1. Heterotrimeric G proteins and the role of lipids in signaling John Sondek, Ph.D. Depts. of Pharmacology and Biochemistry & Biophyscis

2. The GTPase cycle – molecular switch

3. A GTPases is NOT a kinase

5. Two major regulators of GTPase cycle

6. Specific GEFs and GAPs for heterotrimeric G proteins

7. Active heterotrimer dissociates into a G  subunit and a G  dimer

8. G protein-coupled receptors as GEFs for heterotrimeric G proteins GPCRs are the largest family of cell-surface receptors for extracellular signals (superfamily of >1000 members) GPCRs respond to a wide range of inputs: hormones, neurotransmitters, odorants, tastants, photons of light, etc. ~60% of all clinical therapeutics act by affecting some aspect of GPCR signaling (e.g., agonist, antagonist, inhibition of natural ligand metabolism)

9. GPCRs are seven transmembrane (7TM) receptors

10. G proteins sense conformational changes of intracellular loops

12. Active receptor stabilized nucleotide-depleted heterotrimer

13. GTP-loading catalyzes heterotrimer dissociation

14. Figure 10-20 Molecular Biology of the Cell (© Garland Science 2008)

15. Rasmussen et al., Nature 450, 383 (2007)

16. Bockaert & Pin (1999) EMBO J. 18:1732 R G E

17. Rhodopsin-like:Ligand-bindingpocket IL8, fMLP PAF Thrombin: - dopamine, adrenaline, serotonin (5HT) Special case of protease-activated GPCRs - Irreversible activation by N-terminal cleavage Bockaert & Pin (1999) EMBO J. 18:1732 “DRY”-motif critical for G-protein activation Palmitoylated cysteine

18. Bockaert & Pin (1999) EMBO J. 18:1732 High-mol.wt. hormones: Glucagon Metabotropic glutamate/GABA/pheromone: Large “Venus flytrap” disulfide-linked ectodomain

19. Figure 10-33 Molecular Biology of the Cell (© Garland Science 2008) Crystal structure of rhodopsin Nature, 289, 739 (2000)

20. The G  subunit : Lipid modifications for membrane binding Exotoxin from Bordetella pertussis (whooping cough) catalyzes ADP-ribosylation of i-class G  : Result? De-coupling from receptor Exotoxin from Vibrio cholerae (cholera diarrhea) catalyzes ADP-ribosylation of s-class G  Result? Constitutive activity since crippled as a GTPase

21. G  contains a Ras-like domain and an all-helical domain Three “switch regions” have conformations that depend on which nucleotide is bound

22. G  contains a Ras-like domain and an all-helical domain

23. G  t. GDP. AlF 4 Ras/p120 GAP. GDP. AlF 3 S1 S2 S3 S1 S2 p120 GAP Ras Helical domain Ras-like domain “Arginine finger” is critical for GTP hydrolysis. R174 R789 transducin Aluminum tetrafluoride ion mimics third phosphate of GTP and thereby “activates” GDP-bound G  subunits.

24. Mg 2+ Q200 R174 AlF 4 S2 S1 H2OH2O Q61 R789 S1 S2 AlF 3 Mg 2+ H2OH2O GDP G  t. GDP. AlF 4 Ras/p120 GAP. GDP. AlF 3 For Ras-like GTPases, arginine finger is supplied in trans by GAPs. For heterotrimeric G proteins, the catalytic arginine is part of the alpha subunit. “Arginine finger” is critical for GTP hydrolysis Site of cholera activation; R>C mutation activates. Q>L mutation also activates by crippling GTPase function.

25. Primary sequence characteristics of G  dimers

26. G  forms a propeller; G  is an extended helical peptide. CAAX Sondek et al. Nature (1996) 379: 369

27. The switches of G  are primary interaction sites with G  (no interactions between G  and G  !) Lambright et al. Nature (1996) 379: 311.

28. The switches of G  are primary interaction sites with G  (the N-terminal helix of G  is also used) Lambright et al. Nature (1996) 379: 311.

29. Portions (purple) of all three subunits contact receptor Area of ADP-ribosylation via pertussis toxin (  i-class) Swap receptor specificity by switching C-termini? Inhibit coupling via C-terminal minigenes? Gilchrist et al. (2001) JBC 276:25672

30. m retinal sugars rhodopsin GGGG GGGG S2 S1 S3 S-Prenyl (GG or F) N-Myristoyl, S-palmitoyl GDP Interface defined by structural features and biochemical evidence Regions of protein in purple are implicated in receptor interactions membrane

31. G  switches (I, II, III) are sensitive to bound nucleotide

32. G  switch regions (I, II) directly interact with GTP

33. The four families of G  subunits Adenylyl cyclase (+) (-)(-) Phospholipase C-beta (+) Rho-GTP (+) Pertussis-toxin sensitive G  subunits

34. Examples of G-protein effector systems G-protein subunitEffector“Second messenger” G  s, G  olf  adenylyl cyclase  [cAMP] G  i1/i2/i3  adenylyl cyclase  [cAMP] G  q/11  phospholipase-C  [IP 3 ] &  [DAG] G  12/13  RGS-box RhoGEFs  [RhoA-GTP]  phospholipase-C  [IP 3 ] &  [DAG] G  (transducin)  cyclic GMP phosphodiesterase  [cGMP] G  dimer  or  adenylyl cyclase  or  [cAMP]  PLC-  PLC-  [IP 3 ] &  [DAG]  or  ion channel flux K+ and Ca2+

35. Four G  subunits and one oddball

36. Blake et al. (2001) JBC 276:49267 Plenty o’ G  subunits

37. Gs = “stimulatory” G-protein linked to adenylyl cyclase activation (2nd-messenger generation)  s GTP   AC Adenylyl cyclase  2-adrenergic receptor on vasculature of skeletal muscles Adrenaline or Isoproterenol (“1st messenger”) + ATPcAMP Second messenger Net result: Five-fold increase in [cAMP] i in seconds

38. S3 S2 S1 GsGs IIC2 VC1 forskolin GTP  S Forskolin favors dimerization of cyclase domains G  s interacts with cyclase primarily through switches 1 and 2 Complex of G  s with cytoplasmic portions of Adenylyl cyclase Tesmer (1997) Science 278:1907

39. Turning off the signal Multiple levels & multiple time-frames Reuptake/destruction of agonist (~millisec) Hydrolysis of GTP bound to G  subunit (~sec) Reuptake/destruction of second messenger (~sec) Uncouple receptor from signal machinery (~sec/min) Remove receptor from cell-surface (~min/hr)

40. Adapted from Hollinger & Hepler (2002). The R7 family

41. RGS proteins stabilize the transition state for GTP hydrolysis

42. Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008)

43. Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008)

44. Figure 10-3 Molecular Biology of the Cell (© Garland Science 2008)

45. Figure 10-4 Molecular Biology of the Cell (© Garland Science 2008)

46. Figure 10-5 Molecular Biology of the Cell (© Garland Science 2008)

47. Figure 10-7b Molecular Biology of the Cell (© Garland Science 2008)

48. Figure 10-11 Molecular Biology of the Cell (© Garland Science 2008)

50. Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)

51. PLCs are phospholipases

52. PLC-  isozymes are classic effectors of heterotrimeric G proteins