Lecture 11: GPCR Lecture 11: GPCR pathwayspathways

Fain Chapter 410/7/09

Kao - high transmission fiber Kao - high transmission fiber optic cables from pure optic cables from pure

materialsmaterials

Fiber optic networksFiber optic networks

Current amount of fiber goes around world 25,000 times

Boyle and Smith - CCDBoyle and Smith - CCD

Central dogmaCentral dogmaDNA DNA mRNAmRNAproteinprotein

The amazing ribosome creates proteins

Ada Yonath crystallizes ribosome

Small subunit - 32 proteins

Large subunit - 46 proteins

Geobacillus stearothermophilus - hot springs

Haloarcula marismortui - Dead Sea

Homework #5Homework #5

Gene duplicationBuild a tree (Pasteur)Think about gene function (OMIM)Locate genes (Map viewer)

Homework #1: Homework #1: GNA treesGNA trees

GNAI2

GNAI3

GNAI1

GNAT1

GNAT2

GNAT3 Taste

Cone

Rod

Search for gene of Search for gene of interestinterest

Link to chromosome Link to chromosome locationlocation

Click here

Can control which maps Can control which maps are shownare shown

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

Find chromosomal Find chromosomal location of gene - many location of gene - many

linkslinks

You can zoom in or out to You can zoom in or out to see more detailsee more detail

Location of GNAT and Location of GNAT and GNAIGNAI

GNAT2GNAI3

1p13GNAT1GNAI2

3p21

GNAT3GNAI1

7q21

Location of GNAI and Location of GNAI and GNATGNAT

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

The wonders of G protein The wonders of G protein signalingsignaling

Signal amplification Control, regulation and specificity Evolution of diversity Gene expression

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

Metabotropic sensory Metabotropic sensory transductiontransduction

Figure 4.1

Channel

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

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

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

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

Xray crystal structure of Xray crystal structure of GPCRGPCR

Palczewski et al 2000

11-cis retinal11-cis retinal

Rhodopsin

Yokoyama and Starmer 1996

GPCR phylogenyN=neurotransmitter

P=peptides

S=sensory

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

G protein=G protein=

Numbers of different versions of subunits in human genome

20-30 G

5 G

12 G

1994 Nobel prize in 1994 Nobel prize in medicinemedicine

GGGPA

Ancient G proteins

GGGs stimulates adenylate cyclase

includes olfactory

GGGs stimulates adenylate cyclase

includes olfactory

Gi/Go Inhibitory and otherIncludes vision and taste

Transducingustducin

GGGs stimulates adenylate cyclase

includes olfactory

Gi/Go inhibitsIncludes vision and taste

Gq

Activates PLC

G G and and tethered to tethered to membranemembrane

tethered by geranyl geranyl (gg) tethered by palmitoyl (p)

GG bound to GDP is bound to GDP is inactiveinactive

Activated GPCR activates Activated GPCR activates G proteinG protein

GDP

GPCR

GTP binding activates GGTP binding activates G

GDP

GTP

GTP

+

Get dissociation of GGet dissociation of G and G and G

GPCR*

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

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)

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?

Evolutionary trace Evolutionary trace analysisanalysis

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

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

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

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

ET methodET method

Key changes Key changes in function in function occur along occur along nodes to nodes to groupsgroups

GTP binding activates GGTP binding activates G

GDP

GTP

GTP

+

Get dissociation of GGet dissociation of G and G and G

GPCR*

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

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

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

Remove Remove subunit to find subunit to find conserved conserved binding binding

regionregion

Conserved sites identified by ET comparisons

Remove GRemove G to conserved to conserved binding site binding site

A2 is binding site of to

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

Onrust et al. 1997Onrust et al. 1997

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

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!

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!