the slope within the linear portion of the sigmoidal graph.

One-way ANOVA with Bonferroni correction post-hoc was used to determine the differences

between data sets.

38

Chapter 3

Results

In order to demonstrate the utility of the βlac assay two well characterized GPCRs were

used: β2AR and GBR1. It was first determined that the trafficking and signalling of the N-

terminal βlac tagged β2AR and GBR1 was not impaired. Using these two receptors the βlac

assay was quantitatively compared to the well established ELISA approach for agonist induced

internalization of the β2AR and the surface expression of the GBR1. In addition the βlac was

also used to determine the Z’ of β2AR internalization, antagonist blocking of agonist induced

internalization, and pharmacological chaperoning.

3.1 Generation of the βlac Plasmids and Stable Cell Lines Expressing βlac-

GPCR Fusion Constructs

The βlac vector was originally created by Ali Salahpour/Stephane Angers where the

Ampr gene was cloned from the pcDNA vector and added in frame to the 3`end of the ORF. In

addition, to facilitate the proper plasma membrane targeting of receptor constructs, the chicken

α7 nicotinic receptor signal sequence (SS) was added 5`to the βlac sequence. Furthermore, an

HA epitope was also added in frame between the signal sequence and the βlac. The GBR1 and

β2AR cDNA were cloned with the appropriate restriction sites and added to 3`to the βlac. DNA

Sequencing confirmed that the receptor sequences were in frame to the βlac, therefore generating

the two following constructs (SS-HA-βlac-β2AR and SS-HA-βlac-GBR1).

Stable HEK293 cell lines were generated by transfecting the cells with either the SS-HA-

βlac-β2AR or SS-HA- βlac-GBR1. After selection, monoclonal cell lines were generated from

evaluating individual colonies. Western blotting was used to confirm the expression of the βlac-

receptor construct (Figure 3.1). Clone 13 of the GBR1 was chosen due to its high expression

level while clone 1 was the only clone that was isolated for the β2AR.

3.2 Trafficking and Signalling of the βlac-β2AR

It is always a concern that the addition of a 30kDa tag (in this case βlac) to the N-terminus of a

GPCR can affect the function of the receptor. However, it was previously shown

39

Figure 3.1: Western blot of stable clonal cell line expression levels of SS-HA-βlac-GBR1

and SS-HA-βlac-β2AR. Stable cell lines for the constructs SS-HA-βlac-GBR1 and SS-HA-βlac-

β2AR were generated with individual colonies expanded. Mock (non-transfected cells), SS-HA-

βlac-GBR1 clones 12-16, and SS-HA-βlac-β2AR clone 1 were lysed in RIPA buffer and boiled

at 95°C in the presence of β-mercaptoethanol. 30μg of protein was loaded for each clone in an

SDS-PAGE gel and transferred overnight. Blanks represent wells that have not been loaded with

protein. Mouse monoclonal anti-HA primary antibody was added with visualization of the blot

by alexafluor-800 secondary antibody. The expected bands for the SS-HA-βlac-β2AR and SS-

HA-βlac-GBR1 were ~70kDa and ~140kDa respectively. The blot was visualized using the

LiCor Odyssey infrared imaging system. SS-HA-βlac-GBR1 clone 13 was chosen due to being

the highest expressing clone. Clone 1 of the SS-HA-βlac-β2AR was confirmed to express the

construct. The bands did not migrate at these molecular weights due to the boiling of the samples

causing the aggregation of the receptors. The low molecular weight bands could be degradation

products of the receptors.

40

that the addition of the SNAP-tag (also 30kDa) to the N-terminus of other GPCRs did not affect

receptor function (Maurel et al., 2008). The trafficking of the SS-HA-βlac-β2AR receptor for

both surface expression and internalization was assessed using immunofluorescence. In addition,

the functionality of SS-HA-βlac-β2AR receptor was investigated in a functional assay utilizing a

BRET cAMP biosensor EPAC.

Trafficking of the SS-HA-βlac-β2AR was assessed with the use of immunofluorescence

(Figure 3.2). Cells were first labelled with anti-HA primary antibody on ice, and subsequently

either treated with vehicle or 10μM isoproterenol. In the absence of agonist (Figure 3.2A) the

SS-HA-βlac-β2AR had a uniform membrane distribution consistent with surface expression at

the plasma membrane (Kim et al., 2008). Stimulation with a dose of 10μM isoproterenol (Figure

3.2B) resulted in receptor internalization marked by the redistribution of receptors to intracellular

endosomes consistent with agonist induced internalization (Cao et al., 2005). Therefore the

addition of the βlac to the N-terminus of the β2AR does not affect the surface expression nor

internalization of the receptors and the results from our experiments (Figure 3.2) are similar to

what has been reported in the literature (Cao et al., 2005; Kim et al., 2008).

Secondly, to verify that the function of β2AR was not hindered by the addition of the βlac to the

N-terminus, we evaluated the ability of β2AR to stimulate cAMP production. For this we used a

previously described EPAC-BRET cAMP biosensor to quantify B2AR function (see section 4.3

for description of this biosensor). Briefly, HEK293 cells stably expressing Rluc-EPAC-YFP

were transfected with 50ng of either SS-HA-βlac-β2AR or HA-β2AR and stimulated with

isoproterenol. We hypothesized that the two transfected receptors would show distinct

pharmacological properties compared to the β2AR that are endogenously expressed in HEK293

cells (see section 4.3). First, the dose response of isoproterenol response in the transfected cells

would be left shifted compared to mock cells that endogenously express β2AR (Zhong et al.,

1996). Therefore by comparing the dose response of both the SS-HA-βlac-β2AR and HA-β2AR,

the effects of the βlac on the signalling of β2AR can be quantified. As seen in figure 3.3A and B,

transfection of 50ng of both SS-HA-βlac-β2AR and HA-β2AR shifted the isoproterenol dose

response to the left compared to the β2AR signalling observed in mock cells (empty vector

transfected cells). Indeed the EC50 of isoproterenol in both the SS-HA-βlac-β2AR and HA-

β2AR, 0.69±0.5 and 0.36 ±0.3 nM respectively, is 170 times lower than EC50 measured in the

41

Figure 3.2: Immunofluorescence of HEK293 cells stably expressing SS-HA-βlac-β2AR.

Cells were seeded in a 6 well plate containing a glass coverslip. Mouse monoclonal anti-HA

antibody (Primary antibody) was added first followed by 30 minute treatment with vehicle (A) or

10μM isoproterenol (B). The cells were then fixed with 4% PFA and Alexafluor 640 conjugated

secondary antibody was added for visualization. The cells were visualized using the Nikon

eclipse 80i fluorescent microscope. (A) Cells treated with vehicle showed a uniform distribution

around the cell. (B) Stimulation with isoproterenol localized the receptors to intracellular

aggregates consistent with internalization into endosomes.

A

B

42

empty vector transfected cells at 140 ± 500 nM. In addition, the endogenous β2AR signal in the

mock transfected cells is almost completely desensitized after 30 minutes while this was not the

case with the transfected cells, in which the signal was persistent even after 30 minutes of

stimulation (Compare empty vector transfected condition in both figure 3.3A and B).

Furthermore, the EC50 of the two transfected cells remained similar after 30 minutes of

isoproterenol stimulation with EC50 values of 0.78±0.3 and 0.34±0.2 nM for the SS-HA-βlac-

β2AR and HA-β2AR respectively, which also indicates a lack of desensitization in the

transfected cells compared to the mock cells. In summary, the addition of the N-terminal βlac

does not affect the signalling of the β2AR when compared to the HA- β2AR as measured using

an EPAC BRET cAMP biosensor.

3.3 βlac-β2AR experiments

Having established that the presence of an N-terminal βlac does not alter β2AR

pharmacology, the experimental conditions for measuring the internalization of SS-HA-βlac-

β2AR using the βlac as a reporter were optimized. We used a concentration of 100μM of

nitrocefin which is above the Km of the enzyme and has previously been used in the literature

(Jones et al., 1982; De Meester et al., 1987; Bouillenne et al., 2000; Bebrone et al., 2001; Tan et

al., 2003). Cells were seeded in a poly-d-lysine coated 96/48 well plate, and incubated for 24h at

37 ˚C. Initial studies showed that seeding stably expressing SS-HA-βlac-β2AR cells at a density

of 100 000 cells/well led to a complete hydrolysis of nitrocefin within 5 minutes, which under

our experimental conditions was considered to be too fast for the internalization studies.

Therefore, cells were seeded at a lower density of 10 000 cells/well for future experiments using

the SS-HA-βlac-β2AR stable cell line.

3.3.1 Comparison of Isoproterenol stimulated β2AR Internalization using the βlac and

the ELISA Assays

Internalization of the β2AR by isoproterenol has been used by others in the validation of

their assays for the quantification of surface expression (Hammer et al., 2007; Fisher et al.,

2010; Wu et al., 2012b). Having optimized the conditions for measuring βlac activity for the SS-

HA-βlac-β2AR stable cell line, the effects of isoproterenol on internalization of the SS-HA-βlac-

β2AR were investigated in parallel to well established classical ELISA.

43

5 Minute Agonist Stimulation

-12 -10 -8 -6 -4

-0.05

0.00

0.05

0.10

0.15

0.2050ng Blac-B2AR

50ng HA-B2AR

Empty Vector

log [Isoproterenol] M

cA

MP

levels

(

BR

ET

)

30 Minute Agonist Stimulation

-12 -10 -8 -6 -4

-0.05

0.00

0.05

0.10

0.15

0.2050ng Blac-B2AR

50ng HA-B2AR

Empty Vector

log [Isoproterenol] M

cA

MP

levels

(

BR

ET

)

Figure 3.3: β2AR functional assay using the BRET cAMP EPAC biosensor. HEK293 cells

stably expressing EPAC were transfected with the following: SS-HA-βlac-β2AR (red), HA-

β2AR (blue), or empty vector (black). Cells were plated in white 96-well plates at 100,000 cells

per well. Cells were washed once with PBS and coelenterazine H was added. After 5 minutes

isoproterenol was added and the plate read once every 5 minutes on the Mithras luminometer.

(A) After 5 minutes isoproterenol stimulation the empty vector transfected cells had an EC50 of

120 nM. The SS-HA-βlac-β2AR and HA-β2AR EC50 were left shifted at 0.68±1.6 and 0.31 ±0.8

nM respectively. (B) After 30 minutes of isoproterenol stimulation the empty vector transfected

cells had almost completely desensitized. The SS-HA-βlac-β2AR and HA-β2AR maintained

their EC50 at 0.78±0.7 and 0.34±0.4 nM respectively. The empty vector transfected cells was a

representative curve while the SS-HA-βlac-β2AR and HA-β2AR transfected cells were three

independent experiments (n=3). All data are represented as mean of the background subtracted

Rluc/YFP ratio ± S.E.M.

A

B

44

0 20 40 60

60

80

100

120

Time (min)

% C

ell S

urf

ace

Exp

ressio

n

0 20 40 60

60

80

100

120

Time (min)

% C

ell S

urf

ace

Exp

ressio

n

-12 -10 -8 -6 -4 -240

50

60

70

80

90

100

110

log [Isoproterenol] M

% C

ell S

urf

ace

Exp

ressio

n

-12 -10 -8 -6 -4 -240

50

60

70

80

90

100

110

log [Isoproterenol] M

% C

ell S

urf

ace

Exp

ressio

n

Figure 3.4: Comparison of βlac and ELISA with the SS-HA-βlac-β2AR stable cell line. Cells

for the βlac assay were seeded into 48 well plates at 10 000 cells/well and the ELISA was seeded

at 100 000 cells/well. Time course of internalization of the SS-HA-βlac-β2AR was done on the

βlac (A) and ELISA (B) where the cells were stimulated with a dose of 10μM isoproterenol once

every five minutes. The half life values for βlac and ELISA internalization were 6.54±1.13 and

6.39±1.3 min respectively. The dose response for internalization for the SS-HA-βlac-β2AR was

also done with the βlac assay (C) and ELISA (D). Cells were treated with a dose range of

isoproterenol from 10-4

to 10-11

M and incubated for 30 minutes. An EC50 of 14.54±12.7 nM was

obtained for internalization using the βlac assay; the ELISA yielded a linear relationship. All

data are represented as mean of % vehicle treated ± S.E.M or three independent experiments

(n=3).

A B

C D

45

-12 -10 -8 -6 -4 -260

80

100

120

log [Alprenolol] M

% C

ell S

urf

ace

Ex

pre

ssio

n

-12 -10 -8 -6 -4 -260

80

100

120

log [Propranolol] M

% C

ell S

urf

ace E

xp

ressio

n

Figure 3.5: Blocking of isoproterenol induced internalization with pre treatment of

antagonists. SS-HA-βlac-β2AR cells were seeded in 48 well plates at 10 000 cells/well.

Antagonists alprenolol (A) and propranolol (B) were added to the cells for 30 minutes from a

dose range of 10-4

- 10-10

M. After antagonist incubation, cells were subsequently stimulated with

a dose of 10μM isoproterenol for 30 minutes. Alprenolol and propranolol yielded IC50 values of

16.3±18.0 and 66.1±35.5 nM respectively. All data are represented as mean of % vehicle treated

± S.E.M of four independent experiments (n=4).

A

B

46

Parallel experiments were done to investigate the differences between the ELISA and

βlac assay by looking at the time course and dose response of isoproterenol induced

internalization (Figure 3.4). The time course of receptor internalization was measured after

stimulating cells with a dose of 10μM isoproterenol in 5 minute intervals for 60 minutes. Both

the βlac assay and ELISA yielded similar half-lives (6.54±1.13 and 6.39±1.3 minutes

respectively) for internalization of β2AR (Figure 3.4A–B). In a second set of studies, the dose

response of β2AR internalization was evaluated with a dose range of 10-4

to 10-11

M of

isoproterenol. After stimulating the cells for 30 minutes with various doses of isoproterenol an

EC50 of 14.54±12.7 nM was obtained for the βlac assay (Figure 3.4C). In comparison the ELISA

yielded a linear relationship which does not allow us to calculate an EC50 value (Figure 3.4D). In

summary, the parallel experiments showed the βlac assay was equally as quantitative as the

ELISA in the time course experiments. However for the dose response of isoproterenol induced

internalization, the βlac assay was able to yield an EC50 consistent to what has been reported in

the literature (Hammer et al., 2007; Fisher et al., 2010) while the ELISA yielded a linear

relationship for which an EC50 cannot be calculated.

3.3.2 Antagonist Blocking of Isoproterenol Induced Internalization of the β2AR

It is well established that agonist induced internalization can be blocked with the

presence of antagonists. We investigated the ability of propranolol and alprenolol, two known

β2AR antagonists, in blocking isoproterenol induced internalization of the SS-HA-βlac-β2AR.

Cells were pre-treated for 30 minutes with the antagonists followed by the addition of 10μM

isoproterenol. Both alprenolol and propranolol blocked isoproterenol mediated internalization in

a dose dependent manner (Figure 3.5A and B). The IC50 values for alprenolol and propranolol

for inhibition of isoproterenol induced internalization were 16.3±18.0 and 66.1±35.5 nM

respectively.

3.3.3 Z’ Determination of the SS-HA-βlac-β2AR Internalization

In order to evaluate the robustness of the βlac assay for measuring β2AR internalization, the Z’

Factor of this specific assay was evaluated (Figure 3.5). The Z’ is a statistical measure regarding

the quality of an assay where the Z’ takes into account both the signal to noise ratio as well as the

variation between positive and negative controls. A Z’ value greater than 0.5 indicates the assay

is suitable for screening purposes. These studies were carried out in a 96 well plate where the SS-

47

Figure 3.6: Z’ of the βlac assay using the SS-HA-βlac-β2AR stimulated with isoproterenol.

SS-HA-βlac-β2AR cells were seeded into 96 well plates at 5000 cells/well. Evaluation of the Z’

of SS-HA-βlac-β2AR internalization was done after stimulation with isoproterenol. Cells were

treated with either vehicle (blue) or 10µM isoproterenol (red) for 30 minutes and internalization

was assessed using the using the βlac assay. The solid horizontal line represents the mean of the

two conditions while the dotted liens represent 3 standard deviations away from the mean. The

calculated value is Z’=0.52.

Z’=0.52

48

HA-βlac-β2AR stable cells were seeded at a density of 5000 cells/well. Cells were treated with

either 10μM isoproterenol (Positive control) or vehicle (Negative control) for 30 minutes. Using

the equation described in the methods sections (Section 2.14), the Z’ was determined to be 0.52,

indicating the assay is robust enough to be suitable for screening purposes (Zhang et al., 1999b).

3.3.4 Pharmacological Chaperoning using β2AR Antagonists

In the last set of experiment with SS-HA-βlac-β2AR, the effects of alprenolol and

propranolol as potential pharmacological chaperones of the β2AR were evaluated. It has been

shown previously that both alprenolol and propranolol act as pharmacological chaperones for the

β1AR (Kobayashi et al., 2009) and given that both alprenolol and propranolol are high affinity

antagonists for β2AR as well as being lipophilic (logp = 2.9, ACD logP), it is proposed that these

antagonists could potentially act as pharmacological chaperones for the β2AR as well. Using the

βlac assay, the effects of an over-night treatment of alprenolol and propranolol on surface

expression of β2AR were measured (Figure 3.7). Treatment with both antagonists (10μM)

increased the surface expression of the SS-HA-βlac-β2AR. Alprenolol (Figure 3.7A) induced

increase in surface expression could be measured starting at a dose of 1μM compared to

propranolol (Figure 3.7B) which showed initial effects at a dose of 10μM. This result indicates

that alprenolol has a greater potency than propranolol for increasing the surface expression,

potentially through pharmacological chaperoning. Therefore the βlac assay has the ability to

quantify increases in surface expression of β2AR after treatment with alprenolol and propranolol.

In summary the βlac assay was able to quantify the internalization profile of the SS-HA-

βlac-β2AR. Specifically when the βlac assay is compared to the ELISA, both assays show

similar results for the time course of β2AR internalization upon stimulation with isoproterenol.

The dose response for internalization yielded an EC50 for the βlac assay while the ELISA yielded

a linear trend for internalization. Furthermore the βlac assay was also able to quantify antagonist

blockade of agonist induced internalization. In addition the βlac assay was able to produce a Z’ =

0.52 for SS-HA-βlac-β2AR internalization; indicating this assay is suitable for screening

purposes. Finally chronic/overnight treatment with the antagonists alprenolol and propranolol

also increased the surface expression of the SS-HA-βlac-β2AR receptors consistent with the

possibility of pharmacological chaperoning. Therefore these results indicate that βlac assay is a

49

0 -9 -8 -7 -6 -5 -4

90

100

110

120

130

140

150**

***

*

log [Alprenolol] M

% C

ell S

urf

ace

Exp

ressio

n

0 -9 -8 -7 -6 -5 -4

90

100

110

120

130

140

150*

**

log [Propranolol] M

% C

ell S

urf

ace

Exp

ressio

n

Figure 3.7: Overnight incubation with alprenolol and propranolol increases surface

expression of SS-HA-βlac-β2AR. SS-HA-βlac-β2ARcells were seeded into 48 well plates at 10

000 cells/well and were incubated overnight with antagonists (n=3). (A) Overnight incubation

with alprenolol showed significant differences in surface expression were detected with doses of

1, 10, and 100 μM. (B) Overnight incubation with propranolol showed significant differences in

surface expression were detected with doses of 10 and 100 μM. One-way ANOVA with

bonferonni correct post-hoc were used to determine the differences between data sets * P<0.05,

** P<0.01, *** P<0.001. All data are represented as mean of % vehicle treated ± S.E.M.

A

B

50

suitable assay for measuring the effects of agonist induced internalization as well as the potential

for pharmacological chaperoning.

3.4 Comparison of GBR1 Surface Expression using the βlac and the ELISA

GBR1 is a well characterized class C GPCR that requires the dimerization of the

molecular chaperone GBR2 for its proper surface expression (see section 1.4.3). Therefore by

using the SS-HA-βlac-GBR1 construct we investigated the ability of the βlac assays to quantify

increases in surface expression due to molecular chaperoning. As with the internalization

experiments, both the βlac and the ELISA experiments were done in parallel. The SS-HA-βlac-

GBR1 stable cell line was transfected with an increasing amount of GBR2 and plated in a 48

well plate with 100,000 cells/well for both the βlac and ELISA assays. As shown in figure 3.8,

the resulting increase in surface expression of the SS-HA-βlac-GBR1 after GBR2 transfection

was quantitatively very similar between the ELISA and βlac assay. For both assays, the βlac and

ELISA (Figure 3.8A and B) there was a statistically significant increase in surface expression of

SS-HA-βlac-GBR1 at 0.22 μg of GBR2 transfection and above. In summary the βlac assay is

equivalent to the ELISA in quantifying the increase in GBR1 surface expression due to

molecular chaperoning from GBR2 co-expression.

51

g GBR2

0.00

g G

BR2

0.02

g G

BR2

0.07

g G

BR2

0.22

g G

BR2

0.66

g G

BR2

2.00

0.5

1.0

1.5

2.0

2.5

3.0

***

**

Fo

ld o

f 0

g G

BR

2

g GBR2

0.00

g G

BR2

0.02

g G

BR2

0.07

g G

BR2

0.22

g G

BR2

0.66

g G

BR2

2.00

0.5

1.0

1.5

2.0

2.5

3.0

Fo

ld o

f 0

g G

BR

2

**

***

*

Figure 3.8: Comparison of βlac assay and ELISA Surface expression of GBR1 with GBR2

co-expression. SS-HA-βlac-GBR1 cells were transfected with differing amounts of GBR2 and

seeded into 48 well plates at 100 000 cells/well (N=3). Quantification of the increase of GBR1

surface expression using the βlac assay (A) or ELISA (B) Both assays showed significant

differences in surface expression when transfected with 0.22, 0.66, and 2.00 μg of GBR2. One-

way ANOVA with bonferonni correct post-hoc were used to determine the differences between

data sets * P<0.05, ** P<0.01, *** P<0.001. All data are represented as mean of % mock

transfected cells ± S.E.M.

A

B

52

Chapter 4

Discussion

4.1 Summary of Key Findings

In the present study, we show that the βlac assay is a novel assay for the quantification of

GPCRs. Our results show that the βlac assay is equally quantifiable as the classical approach,

ELISA, when monitoring the time course of isoproterenol mediated internalization of the β2AR

or the surface expression of GBR1. Furthermore, the βlac assay was able to quantify changes in

β2AR surface expression after antagonist treatment hinting to the potential pharmacological

chaperoning effects of these antagonists on β2AR.

4.2 Nitrocefin Permeability

One important aspect of the βlac assay that has not been addressed thus far is whether or

not nitrocefin is cell impermeable. Although this has not been well established in the literature

for eukaryotic cells, there are two lines of evidence that strongly suggest that nitrocefin is cell

impermeable. First, the results obtained from βlac and ELISA experiments conducted in parallel

are quantitatively and qualitatively similar, which would have not been the case if nitrocefin was

cell permeable. Second, the cytoplasmic βlac-arrestin construct was utilized to experimentally

measure cell permeability of nitrocefin (Pieter Beerepoot, Salahpour lab; Appendix Figure 1).

Since βlac-arrestin is localized in the cytoplasm, the βlac is therefore not present at the plasma

membrane, resulting in no nitrocefin hydrolysis if nitrocefin is cell impermeable. In this

experiment there is little to no signal when nitrocefin is added to cells transfected with different

amounts of βlac-arrestin. However when the cells are lysed using mechanical force and

hypotonic shock, there is a large increase in signal upon addition of nitrocefin, indicating that

nitrocefin did not cross the plasma membrane in the non-lysed, intact cells. Therefore based on

the data presented in this experiment, nitrocefin is cell impermeable and is a suitable substrate

for use in the quantification of surface expression of GPCRs.

4.3 Functional Experiments with the SS-HA-βlac-β2AR

The addition of a large N-terminal tag to GPCRs can potentially affect the function of the

receptor. This is especially important when attempting to quantify effects of GPCR trafficking

53

(ie internalization) that is a direct result of GPCR signalling and ligand binding. It is therefore

important to determine the effects that the βlac might have on the β2AR. The first experiment

carried out was immunoflouresence of the SS-HA-βlac-β2AR, which showed a uniform

distribution of the receptor at the cell surface when treated with vehicle, consistent with surface

expression as shown in the literature(Kim et al., 2008). Upon stimulation with isoproterenol the

receptor localizes to intracellular aggregates which is consistent with localization within

endosomes as reported before (Cao et al., 2005).

To complement the immunofluoresence data, the signalling of the SS-HA-βlac-β2AR

was compared in parallel with the HA-β2AR receptor. For these studies a BRET EPAC cAMP

biosensor was used. This biosensor has been established as a robust method for assessing GPCR

signalling through the Gαs pathway (Barak et al., 2008; Salahpour et al., 2012). The biosensor is

formed by the addition of Renilla-luciferase (Rluc) and YFP to the N- and C-terminus of the

EPAC protein. In its basal state the EPAC adopts a conformation such that the Rluc and YFP

moieties are in close proximity resulting in high transfer of energy from the Rluc to the YFP and

therefore high BRET levels. Upon binding of cAMP the EPAC adopts a conformation that

moves the Rluc and YFP apart leading to a decrease in the BRET signal (Figure 4.1). Using this

cAMP biosensor the signalling of the SS-HA-βlac-β2AR and HA-β2AR were assessed. Our

results show that SS-HA-βlac-β2AR and HA-β2AR produce similar dose response curves and

EC50s with regards to isoproterenol induced cAMP signalling. It is important to note that it is

possible that the HA tag could affect the function of the β2AR. However it has been shown

previously that other small N-terminal epitopes (ie c-myc) on the β2AR did not affect binding of

[I125

]cyanpindolol compared to WT receptors(Angers et al., 2000). Lastly, it was observed that

isoproterenol response in both the SS-HA-βlac-β2AR and HA-β2AR transfected cells did not

desensitize after 30 minutes. It is possible that the heterologous expression of these receptors

saturates the desensitization/internalization machinery of the HEK 293 cells. Indeed, this has

been previously observed with the β3AR in other cell lines (Chaudhry and Granneman, 1994)

and the AT1R (Violin et al., 2006a). Furthermore, studies with TAAR1 in HEK293 cells showed

that transfection of β-arrestin 2 enables proper desensitization of this receptor (Barak et al.,

2008). Therefore the cause of the lack of desensitization of recombinant β2AR in HEK 293 cells

can be studied by either using different cell lines or by transfecting additional arrestins or GRKs

into HEK 293 cells. In summary the SS-HA-βlac-β2AR and HA-β2AR dose response curves

54

Figure 4.1: Representative diagram for the BRET EPAC cAMP Biosensor. The EPAC

biosensor contains an N- and C-terminal Rluc and YFP respectively. In its resting state the

EPAC adopts a conformation such that the Rluc and YFP are within 100Å, allowomg for the

transfer of energy from the Rluc to the YFP, resulting in a high BRET level (left panel). Upon

binding of cAMP the EPAC adopts a conformation that moves the Rluc and YFP away

decreasing the BRET (right panel).

cAMP

EPAC Rluc

YFP

EPA

C

Substrate

YFP

Rluc Substrate

475nm

525nm

475n

m

55

have nearly identical EC50 values indicating that the addition of βlac at the N-terminus of the

receptor does not affect its ability to signal through Gs. Additional experiments can be performed

to further determine if the additional of the N-terminal βlac has any effect on β2AR function.

Such experiments include radioligand binding of the receptor and determining the intracellular

and extracellular pools of receptors for both the SS-HA-βlac-β2AR and HA-β2AR cells to

determine if there is a difference in expression level.

4.4 SS-HA-βlac-β2AR Internalization

Our assay operates under the assumption that the enzyme reaction is operating under

Michaelis-Menten kinetics. The substrate concentration used is above Km and presumably in

excess of the enzyme, allowing for zero order kinetics. The assay is measured kinetically once

every minute upon addition of substrate (nitrocefin) where the readout for the assay is the initial

velocity that is measured as the initial slope of the kinetics of the reaction. In the future,

additional characterization will be done on the enzyme kinetics in order to insure that the

experimental conditions are appropriate.

In order to validate the βlac assay, the β2AR internalization profile of β2AR was

evaluated using the βlac assay and ELISA in parallel. The isoproterenol induced internalization

of the SS-HA-βlac-β2AR with the βlac assay yielded results that were consistent with the values

in the literature. The dose response of isoproterenol induced β2AR internalization with βlac

assay yielded EC50 values that were nearly identical to what has been reported with other surface

expression assays (Hammer et al., 2007; Fisher et al., 2010; Takeda et al., 2012). In addition, the

EC50 of the βlac assay is similar to the EC50 (24.54 ± 12.7 nM) as measured by flow cytometry

(Pieter Beerepoot, Salahpour lab; Appendix Figure 2). However when the dose response of

internalization was done in parallel with an ELISA, the ELISA yielded a linear trend. The linear

trend for the ELISA could potentially be due to an assay artefact. When looking at flow

cytometry, a relatively similar assay protocol, the results yielded similar EC50 values to the βlac

assay. The specific cause of this linear relationship remains elusive where additional experiments

need to be done to determine the cause. One additional experiment to examine the cause of the

linear trend in the ELISA would be to increase the dose range of isoproterenol in order to see if

the response returns to a sigmoidal function.

56

The time course of isoproterenol induced internalization of the βlac-β2AR yielded very

similar half-lives for the βlac and ELISA assays. Furthermore these values are in agreement with

what has been previously reported for β2AR internalization (Moore et al., 1995). In addition

these half-lives are also very similar to the half-life (4.52±0.74 minutes) that was obtained from

flow cytometry (Pieter Beerepoot, Salahpour lab; Appendix Figure 3). However, in some other

studies, using other surface expression assays, the half-lives of isoproterenol induced β2AR

internalization is at least 2 fold higher than what we observed. These assays include the FAP

biosensor, β-galactosidase complementation, and a endosomal BRET assay (Hammer et al.,

2007; Fisher et al., 2010; Lan et al., 2011). It is important to note the experimental differences

between these and the βlac assay. Two of the reported studies and assays quantify receptor

internalization through the recruitment of the receptors into endosomes (Hammer et al., 2007;

Lan et al., 2011) while the βlac assay and ELISA quantifies internalization through the absence

of receptors on the cell surface. It is therefore possible that the time point for targeting a GPCR

to an endosome is greater than that of internalization. However, a direct comparison between

these assays and the βlac assay needs to be done in order to further examine why the half-life

values are different for these different assays. All three assays have different maximum

internalization values. While the ELISA and βlac assay have similar maximum internalization

value (~40%), flow cytometry yielded ~60% internalization (see Appendix figure 2 and 3). It is

probable that this difference is due to the fact that in flow cytometry, the antibody is done on

cells in suspension while for ELISA and βlac the assays are carried out on adherent cells.

Lastly SS-HA-βlac-β2AR internalization by isoproterenol can be blocked through pre-

treatment with antagonists alprenolol or propranolol in a dose dependent manner. The IC50 for

alprenolol and propranolol were 16.3±18.0 and 66.1±35.5 nM respectively. The IC50 value for

propranolol is similar to what has been reported in the literature (Hammer et al., 2007; Fisher et

al., 2010), and it appears that our study is the first to report an IC50 value for alprenolol blocking

of isoproterenol induced β2AR internalization. It is important to note that after 30 minutes of

alprenolol treatment there was an increase in surface expression compared to vehicle treated cells

(Figure 3.5). Since alprenolol is an inverse agonist for the β2AR (Varma et al., 1999) it is

possible that the 30 minute treatment could have stabilized the receptors present on the plasma

membrane. Interestingly however, this effect was not seen with propranolol treatment which is

also an inverse agonist as well (Varma et al., 1999).

57

4.5 Pharmacological chaperoning

Using the βlac assay we observed that overnight treatment with either alprenolol or

propranolol increased the surface expression of the SS-HA-βlac-β2AR. This experiment also

showed that alprenolol increased the surface expression of the β2AR at a lower dose (1μM)

compared to propranolol (10µM). The mechanism for why alprenolol is more potent than

propranolol in increasing surface expression is not known. Although these doses for

pharmacological chaperoning are rather high for the antagonists, the relationship between

antagonist affinity and its ability to act as a pharmacological chaperone is not currently known. It

is possible that the dose reported in this study is a function of both the affinity of the antagonist

for the receptor as well as the ability of the compound to cross the plasma membrane. The

affinity of these two antagonists are similar with Ki values of 0.4 and 1nM for propranolol and

alprenolol respectively (Fraundorfer et al., 1994; Hoffmann et al., 2004). One potential

explanation for this effect could be in relation to the off rate for these two antagonists where the

relative off rate of alprenolol could be higher than propranolol; however this has not been shown

in the literature.

In addition while this increase in surface expression is consistent with pharmacological

chaperoning, additional experiments need to be done in order to determine the exact mechanism

by which treatment with these antagonists leads to increased surface expression. Indeed, there

could be multiple effects that increase surface expression of GPCR. For example, compounds

could act by stabilizing the receptor in its inactive state decreasing constitutive activity and

therefore stabilizing the receptor on the plasma membrane. Therefore, it is possible that inverse

agonists may be more potent at stabilizing receptors at the cell surface than neutral antagonists.

Indeed this mechanism of increasing surface expression through stabilization of the receptor at

the plasma membrane has previously been reported for the histamine H2 (Smit et al., 1996)

receptor as well as other constitutively active mutant receptors (Heinflink et al., 1995; Gether et

al., 1997; Lee et al., 1997; Takeda et al., 2012). Indeed, since both alprenolol and propranolol act

as inverse agonists on the β2AR they could potentially increase surface expression through this

mechanism (Chidiac et al., 1994). This mechanism has been proposed for the in vivo action of

nadolol, a hydrophilic β2AR antagonist (Walker et al., 2011). Second, the increase in surface

expression could be due to pharmacological chaperoning since there is some percentage of

GPCRs that are misfolded and subsequently degraded through the ER quality control mechanism

58

(Dong et al., 2007). A number of experiments could be performed in order to assess whether or

not alprenolol and propranolol are pharmacological chaperones for the β2AR. First, the most

established way of determining pharmacological chaperoning is the use of misfolding mutants

that do not traffic to the plasma membrane (Morello et al., 2000; Kobayashi et al., 2009). If

treatment of these mutants with alprenolol or propranolol rescued their surface expression, it

would be good evidence of a pharmacological chaperoning effect of these compounds. Secondly

incubation with a hydrophilic antagonist such as nadolol (logp = 0.56) should not increase the

surface expression of the β2AR if the mechanism is exclusively through pharmacological

chaperoning of nascent receptors within the ER (Morello et al., 2000).

In sum, we suggest that the effects of alprenolol and propranolol on surface expression of β2AR

are possibly due to both the stabilization of the receptor at the plasma membrane as well as

pharmacological chaperoning.

4.6 Z’ Factor of βlac-β2AR internalization

The Z’ is a statistical measure regarding the quality of an assay. The Z’ takes into account

both the signal to noise ratio as well as the variation between positive and negative controls. A Z’

> 0.5 indicates an assay is suitable for screening purposes. Utilizing the internalization of the SS-

HA-βlac-β2AR as a readout, a Z’=0.52 was obtained in a 96 well format. This Z’ indicates that

the βlac assay, under these conditions, can be used to screen for compounds that induce

internalization of the SS-HA-βlac-β2AR. It is important to note that we did not attempt to

determine the Z’ for an ELISA using the SS-HA-βlac-β2AR cells. However we hypothesize that

the Z’ for the ELISA would be lower than the βlac assay due to the variability as a result of the

multiple wash steps.

Other studies have looked at the ability of other assays to yield a suitable Z’ for β2AR

internalization. For the coiled coil tag assay, a Z’= 0.30 in a 96 well plate for β2AR

internalization was reported, which indicates that this assay is not suitable for screening (Takeda

et al., 2012). Next, the β-galactosidase complementation assay yielded a Z’=0.6 in a 384 well

plate, a similar Z’ to the βlac assay (Hammer et al., 2007). The higher Z’ of the β-galactosidase

assay is most likely due to automation of that assay which would decrease the variability of the

assay and improve the Z’. Next, the FAP biosensor yielded a Z’= 0.72 in a 384 well plate (Wu et

al., 2012b). The higher Z’ factor for this assay is most likely due to the increase in the maximum

59

measured internalization to 100% compared to 40% for the βlac assay. This increase in the

maximal internalization could be a result of two factors. First the U937 cell line was used for the

FAP biosensor experiments. Therefore, it is possible that the U937 cell line expressed a higher

level of GRKs and/or arrestins leading to increased receptor phosphorylation and internalization.

Indeed expression levels of arrestins and specific GRKs in HEK293 cells affect the

internalization of the β2AR (Violin et al., 2006b, 2008). However the expression levels of

arrestin and GRKs in U937 cells has not been studied. Secondly because the FAP biosensor is

quantified using flow cytometry, it is possible that this method of measurement reports a higher

rate of internalization than other methods. Indeed this is seen within our lab (Pieter Beerepoot,

Salahpour lab; Appendix Figure 2 and 3) where using flow cytometry we have measured a

greater percentage of internalized receptors compared to ELISA or βlac. Therefore the increase

in Z’ for the FAP biosensor could potentially be due to these two factors listed above.

In order to improve the Z’ of the βlac assay for further miniaturization, several

modifications to the system can be done. As listed above, transfecting arrestins and GRKs could

increase the internalization of the receptors therefore increasing the signal/noise ratio.

Furthermore, automation could be introduced to decrease the variability associated with manual

pipetting. Lastly using different cell lines could increase the internalization of the receptors as

seen with other assays (Hammer et al., 2007; Fisher et al., 2010; Wu et al., 2012b). These

modifications could potentially further improve the Z’ of the βlac assay for internalization of

β2AR.

Our future experiments include determining the Z’ for both molecular and

pharmacological chaperoning using the βlac assay. We expect that these two measurement

outputs should yield a higher Z’ than that of β2AR internalization. For molecular chaperoning,

the negative non-transfected control should have little to no surface expression yielding a higher

signal to background ratio for this assay in comparison to the β2AR internalization assay. Indeed

our results show that transfecting 2μg of GBR2 into the SS-HA-βlac-GBR1 stable cell line

yielded a 2.5 fold higher signal compared to mock transfected SS-HA-βlac-GBR1 cells. This is

considerably higher than the 40% difference measured in internalization assays.

To determine the Z’ for pharmacological chaperoning, a misfolding GPCR mutant would

be used. For example the vasopressin V2R del62-64 mutant has been previously shown to be

60

misfolded in the ER leading to a lack of surface expression of this receptor (Morello et al., 2000).

Treatment of cells with SR 49059, a selective cell permeable antagonist of V2R, results in the

rescue of surface expression of this mutant through a pharmacological chaperone effect.

Therefore this mutant V2R receptor in conjunction with SR 49059 treatment could be used to

determine the Z’ of the βlac assay for pharmacological chaperoning.

4.7 SS-HA- βlac-GBR1 Surface Expression by Co-expression with GBR2

In our last set of experiments we show that the βlac assay is able to quantify GBR1 cell

surface expression in an equivalent manner to the ELISA. Although the ELISA appears to have a

slightly greater magnitude of change compared to the βlac assay (Figure 3.8), the ELISA is also

more variable as evidenced by the larger error bars.

It is important to note that studies within the Salahpour lab have shown that SS-HA- βlac-

GBR1 receptor is able to signal in a similar manner to WT-GBR1 upon stimulation with

baclofen, a GBR1 agonist (Pieter Beerepoot, Salahpour lab; Appendix Figure 4). This

observation is in line with studies that have shown that the addition of the 30kDa SNAP tag to

the N-terminus of GBR1 does not affect its function when compared to WT receptors (Maurel et

al., 2008). It is important to note that interaction sites of GPCRs with molecular chaperones (in

this case GBR2) reside in the transmembrane domain as well as the C-terminus of the GPCR

(White et al., 1998; Wu et al., 2012a). Therefore tagging GPCRs on the N-terminus would seem

an appropriate approach when investigating molecular chaperones.

4.8 Cost Analyses

The main advantages on the βlac assay are the simplicity and low cost relative to other

assays that quantify surface expression of GPCRs (Table 1). When compared to an ELISA, the

βlac assay is almost 10 fold less expensive in material agents alone. Furthermore, the βlac assay

compares favorably to other surface expression assays such as the FAP biosensor and the

SNAP/CLIP-tag assays due to the lack of commercial availability of the substrates for these

assays. Indeed both the FAP and SNAP/CLIP-tag assays require chemical synthesis of their

respective compounds before the assay can be carried out (Maurel et al., 2008; Szent-Gyorgyi et

al., 2008). In summary, the βlac assay is a simple and cost effective assay for the quantification

of surface expression of GPCRs.

61

Future Directions

Future experiments for βlac assay are split into two groups. First, we would continue to

validate the assay and determine the Z’ factor for both pharmacological and molecular

chaperoning as described section 4.6. Secondly once the Z’ factors are found we would like to

screen for either molecular or pharmacological chaperones for receptors that do not express on

the plasma membrane (ie. TAAR1).

Conclusion

In this study we have demonstrated that the creation of an N-terminal βlac-GPCR fusion

protein allows for the quantification of surface expression. Through the use of the SS-HA-βlac-

β2AR and SS-HA-βlac-GBR1, the βlac assay has been validated for use in quantifying

internalization (dose response, time course, and antagonist blockade), increase in surface

expression due to molecular chaperoning and potentially pharmacological chaperoning. When

comparing the βlac assay with an ELISA, the assays performed very similarly. The main

advantages of the βlac assay are its low cost and simplicity compared to ELISA or other

traditional assays (Table 1). Given the advantages in cost, simplicity and most importantly

robustness, the βlac assay is now the standard assay for quantification of surface expression of

GPCRs within the Salahpour lab, where ELISAs are no longer performed. Therefore to conclude

this study, the βlac assay is a novel assay that can be readily adopted in any laboratory due to its

fast, robust, reproducible and cost effective qualities compared to all other assays currently

available for quantifying surface expression.

62

Table 1: Comparison of time and cost of ELISA and βlac assays

steps ELISA βlac

1 Wash (1 time) Wash (1 time)

2 Block Add nitrocefin

3 Add 1° antibody Read (Abs 486nm)

4 Wash (3 times)

5 Fix (4% PFA)

6 Wash (3 times)

7 Block

8 Add 2° antibody

9 Wash (3 times)

10 Add substrate

11 Stop reaction

12 Read (Abs 496nm)

time 4- 6 hours 15-60 minutes

cost 30-50 cents/well 4 cents/well

63

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Appendices

O2

- ddH

O2

+ ddH

0

10

20

30

40

50Mock Transfected

0.5 mg blac-bArrestin

1.0 mg blac-bArrestin

2.0 mg blac-bArrestin

Fo

ld In

cre

ase in

Sig

nal **

Appendix Figure 1: Cell permeability of the βlac substrate nitrocefin. A rat β-arrestin2-βlac

construct was created by replacing the YFP in the β-arrestin-YFP construct with βlac (Angers et

al., 2000). Cells were transfected with 0, 0.5, 1, and 2 μg of βarrestin- βlac DNA, and were

plated in a 48-well plate 24 hours post-transfection. At 48 hours post-transfection, cells were

washed and incubated for 15 minutes in either PBS or ddH20. After incubation, cell solutions in

the ddH20 treated wells were mechanically lysed by pipetting up and down. PBS was aspirated

from the PBS-treated wells and nitrocefin in PBS was added. To the ddH2O treated wells, a

solutioin of 2X nitrocefin in 2X PBS was then added, and nitrocefin hydrolysis was monitored.

Unlysed cells showed no increase in signal compared to mock transfected cells, regardless of the

amount of βarrestin-βlac DNA that was transfected. Cell lysis resulted in increases in signal with

increasing amounts of βarrestin-βlac DNA reaching 33±6.8.fold compared to 2±0.9 fold for

lysed mock cells. Two-way ANOVA with Bonferroni correct t-test post-hoc were used to

determine differences between data sets ** P<0.01. All data represented as mean fold increase in

signal from mock transfected HEK 293 cells ± S.E.M, n=3.

84

-12 -10 -8 -6 -4 -240

60

80

100

log [Isoproterenol] M

% C

ell S

urf

ace

Exp

ressio

n

Appendix Figure 2: Dose response of isoproterenol mediated internalization quantified

with flow cytometry. Cells were seeded into 6 well plates at 4 000 000 cells/well. The cells

were stimulated with isoproterenol in a dose range of 10-4

0 10-11

M. Immediately after

completion of drug treatment cells were put on ice and washed with cold PBS. The cells were

lifted off by incubation for 10 min with PBS 0.02%EDTA.The EDTA was neutralized by adding

cell culture media (DMEM 10% FBS). The suspensions were then spun down in a centrifuge

with a swinging bucket rotor at 1500 rpm for 5 minutes and washed twice with PBS 2% FBS.

Cells were spun down and resuspended in PBS 2%FBS with 1:250 primary antibody and

incubated for 30 minutes. Subsequently, cells were spun down and washed twice in PBS

2%FBS. After centrifugation cells were incubated in 1:100 Alexa Fluor 647 Donkey anti-mouse

IgG antibody (Invitrogen) in 100 μL PBS 2% FBS for 15 minutes. After 2 more PBS 2% FBS

washes, the cells were fixed in PBS 2% PFA for 30 minutes, spun down, and resuspended in

PBS 2%FBS. Subsequently, the cell suspensions were strained through cell strainer caps (BD

Falcon) into round bottom tubes. Fluorescence was then acquired on a BD LSR Fortessa flow

cytometer with an excitation wavelength of 640 nm, and a 670/14 bandpass filter, collecting

10000 events per sample. All steps were performed on ice and samples were protected from light

after addition of secondary antibody. The EC50 for SS-HA-βlac-β2AR internalization was 24.54

± 12.66 nM. All data are represented as mean of % vehicle treated ± S.E.M or three independent

experiments (n=3).

85

0 20 40 6040

60

80

100

Time (min)

% C

ell S

urf

ace

Exp

ressio

n

Appendix Figure 3: Time course of isoproterenol mediated internalization quantified with

flow cytometry. Cells were seeded into 6 well plates at 4 000 000 cells/well. The cells were

stimulated with isoproterenol at a dose of 10μM at time points of 0, 5, 10, 15, 30, and 50

minutes. Immediately after completion of drug treatment cells were put on ice and washed with

cold PBS. The cells were lifted off by incubation for 10 min with PBS 0.02%EDTA.The EDTA

was neutralized by adding cell culture media (DMEM 10% FBS). The suspensions were then

spun down in a centrifuge with a swinging bucket rotor at 1500 rpm for 5 minutes and washed

twice with PBS 2% FBS. Cells were spun down and resuspended in PBS 2%FBS with 1:250

primary antibody and incubated for 30 minutes. Subsequently, cells were spun down and washed

twice in PBS 2%FBS. After centrifugation cells were incubated in 1:100 Alexa Fluor 647

Donkey anti-mouse IgG antibody (Invitrogen) in 100 μL PBS 2% FBS for 15 minutes. After 2

more PBS 2% FBS washes, the cells were fixed in PBS 2% PFA for 30 minutes, spun down, and

resuspended in PBS 2%FBS. Subsequently, the cell suspensions were strained through cell

strainer caps (BD Falcon) into round bottom tubes. Fluorescence was then acquired on a BD

LSR Fortessa flow cytometer with an excitation wavelength of 640 nm, and a 670/14 bandpass

filter, collecting 10000 events per sample. All steps were performed on ice and samples were

protected from light after addition of secondary antibody. The half life of internalization for SS-

HA-βlac-β2AR internalization was 4.52±0.74 min. All data are represented as mean of %

vehicle treated ± S.E.M or three independent experiments (n=3).

86

Appendix Figure 4: Functionality of SS-HA-βlac-GBR1 using the BRET EPAC cAMP

biosensor. HEK293 cells stably expressing EPAC were transfected with the following: WT-

GBR1 or SS-HA-βlac-GBR1 with 0.5-5μg GBR2. Cells were plated in white 96-well plates at

100,000 cells per well. Cells were washed once with PBS and coelenterazine H was added. After

5 minutes isoproterenol was added and the plate read once every 5 minutes on the Mithras

luminometer. Treatment with forskolin results in formation of cAMP and therefore a change in

BRET. This forskolin signal is partially blocked by -4M but not –9M baclofen treatment in the

wildtype and βlac tagged GBR1. t-tests were performed to compare the effect of -4M baclofen to

forskolin only treated cells n=4, * P<0.05, ** P<0.01