A Primer on G Protein Signaling

Elliott RossUT-Southwestern Medical Center

Receptor G Effector

RhodopsinsAdrenergicsMuscarinicsSerotonin, DopamineHistamine, GABAb, GlutamateEiscosanoidsPAF, SphingolipidsPurinergicsPeptides(kinins, angiotenisin, opioids,endothelin, glucagon, etc.)

Glycoprotein hormonesMembrane proteins

(Smoothened, BOSS)Fungal pheromonesOdorants, tastants

Gs (>3), GolfGi (3)Gt (2), GgusGo (2)GzGq (4)G12/13

Gβ (5)Gγ (11)

Adenylyl cyclasecGMP PhosphodiesterasePhospholipase C-βChannels (K+, Ca2+, Na+)PI-3-KinaseRho GEFsRap GAPsProtein kinases (S/T and Y)

(phosphatases ?)Transporters (Mg2+, glucose ?,

biogenic amines?,)Vesicle trafficking

The MODULE

The Gq-PLC Module – An example

RHODOPSIN – a G Protein-Coupled Receptor

Extracellular

Cytoplasmic

Gα β γi1 1 2Mark Wall, Steve Sprang, et mult.

The G Protein αβγ Trimer

N. Gautam

How these proteins MAY be arranged in space

R

G

E

R1

G1

E1

R2

G2

E2

R3

G3

E3

E1

R

G

E2 E3

G1

E1

R

G2

E2

G3

E3

R1 R2

G

E

R3 R1

G1

R2

G2

E

R3

G3

But the pathwayscan branch

Gα-GDP Gα∗-GTP

GDP GTP

Pi

k1

k-1

G PROTEINS ARE TWO-STATE SWITCHES

A Gα subunit can assume active and inactive conformations.G proteins are activated when they bind GTP.“Activated” means that they can regulate an effector, either positively or negatively.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

G proteins are two-state switches; they have active and inactive conformations.G proteins are activated when they bind GTP. “Activated” means that theycan regulate an effector either positivelyor negatively.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Gα subunits hydrolyze bound GTP to GDP. Hydrolysis is slow, but faster thandissociation of GTP, so a GTPase cycleis created.The “steady-state” fraction of G protein in the GTP-bound state constitutes the fractional activity of the system.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Receptors accelerate the release of GDPand the binding of GTP; they thus “activate”G proteins. Receptors can act catalytically (sequentially)on many G proteins, which results in signal amplification.Receptors are exchange catalysts.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Hydrolysis of bound GTP is slow; t1/2 ~ 10 s - 5 min. GTPase-activating proteins (GAPs)accelerate hydrolysis ~2000-fold.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

These proteins constitute a G proteinsignaling module. Specific components can be chosen from the parts list. You getone of each in each module.

G Protein Activation Creates Two Signaling Molecules

Gα-GTP

Gβγ

Gαβγ-GDP Gαβγ-GTP

E1E2 E3

E4E5E6

Gβγ is a stable dimer from which Gα can dissociate when it binds GTP.

Activation of Gα “releases” active Gβγ

Gβγ independently regulates its own effector proteins

This means that there is also a Gβγ cycle

Pi

GDP

R

R

GTP

Gα∗-GTP

R-Gαβγ

Gαβγ-GDP

Gα-GDP

Gαβγ-GTP

Gβγ

Gβγ Cycle

G Protein Activation Creates Two Signaling Molecules

Gα-GTP

Gβγ

Gαβγ-GDP Gαβγ-GTP

E1E2 E3

E4E5E6

Activation of Gα “releases” active Gβγ

Therefore

Gβγ drives deactivation of Gαprobably not very important

G Protein Activation Creates Two Signaling Molecules

Gα-GTP

Gβγ

Gαβγ-GDP Gαβγ-GTP

E1E2 E3

E4E5E6

Activation of Gα “releases” active Gβγ

ThereforeGβγ drives deactivation of Gα

probably not very important

Therefore, too, with a lot of data and some thermodynamics thrown in,

Gβγ stabilizes the binding of GDP from GαIt’s a GDP-dissociation inhibitor, or GDI

Gβγ both inhibits G protein activation and suppresses spontaneous background noise

Functions of Gβγ

Regulates effectors when released by activated GαInhibits Gα activation (by GDI effect)

Suppresses spontaneous noiseGβγ release by one trimer may inhibit activation of another

Anchors Gα to membranesFacilitates activation of Gα by receptor

Nearly obligate Maybe by anchoring

Inhibits GAPs

G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Nucleotide exchange is slow, but receptor catalyzes exchange by convertingthe GTP-binding site on Gα from the closed to the open configuration.

Gαo*-GTP

Gαo-GDP Gαc-GDP

Gαc*-GTP

RNegative heterotropic binding of R and nucleotide

Quantitatively reciprocalLigand-mediated ligand exchange

G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Gαo*-GTP

Gαo-GDP Gαc-GDP

Gαc*-GTP

RTwo major factors drive this cycle clockwise:

Hydrolysis of GTP is favoredCellular [GTP] > [GDP]

GTP also binds much tighter than GDPAffinities have never been measured at equilibrium

(really ! )

G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

Gαo*-GTP

Gαo-GDP Gαc-GDP

Gαc*-GTP

R

If R can exchange nucleotide in less than the lifetime of the activated state,then it can catalytically (sequentially) act on multiple G proteins.

G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange

If R can exchange nucleotide in less than the lifetime of the activated state,then it can catalytically (sequentially) act on multiple G proteins.

Gα*-GTP

Gα-GDP

[R-Gα-GDP]R-Gα

Pi

GDP

GTPR

[R-Gα*-GTP]

R

The diffusion-limited rate of encounter of receptor with Gα-GDP can limit the rate of activation (“collisional coupling”).Scaffolding proteins increase the encounter rate (a lot) but limit amplification.

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

GAPsYou can’t turn off a signal upon removal of hormone any faster than you can hydrolyze GTP.

GAPs for Heterotrimeric G Proteins

ACCELERATE GTP HYDROLYSIS 2000-FOLDEFFECTORS

Phospholipase C-β : Gq GAPRho GEF p115: G13 GAP (with RGS domain)cGMP phosphodiesterase : “co-GAP” for GtGPCR kinases (RGS domain)

RGS PROTEINSMost not effectors ~30 genes in mammalsConserved RGS box, diverse functional endsFor Gi and Gq

p115’s for G12/13, maybe a new group for Gs

G-GDPReceptor

GAPG*-GTP

Regulatory Functions of G Protein GAPs

AttenuateSharpen

Turn offLowerBackground

Time

Res

pons

eG-GDP

Receptor

GAPG*-GTP

Regulatory Functions of G Protein GAPs

Time

Res

pons

e

Inhibit

Changeselectivity

Steepen

log [Agonist]

GIRK Channels in Xenopus Oocytes

Kir 3.1/3.2; m2 MAChR

Doupnik et al., PNAS 94:10461

GAPs Need Not Attenuate the Signal

Chen et al., Nature 403, 557 (2000)

Single Photon Responses of RGS9- Mice

Reconstitution of Gq-Phospholipase Signaling Pathway

M1 MuscarinicAcetylcholine Receptor

Gαqβγ Phospholipids(including PIP2)

Phospholipase C-β(± other GAP)

(purified, in detergent solution)

Slowly remove detergent

Unilamellar vesicles, ~100 nm diameter, scrambled

Measure binding and release of hormone and nucleotides, hydrolysis of GTP, hydrolysis of PIP2;both at steady state and in single catalytic cycles.

FIG. 4.concentrvesicles thmeasuredand eithe

FIG. 2. Reconstitution of Gq-mediated activation of PLC-�1 bym1AChR. m1AChR and Gq were co-reconstituted with [3H]PIP2 asdescribed under “Experimental Procedures.” The activity of addedPLC-�1 was measured in the presence of guanine nucleotide and/ormuscarinic ligands. A, GTP-dependent PLC activity; B, GTP�S-stimu-lated PLC activity. Conditions were as follows: no addition (�), 10 �M

GTP (GTP), 1 mM carbachol (Cch), carbachol plus 10 �M atropine

m1 Muscarinic Receptor-Gq-PLC8002

FIG. 3. Effect of Ca2� on Gq-stimulated PLC-�1 activim1AChR and Gq were co-reconstituted with [3H]PIP2, and the activof added PLC-�1 was measured at various free Ca2� concentratioThe data plotted are the initial rates determined from time coursesPLC activity conducted at each Ca2� concentration in the presence of�M GTP and either 1 mM carbachol (filled circles) or 10 �M atrop(empty circles). Reactions contained 0.35 nM m1AChR, 3.0 nM Gq, 0�M accessible PIP2, and 1.0 nM PLC-�1.

PLC-�1 was measured in the presence of guanine nucleotide andmuscarinic ligands. A, GTP-dependent PLC activity; B, GTP�S-stimlated PLC activity. Conditions were as follows: no addition (�), 10GTP (GTP), 1 mM carbachol (Cch), carbachol plus 10 �M atrop(C�A), and 100 nM GTP�S (�S). All samples contained 10 nM free Ca2.4 nM Gq, 0.33 nM m1AChR, 1 nM PLC-�1, and 0.56 �M accessible PIA and B show data (mean � S.D.) from the same experimentdifferent scales.

Phospholipase C Regulation in Receptor-Gq-PLC Vesicles

- GTP - - GTP GTP- - CCh +At CCh +At [Ca2+] (nM)

0 100 200 300

Phos

phol

ipas

e A

ctiv

ity (

pmol

IP3/

min

)

[GAP] (nM)

GTP

ase

(mol

/ min

/m

ol G

q)

0

10

20

30

40

50

60

RGS4PLC-β1

0.1 1 10 100 1000

M1AChR - Gq Vesicles

S. Mukhopadhyay

What kind of mechanistic information

can you get out of a system like this?

G. Berstein

Synergistic Action of Receptors and GAPs

Carbachol-Stimulated GTP Binding to Gq

G. Biddlecome

G. Biddlecome

PLC Activation Displays a Lag When Initiated by Carbachol

Gα*-GTP

Gα-GDP

[R-Gα-GDP]R-Gα

R-Gα*-GTP-G

R-Gα-GDP-GR-Gα-G

Pi

GDP

GTPR

[R-Gα*-GTP]

R

Quench-Flow Assay of GTP Hydrolysis Rate1. Receptor-G protein vesicles, GAP, agonist, GTP

Incubate to steady-state 2. Equilibrate with [γ-32P]GTP3. Add excess unlabeled GTP, antagonist: t = 04. Terminate with H3PO4 at time t

VesiclesGAPGTP

Agonist

[γ-32P]GTP Cold GTP

A B CBuffer

0.0 0.2 0.40

150

175

200

0.0 0.2 0.40

150

200

250

300

0 2 4 60

100

150

200

250

0 2 4 60

100

200

300

400

Time (s)

GTP

Hyd

r oly

zed

(fm

ol)

Hydrolysis of Gαq-Bound GTP

t1/2=25 ms t1/2=57 msRGS4

PLC-β1

S. Mukhopadhyay

Time (s)0 2 4 6 8 10

[ α-32

P]G

DP

Bou

nd (f

mol

)

0

5

10

15

20

25

30o

GDP Dissociation from Gq

t1/2 = 460 ms

S. Mukhopadhyay

Gα-GDP

[R-Gα-GDP]R-Gα

R-Gα*-GTP-G

R-Gα-GDP-GR-Gα-G

Pi

GDP

GTPR

[R-Gα*-GTP]

R

2 s-1

2 s-1

20 s-1105 M-1s-1

Gα*-GTP

Receptor-G Protein-GAP Complex

Kinetically limited by receptor-G protein binding or other receptor eventSlow: t1/2 ~ 20 s; agonist dependent, GTP-independent

Scaffolded in cells ? Association with GAP is fast

Stability during steady-state turnoverrequires agonist and GTP (not GTPγS)

Complex dissociates slowly upon removal of agonistt1/2 30-90 sec by quenching assay kdissoc for Gα-GTP ~ 0.05 - 0.1 s-1 τ ~ 10-20 s

One more aspect to maintaining a relatively stable R - G - GAP module:

Only a receptor that can bind G-GTP tightly enough to traverse the cycle can signal; these receptors will be kinetically tuned by the GAP.

Signals from receptors that bind less tightly will simply be inhibited.

So what’s wrong with this picture?Good:Predicts Km and Vmax at

steady-statePredicts reasonable amount of

Gα*-GTPHas physiologically fast rates

Not so good:Amplitude of Pi release in single-

turnover hydrolysis ~8X higherthan the predicted steady-state amount

Likely explanation: During rapid turnover, the binding site on Gαnever relaxes to the closed configuration because receptor is always bound. And GAPs amplify the ability of receptors to drive activation. And a bunch of other stuff.

Gα-GDP

[R-Gα-GDP]R-Gα

R-Gα*-GTP-G

R-Gα-GDP-GR-Gα-G

Pi

GDP

GTPR

[R-Gα*-GTP]

R

2 s-1

2 s-1

20 s-1105 M-1s-1

Gα*-GTP

G*-GTP

G-GDP

G*-GTP-E*

E

GR + H R*-HPiGTP

GDP

GAP

So here’s a module.

PhosphorylationDephosphorylation

PalmitoylationDe…

EndocytosisDegradationArrestin bindingOther proteins…Scaffold assemblyOther GDP/GTP exchange

catalystsOther GDIs

There are lots of extra-modular regulatory inputs --feedback, off-pathway, and/or cell type-dependent ...

What is extramodular ???All this stuff, plus the products of the effector and the incoming signal.