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GTP-Binding Proteins • Heterotrimeric G Proteins

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GTP-Binding Proteins • Heterotrimeric G Proteins GTP-binding proteins • Heterotrimeric G proteins • Small GTPases • Large GTP-binding proteins (e.g. dynamin, guanylate binding proteins, SRP- receptor) GTP-binding proteins Heterotrimeric G proteins Subfamily Members Prototypical effect Gs Gs, Golf cAMP Gi/o Gi, Go, Gz cAMP , K+-current Gq Gq, G11, G14, Inositol trisphosphate, G15/16 diacylglycerol G12/13 G12, G13 Cytoskeleton Transducin Gt, Gustducin cGMP - phosphodiesterase Offermanns 2001, Oncogene GTP-binding proteins Small GTPases Family Members Prototypical effect Ras Ras, Rap, Ral Cell proliferation; Cell adhesion Rho Rho, Rac, CDC42 Cell shape change & motility Arf/Sar Arf, Sar, Arl Vesicles: fission and fusion Rab Rab (1-33) Membrane trafficking between organelles Ran Ran Nuclear membrane plasticity Nuclear import/export The RAB activation-inactivation cycle REP Rab escort protein GGT geranylgeranyl-transferase GDI GDP-dissociation inhibitor GDF GDI displacementfactor Lipid e.g. RAS e.g. ARF1 modification RAB, Gi/o of GTP-binding proteins Lipid modification Enzyme Reaction Myristoylation N-myristoyl-transferase myristoylates N-terminal glycin Farnesylation Farnesyl-transferase, Transfers prenyl from prenyl-PPi Geranylgeranylation geranylgeranyltransferase to C-terminal CAAX motif Palmitoylation DHHC protein Cysteine-S-acylation General scheme of coated vesicle formation T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Published by AAAS General model for scission of coated buds T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Published by AAAS Conformational change in Arf1 and Sar1 GTPases, regulators of coated vesicular transport This happens if protein is trapped in the active conformation Membrane tubules formed by GTP- bound Arf1 Membrane tubules formed by GTP-bound Sar1 Published by AAAS T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Conformational switch in ARF1 T. J. Pucadyil et al., Science 325, 1217-1220 (2009) Schematic diagram of RAS G-box regions (G-1-5) form the guanine nucleotide binding pocket From Sprang and Gilman, Annu. Rev. Pharmacol. 1997 Mg2+ Mechanism of GDP release Structure shows a nucleotide-depleted Rho (CDC42) bound to its GEF (DBS) and modelled at a membrane surface. For Cdc42 that is bound to Dbs, both the DH (Dbl homology) and PH (pleckstrin-homology) domains must directly engage Cdc42 for maximal nucleotide exchange. The subsequent loading of GTP•Mg2+ results in the activation of Rho proteins. In this way GEFs function as coincidence detectors that are designed to integrate information regarding local concentrations of Rho GTPases that are released from guanine nucleotide-dissociation inhibitors as well as the membrane composition. GEFs interact with switch 1 and 2 to break interactions with GDP and to disrupt contact of GDP with the required Mg2+ cofactor. From Rossman et al., Nature Reviews 2005 Mechanism of GTP-hydrolysis by GAP proteins: External amino acid side chains supplied by GAP to activate GTPase. Catalytic core of GTPase is shown in the transition state (transition from GTP to GDP bound). From Farfel et al., N Engl J Med. 1999 Structure of the G protein alpha-subunit RAS G surface facing the receptor From Sprang and Gilman, Annu. Rev. Pharmacol. 1997 Biochemical Properties of G Protein -subunits Structure RAS-like and helical domain Guanine nucleotide GDP/GTP-bound in cleft between the two domains binding inactive in the GDP-bound form 2+ active in the GTP.Mg -bound form or when liganded to GDP.Mg.AlF4. (mimics transition conformational state of GTP-hydrolysis) switch accommodated by changes in three non-contiguous regions (termed switch I-III) via co- (aminoterminal myristoylation, e.g. G i) and post-translational modification (palmitoylation on cystein Membrane anchoring close to the N-terminus) to C-terminus of G plus other:, loops between strands 2/3, N--helix/strand 1, strand 6/helix 4, Receptor binding & strands 4/ 2 Activation GDP/GTP-exchange reaction catalyzed by the appropriate receptor in a manner dependent on -dimers class specific for -subunits Effector regulation Effector binding to three discontiguous regions (switch II; the region adjacent to switch III; helix 4 and the loop connecting 4 and 6) by intrinsic GTPase and by reassociation with -dimers (which bind to the aminoterminus and to switch II of Deactivation G) GTPase accelerated and effector regulation blocked by RGS-proteins (=Regulators of G protein signalling) Regulation through binding to switch I-III Bacterial toxins catalyze NAD-dependent ADP-ribosylation 187/188,201/202 Cholera toxin of arg in splice variants of Gs, (Gt) persistent activation due to impaired GTP-hydrolysis Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt blocks receptor interaction Pasteurella toxin attacks Gq, (mechanism unknown) sensitizes to receptor agonist action Biochemical Properties of G Protein -subunits Structure RAS-like and helical domain Guanine nucleotide GDP/GTP-bound in cleft between the two domains binding inactive in the GDP-bound form 2+ active in the GTP.Mg -bound form or when liganded to GDP.Mg.AlF4. (mimics transition state of GTP- conformational hydrolysis) switch accommodated by changes in three non-contiguous regions (termed switch I-III) via co- (aminoterminal myristoylation, e.g. G i) and post-translational modification (palmitoylation on cystein Membrane anchoring close to the N-terminus) to C-terminus of G plus other:, loops between strands 2/3, N--helix/strand 1, strand 6/helix 4, Receptor binding & strands 4/ 2 Activation GDP/GTP-exchange reaction catalyzed by the appropriate receptor in a manner dependent on -dimers class specific for -subunits Effector regulation Effector binding to three discontiguous regions (switch II; the region adjacent to switch III; helix 4 and the loop connecting 4 and 6) by intrinsic GTPase and by reassociation with -dimers (which bind to the Deactivation aminoterminus and to switch II of G) GTPase accelerated and effector regulation blocked by RGS-proteins (=Regulators of G protein signalling) Regulation through binding to switch I-III Bacterial toxins catalyze NAD-dependent ADP-ribosylation 187/188,201/202 Cholera toxin of arg in splice variants of Gs, (Gt) persistent activation due to impaired GTP-hydrolysis Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt blocks receptor interaction Pasteurella toxin attacks Gq, (mechanism unknown) sensitizes to receptor agonist action Uptake of GTPS by Gs: Kinetics determined by the release of GDP From: Graziano, Freissmuth and Gilman, JBC 1989 GTP hydrolysis by Gs Steady-state GTPase Determination of kcat From: Graziano, Freissmuth and Gilman, JBC 1989 The activation/deactivation cycle of heterotrimeric G proteins basal state receptor activation GTP GDP subunit dissociation deactivation and effector regulation Biochemical Properties of G Protein -subunits Structure RAS-like and helical domain Guanine nucleotide GDP/GTP-bound in cleft between the two domains binding inactive in the GDP-bound form 2+ active in the GTP.Mg -bound form or when liganded to GDP.Mg.AlF4. (mimics transition state of GTP-hydrolysis) conformational switch accommodated by changes in three non-contiguous regions (termed switch I-III) via co- (aminoterminal myristoylation, e.g. G i) and post-translational modification (palmitoylation on cystein Membrane anchoring close to the N-terminus) to C-terminus of G plus other loops between strands 2/3, N--helix/strand 1, Receptor binding & strand 6/helix 4, strands 4/ 2 Activation GDP/GTP-exchange reaction catalyzed by the appropriate receptor in a manner dependent on -dimers class specific for -subunits Effector regulation Effector binding to three discontiguous regions (switch II; the region adjacent to switch III; helix 4 and the loop connecting 4 and 6) Deactivation by intrinsic GTPase and by reassociation with -dimers (which bind to the aminoterminus and to switch II of G) GTPase accelerated and effector regulation blocked by RGS-proteins (=Regulators of G protein signalling) Regulation through binding to switch I-III Bacterial toxins catalyze NAD-dependent ADP-ribosylation 187/188,201/202 Cholera toxin of Arg in splice variants of Gs (G t) persistent activation due to impaired GTP-hydrolysis Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt blocks receptor interaction Pasteurella toxin attacks Gq, (mechanism unknown) sensitizes to receptor agonist action From Farfel et al., N Engl J Med. 1999 A single case of PHP1A plus precocious puberty Dissociation of GDP from wild-type Mutation of arginine 187/201, the site and A326S Gialpha1 of cholera toxin catalyzed ADP- ribosylation of Gs eliminates the GTPase Aktivität Freissmuth M et al. J. Biol. Chem. 1989 Posner, B. A. et al. J. Biol. Chem. 1998;273:21752-21758 Problem in receptor mediated G protein activation = protein geometry Aus: Kisselev OG, Meyer CK, Heck M, Ernst OP, Hofmann KP Proc Natl Acad Sci U S A (1999) 96:4898-4903 Agonist-bound 2AR-mediated activation of GDP release . Shown is the step-wise dissociation of GDP from Gαs (orange) by agonist-activated β2AR that involves the engagement of both the N and the C terminus of Gαs. The activation of Gs through an activated β2AR (green) results in GDP release and subsequent GTP binding. The activated receptor engages the C terminus of the α5-helix of Gαs which undergoes a rigid-body translation upward into the receptor core and reorganizes the β6-α5 loop, a region that participates in purine ring binding. In a simultaneous or sequential event, ICL2 of the β2AR engages the N terminus of the Gαs, leading to reorganization of its β1-strand/P-loop, the loss of coordination of the β-phosphate of GDP (blue), and subsequently GDP release. The position of the N-terminal helix is aided by the Gβγ-subunits (not shown). The concomitant disruption of the interaction between the P-loop and GαsAH, primarily through the highly conserved R201 in the GαsAH and E50 in the P-loop, opens GαsAH allowing GDP to freely dissociate. Formation of the nucleotide-free form allows GTP (grey) to bind, resulting in reformation of the ‘closed’ conformation, and activation of the G protein through functionally dissociating from Gβγ and uncoupling from β2AR.
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