A Primer on G Protein Signaling. Elliott Ross UT-Southwestern Medical Center

Transcription

1 A Primer on G Protein Signaling Elliott Ross UT-Southwestern Medical Center

2 Receptor G Effector The MODULE Rhodopsins Adrenergics Muscarinics Serotonin, Dopamine Histamine, GABA b, Glutamate Eiscosanoids PAF, Sphingolipids Purinergics Peptides (kinins, angiotenisin, opioids, endothelin, glucagon, etc.) Glycoprotein hormones Membrane proteins (Smoothened, BOSS) Fungal pheromones Odorants, tastants G s (>3), G olf G i (3) G t (2), G gus G o (2) G z G q (4) G 12/13 Gβ (5) Gγ (11) Adenylyl cyclase cgmp Phosphodiesterase Phospholipase C-β Channels (K +, Ca 2+,Na + ) PI-3-Kinase Rho GEFs Rap GAPs Protein kinases (S/T and Y) (phosphatases?) Transporters (Mg 2+, glucose?, biogenic amines?,) Vesicle trafficking

3 The Gq-PLC Module An example

4 RHODOPSIN a G Protein-Coupled Receptor Cytoplasmic Extracellular

5 The G Protein αβγ Trimer Gα β γ i1 1 2 Mark Wall, Steve Sprang, et mult.

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7 How these proteins MAY be arranged in space N. Gautam

8 R R 1 R 2 R 3 R 1 R 2 R 3 G G G 1 G 2 G 3 E E E R R G 1 G 2 G 3 G E 1 E 2 E 3 E 1 E 2 E 3 But the pathways can branch R 1 R 2 R 3 G 1 G 2 G 3 E 1 E 2 E 3

9 G PROTEINS ARE TWO-STATE SWITCHES Gα-GDP GDP k 1 k -1 GTP Pi Gα -GTP 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.

10 G proteins are two-state switches; they have 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-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

11 Gα subunits hydrolyze bound GTP to GDP. Hydrolysis is slow, but faster than dissociation of GTP, so a GTPase cycle is created. The steady-state fraction of G protein in the GTP-bound state constitutes the fractional activity of the system. G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

12 Receptors accelerate the release of GDP and 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-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

13 Hydrolysis of bound GTP is slow; t 1/2 ~ 10 s - 5 min. GTPase-activating proteins (GAPs) accelerate hydrolysis ~2000-fold. G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

14 These proteins constitute a G protein signaling module. Specific components can be chosen from the parts list. You get one of each in each module. G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

15 G Protein Activation Creates Two Signaling Molecules Gα-GTP E 1 E 2E3 Gαβγ-GDP Gαβγ-GTP Gβγ E 4 E 5E6 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

16 R Gαβγ-GTP Gα -GTP Pi Gβγ Gα-GDP Gαβγ-GDP GTP R Gβγ Cycle R-Gαβγ GDP

17 G Protein Activation Creates Two Signaling Molecules Gα-GTP E 1 E 2E3 Gαβγ-GDP Gαβγ-GTP Gβγ E 4 E 5E6 Activation of Gα releases active Gβγ Therefore Gβγ drives deactivation of Gα probably not very important

18 G Protein Activation Creates Two Signaling Molecules Gα-GTP E 1 E 2E3 Gαβγ-GDP Gαβγ-GTP Gβγ E 4 E 5E6 Activation of Gα releases active Gβγ Therefore Gβγ 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 Gα It s a GDP-dissociation inhibitor, or GDI Gβγ both inhibits G protein activation and suppresses spontaneous background noise

19 Functions of Gβγ Regulates effectors when released by activated Gα Inhibits Gα activation (by GDI effect) Suppresses spontaneous noise Gβγ release by one trimer may inhibit activation of another Anchors Gα to membranes Facilitates activation of Gα by receptor Nearly obligate Maybe by anchoring Inhibits GAPs

20 G Proteins as Four-State Systems: Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange Nucleotide exchange is slow, but receptor catalyzes exchange by converting the GTP-binding site on Gα from the closed to the open configuration. Gα o * -GTP Gα c * -GTP R + H R*-H G*-GTP-E* E G*-GTP GAP GTP Pi G GDP G-GDP Gα o -GDP R Gα c -GDP Negative heterotropic binding of R and nucleotide Quantitatively reciprocal Ligand-mediated ligand exchange

21 G Proteins as Four-State Systems: Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange Gα o * -GTP Gα c * -GTP G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP Pi Gα o -GDP R Gα c -GDP GDP G-GDP Two major factors drive this cycle clockwise: Hydrolysis of GTP is favored Cellular [GTP] > [GDP] GTP also binds much tighter than GDP Affinities have never been measured at equilibrium (really! )

22 G Proteins as Four-State Systems: Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange Gα o * -GTP Gα c * -GTP G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP Pi Gα o -GDP Gα c -GDP GDP R G-GDP If R can exchange nucleotide in less than the lifetime of the activated state, then it can catalytically (sequentially) act on multiple G proteins.

23 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. R Gα * -GTP [R-Gα * -GTP] Pi Gα-GDP GTP R R-Gα GDP [R-Gα-GDP] 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.

24 GAPs You can t turn off a signal upon removal of hormone any faster than you can hydrolyze GTP. G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

25 GAPs for Heterotrimeric G Proteins Receptor G-GDP G*-GTP GAP ACCELERATE GTP HYDROLYSIS 2000-FOLD EFFECTORS Phospholipase C-β : G q GAP Rho GEF p115: G 13 GAP (with RGS domain) cgmp phosphodiesterase : co-gap for G t GPCR kinases (RGS domain) RGS PROTEINS Most not effectors ~30 genes in mammals Conserved RGS box, diverse functional ends For G i and G q p115 s for G12/13, maybe a new group for G s

26 Regulatory Functions of G Protein GAPs Receptor G-GDP G*-GTP GAP Response Attenuate Turn off Sharpen Lower Background Time

27 Regulatory Functions of G Protein GAPs Inhibit Response Steepen Time Change selectivity log [Agonist]

28 GAPs Need Not Attenuate the Signal GIRK Channels in Xenopus Oocytes Kir 3.1/3.2; m2 MAChR Doupnik et al., PNAS 94:10461

29 Single Photon Responses of RGS9 - Mice Chen et al., Nature 403, 557 (2000)

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31 Reconstitution of G q -Phospholipase Signaling Pathway M1 Muscarinic Acetylcholine Receptor Gα q βγ Phospholipids (including PIP 2 ) (purified, in detergent solution) Phospholipase C-β (± other GAP) Slowly remove detergent Unilamellar vesicles, ~100 nm diameter, scrambled Measure binding and release of hormone and nucleotides, hydrolysis of GTP, hydrolysis of PIP 2 ; both at steady state and in single catalytic cycles.

32 lated PLC activity. Conditions were as follows: no addition ( GTP (GTP), 1 mm carbachol (Cch), carbachol plus 10 M (C A), and 100 nm GTP S ( S). All samples contained 10 nm fr FIG. 2.Reconstitution of G q -mediated activation of PLC- 1 by m1achr. m1achr and G q were co-reconstituted with [ 3 H]PIP 2 as described under Experimental Procedures. The activity of added PLC- 1 was measured in the presence of guanine nucleotide and/or muscarinic ligands. A, GTP-dependent PLC activity; B, GTP S-stimu- Phospholipase C Regulation 2.4 nm G q, 0.33in nmreceptor-g m1achr, 1 PLC- 1, q -PLC andvesicles 0.56 M accessi 8002 A and B show data m1(mean Muscarinic S.D.) fromreceptor-g the same experi q - different scales. Phospholipase Activity (pmol IP 3 /min) GTP - - GTP GTP - - CCh +At CCh +At [Ca 2+ ] (nm) FIG. 3. Effect of Ca 2 on G q -stimulated PLC- 1 a m1achr and G q were co-reconstituted with [ 3 H]PIP 2, and the of added PLC- 1 was measured at various free Ca 2 concen The data plotted are the initial rates determined from time co PLC activity conducted at each Ca 2 concentration in the prese FIG conc vesic

33 GTPase (mol / min / mol G q ) M1AChR - G q Vesicles RGS4 PLC-β [GAP] (nm) S. Mukhopadhyay

34 What kind of mechanistic information can you get out of a system like this?

35 Synergistic Action of Receptors and GAPs G. Berstein

36 Carbachol-Stimulated GTP Binding to G q G. Biddlecome

37 PLC Activation Displays a Lag When Initiated by Carbachol G. Biddlecome

38 R Gα * -GTP [R-Gα * -GTP] R-Gα * -GTP-G Pi Gα-GDP GTP R-Gα-G R-Gα-GDP-G R R-Gα GDP [R-Gα-GDP]

39 Quench-Flow Assay of GTP Hydrolysis Rate 1. Receptor-G protein vesicles, GAP, agonist, GTP Incubate to steady-state 2. Equilibrate with [γ- 32 P]GTP 3. Add excess unlabeled GTP, antagonist: t = 0 4. Terminate with H 3 PO 4 at time t Buffer A B C Vesicles GAP GTP Agonist [γ- 32 P]GTP Cold GTP

40 Hydrolysis of Gα q -Bound GTP GTP Hydrolyzed (fmol) t 1/2 =25 ms RGS Time (s) t 1/2 =57 ms PLC-β S. Mukhopadhyay

41 [α- 32 P]GDP Bound (fmol) GDP Dissociation from G q t 30 o 1/2 = 460 ms Time (s) S. Mukhopadhyay

42 R Gα * -GTP [R-Gα * -GTP] R-Gα * -GTP-G Pi Gα-GDP 10 5 M -1 s s -1 GTP R-Gα-G R-Gα-GDP-G R R-Gα 2 s -1 GDP [R-Gα-GDP] 2 s -1

43 Receptor-G Protein-GAP Complex Kinetically limited by receptor-g protein binding or other receptor event Slow: t 1/2 ~ 20 s; agonist dependent, GTP-independent Scaffolded in cells? Association with GAP is fast Stability during steady-state turnover requires agonist and GTP (not GTPγS) Complex dissociates slowly upon removal of agonist t 1/ sec by quenching assay k dissoc for Gα-GTP ~ s -1 τ ~ s

44 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.

45 So what s wrong with this picture? Good: Predicts K m and V max at steady-state Predicts reasonable amount of Gα*-GTP Has physiologically fast rates R [R-Gα * -GTP] 105 M -1 s -1 GTP R-Gα * -GTP-G R-Gα-G Gα * -GTP Pi 20 s -1 R-Gα-GDP-G Gα-GDP R Not so good: Amplitude of Pi release in singleturnover hydrolysis ~8X higher than the predicted steady-state amount R-Gα 2 s -1 2 s -1 GDP [R-Gα-GDP] Likely explanation: During rapid turnover, the binding site on Gα never 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.

46 So here s a module. G*-GTP-E* E G*-GTP GAP R + H R*-H G GTP GDP Pi G-GDP

47 There are lots of extra-modular regulatory inputs -- feedback, off-pathway, and/or cell type-dependent... Phosphorylation Dephosphorylation Palmitoylation De Endocytosis Degradation Arrestin binding Other proteins Scaffold assembly Other GDP/GTP exchange catalysts Other GDIs What is extramodular??? All this stuff, plus the products of the effector and the incoming signal.

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