Lecture 11: GPCR pathways Fain Chapter 4 10/7/09.
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Transcript of Lecture 11: GPCR pathways Fain Chapter 4 10/7/09.
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Lecture 11: GPCR Lecture 11: GPCR pathwayspathways
Fain Chapter 410/7/09
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Kao - high transmission fiber Kao - high transmission fiber optic cables from pure optic cables from pure
materialsmaterials
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Fiber optic networksFiber optic networks
Current amount of fiber goes around world 25,000 times
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Boyle and Smith - CCDBoyle and Smith - CCD
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Central dogmaCentral dogmaDNA DNA mRNAmRNAproteinprotein
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The amazing ribosome creates proteins
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Ada Yonath crystallizes ribosome
Small subunit - 32 proteins
Large subunit - 46 proteins
Geobacillus stearothermophilus - hot springs
Haloarcula marismortui - Dead Sea
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Homework #5Homework #5
Gene duplicationBuild a tree (Pasteur)Think about gene function (OMIM)Locate genes (Map viewer)
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Homework #1: Homework #1: GNA treesGNA trees
GNAI2
GNAI3
GNAI1
GNAT1
GNAT2
GNAT3 Taste
Cone
Rod
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Search for gene of Search for gene of interestinterest
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Link to chromosome Link to chromosome locationlocation
Click here
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Can control which maps Can control which maps are shownare shown
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Can remove those you Can remove those you don’t want (rna maps)don’t want (rna maps)
Highlight items and then click remove
Then update with OK
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Find chromosomal Find chromosomal location of gene - many location of gene - many
linkslinks
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You can zoom in or out to You can zoom in or out to see more detailsee more detail
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Location of GNAT and Location of GNAT and GNAIGNAI
GNAT2GNAI3
1p13GNAT1GNAI2
3p21
GNAT3GNAI1
7q21
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Location of GNAI and Location of GNAI and GNATGNAT
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Rest of semesterRest of semester Individual senses
Fain chaptersPrimary literature
Midterm projectTrp channel analysis - next week
Individual project topicsDiversity of one sense across organismsSignal transductionSensory diversity within one organism
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The wonders of G protein The wonders of G protein signalingsignaling
Signal amplification Control, regulation and specificity Evolution of diversity Gene expression
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Ch4: Metabotropic signal Ch4: Metabotropic signal transductiontransduction
Indirect link from receptor to channelUse messenger system
Receptor G protein Effector 2nd messenger Channel Neural signal
Receptors are G protein coupledSimilar to hormone and neurotransmitter
signal transduction mechanisms
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Metabotropic sensory Metabotropic sensory transductiontransduction
Figure 4.1
Channel
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G proteins activate G proteins activate effectorseffectors
Adenylyl cyclase = makes cAMP Guanyl cyclase = makes cGMP Phospholipase C = makes DAG and IP3
Phospholipase A = makes arachidonic acid
Phosphodiesterase = hydrolyzes cAMP or cGMP
2nd messengers open/close channelschange ion concentration and membrane
potential
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Diversity of GPCRsDiversity of GPCRs Human genome
1500-2000 GPCRs (3-5% of genome) Kinds
Hormone receptors FSH, Oxytocin, Vasopressin
Synaptic transmittersDopamine, opiates, glutamate
Sensory receptorsOlfactory receptorsVisual pigmentsTaste receptors for bitter, sweet and AA
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Basic GPCR structure• 7 TM regions
•Phosphorylation sites on C terminus
•G protein binds to C terminus and intracellular loops 2 and 3
Figure 4.3
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Basic GPCR structure• 7 TM regions
•Phosphorylation sites on C terminus
•G protein binds to C terminus and intracellular loops 2 and 3
•Ligand binds either- in membrane
- norepinephrine- olfaction
-extracellular site- glutamate- GABA Figure 4.3
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Xray crystal structure of Xray crystal structure of GPCRGPCR
Palczewski et al 2000
11-cis retinal11-cis retinal
Rhodopsin
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Yokoyama and Starmer 1996
GPCR phylogenyN=neurotransmitter
P=peptides
S=sensory
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G proteinsG proteins
Ones that interact with GPCR are trimeric - and
Act like switchBinding site on for GDP If exchange GDP for GTP, becomes
activatedDissociates from
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G protein=G protein=
Numbers of different versions of subunits in human genome
20-30 G
5 G
12 G
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1994 Nobel prize in 1994 Nobel prize in medicinemedicine
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GGGPA
Ancient G proteins
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GGGs stimulates adenylate cyclase
includes olfactory
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GGGs stimulates adenylate cyclase
includes olfactory
Gi/Go Inhibitory and otherIncludes vision and taste
Transducingustducin
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GGGs stimulates adenylate cyclase
includes olfactory
Gi/Go inhibitsIncludes vision and taste
Gq
Activates PLC
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G G and and tethered to tethered to membranemembrane
tethered by geranyl geranyl (gg) tethered by palmitoyl (p)
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GG bound to GDP is bound to GDP is inactiveinactive
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Activated GPCR activates Activated GPCR activates G proteinG protein
GDP
GPCR
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GTP binding activates GGTP binding activates G
GDP
GTP
GTP
+
Get dissociation of GGet dissociation of G and G and G
GPCR*
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GTP binding activates GGTP binding activates G
+
Both GBoth G and G and G can activate effector molecules can activate effector molecules
Effector
AMP
cAMP
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GTP hydrolysis inactivates GTP hydrolysis inactivates GGRecombines with GRecombines with G
+
Hydrolysis toGDP
GDP
+
GDP
G will hydrolyze its own GTP slowlyGTPase activating proteins speed hydrolysis
Regulator of G protein signaling (RGS)
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Lichtarge wanted to Lichtarge wanted to explain properties of G explain properties of G
proteinsproteins How are they kept inactive?
G-G binding How do they interact with receptors?
GPCR binding How are they activated?
GDP-GTP binding pocket How do they interact with effectors? How are they inactivated?
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Evolutionary trace Evolutionary trace analysisanalysis
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Evolutionary trace (ET) Evolutionary trace (ET) methodmethod
ET is a way to compare proteins and identify conserved functional regionsAsk evolution where these regions are?
Hypothesis: Selection acts on AA sequenceParts of molecule which are critical for
function will be highly conservedParts of molecule which vary are not important
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ETET
Compare proteins with same functionSites which are fixed are key to functionIgnore variable sites
Compare proteins with different functionInvariant sites - same for both functionsClass specific - fixed within function and
different between functions
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ET methodET method
1. Gather protein homologs and align sequences
2. Use phylogenetic methods to group them into functional groups
Determine fixed sites in each group
3. Compare fixed sites between groups: Class-specific sites - distinguish functionInvariant sites
4. Map functionally important sites onto 3D structure
If they cluster, this is likely an active site
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Lichtarge compared 120 Lichtarge compared 120 sequences for Gsequences for G
Gs stimulates adenylate cyclase (AC)
Gt stimulates PDE
Gi inhibits AC
Go
Gq activates PLC
Also compare 20 G and 16 G
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ET methodET method
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Key changes Key changes in function in function occur along occur along nodes to nodes to groupsgroups
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GTP binding activates GGTP binding activates G
GDP
GTP
GTP
+
Get dissociation of GGet dissociation of G and G and G
GPCR*
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Conserved sites mapped Conserved sites mapped onto Xray crystal strutureonto Xray crystal struture
G A1 binds to receptor along with C terminal tailGTP is shown in blue
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Conserved sites mapped Conserved sites mapped onto Xray crystal strutureonto Xray crystal struture
9 of 17 sites in A1 class specific A1 likely interacts with effectors too
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Effector can bind to site Effector can bind to site A2 alsoA2 also
14 of 32 sites in A2 are class specific and so specific to effector
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Remove Remove subunit to find subunit to find conserved conserved binding binding
regionregion
Conserved sites identified by ET comparisons
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Remove GRemove G to conserved to conserved binding site binding site
A2 is binding site of to
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Test importance of site for Test importance of site for functionfunction
Mutate sites in G sequence by replacing them one by one with alanine
Express mutant G and combine with G and rhodopsin
Add lightIf G is working:
R+hv R* + G G-GTP-S35
Measure S35 to quantify amount of Gt activation
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Onrust et al. 1997Onrust et al. 1997
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ET sites agree with those found ET sites agree with those found by site directed mutagenesisby site directed mutagenesis
Sites important for interactionsSites important for receptorreceptor binding
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Biochemists can study Biochemists can study protein functionprotein function
Change one site at time and see what happens
Or just let evolution do the testsSee what sites are important!
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HomeworkHomework
Q1 - about Nobel prizes awarded this week
Q2 - about past Nobel prize lectureIf you can’t think of one to do, watch
Roderick Mackinnon talk about ion channels
Lots of good ones!