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