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Heterotrimeric G proteins and the role of lipids in signaling John Sondek, Ph.D. Depts. of Pharmacology and Biochemistry & Biophyscis Slide 2 The GTPase cycle molecular switch Slide 3 A GTPases is NOT a kinase Slide 4 Slide 5 Two major regulators of GTPase cycle Slide 6 Specific GEFs and GAPs for heterotrimeric G proteins Slide 7 Active heterotrimer dissociates into a G subunit and a G dimer Slide 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) Slide 9 GPCRs are seven transmembrane (7TM) receptors Slide 10 G proteins sense conformational changes of intracellular loops Slide 11 Slide 12 Active receptor stabilized nucleotide-depleted heterotrimer Slide 13 GTP-loading catalyzes heterotrimer dissociation Slide 14 Figure 10-20 Molecular Biology of the Cell ( Garland Science 2008) Slide 15 Rasmussen et al., Nature 450, 383 (2007) Slide 16 Bockaert & Pin (1999) EMBO J. 18:1732 R G E Slide 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 Slide 18 Bockaert & Pin (1999) EMBO J. 18:1732 High-mol.wt. hormones: Glucagon Metabotropic glutamate/GABA/pheromone: Large Venus flytrap disulfide-linked ectodomain Slide 19 Figure 10-33 Molecular Biology of the Cell ( Garland Science 2008) Crystal structure of rhodopsin Nature, 289, 739 (2000) Slide 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 Slide 21 G contains a Ras-like domain and an all-helical domain Three switch regions have conformations that depend on which nucleotide is bound Slide 22 G contains a Ras-like domain and an all-helical domain Slide 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. Slide 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. Slide 25 Primary sequence characteristics of G dimers Slide 26 G forms a propeller; G is an extended helical peptide. CAAX Sondek et al. Nature (1996) 379: 369 Slide 27 The switches of G are primary interaction sites with G (no interactions between G and G !) Lambright et al. Nature (1996) 379: 311. Slide 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. Slide 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 Slide 30 m retinal sugars rhodopsin GGGG GGGG 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 Slide 31 G switches (I, II, III) are sensitive to bound nucleotide Slide 32 G switch regions (I, II) directly interact with GTP Slide 33 The four families of G subunits Adenylyl cyclase (+) (-)(-) Phospholipase C-beta (+) Rho-GTP (+) Pertussis-toxin sensitive G subunits Slide 34 Examples of G-protein effector systems G-protein subunitEffectorSecond 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+ Slide 35 Four G subunits and one oddball Slide 36 Blake et al. (2001) JBC 276:49267 Plenty o G subunits Slide 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 Slide 38 S3 S2 S1 GsGs 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 Slide 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) Slide 40 Adapted from Hollinger & Hepler (2002). The R7 family Slide 41 RGS proteins stabilize the transition state for GTP hydrolysis Slide 42 Figure 10-1 Molecular Biology of the Cell ( Garland Science 2008) Slide 43 Figure 10-2 Molecular Biology of the Cell ( Garland Science 2008) Slide 44 Figure 10-3 Molecular Biology of the Cell ( Garland Science 2008) Slide 45 Figure 10-4 Molecular Biology of the Cell ( Garland Science 2008) Slide 46 Figure 10-5 Molecular Biology of the Cell ( Garland Science 2008) Slide 47 Figure 10-7b Molecular Biology of the Cell ( Garland Science 2008) Slide 48 Figure 10-11 Molecular Biology of the Cell ( Garland Science 2008) Slide 49 Slide 50 Figure 10-19 Molecular Biology of the Cell ( Garland Science 2008) Slide 51 PLCs are phospholipases Slide 52 PLC- isozymes are classic effectors of heterotrimeric G proteins