GTP-Binding Proteins • Heterotrimeric G Proteins

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GTP-binding proteins • Heterotrimeric G proteins

• 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, Gi/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 Gq, (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 Gq, (mechanism unknown)  sensitizes to receptor agonist action Uptake of GTPS by Gs: Kinetics determined by the release of GDP

From: Graziano, Freissmuth and Gilman, JBC 1989 GTP hydrolysis by Gs

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 Gs (G t)  persistent activation due to impaired GTP-hydrolysis

Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt  blocks receptor interaction

Pasteurella toxin attacks Gq, (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 Gs 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. From Chung et al., Nature 2011 Fig. 4 a, Changes in HX at the interface between the GαsAH (ribbon diagram) and GαsRas (surface rendering) are mapped onto the ‘open’ conformation of Gαs observed in the β2AR–Gs complex (inset). In this ‘open’ conformation, GαsAH and GαsRas are coloured according to the indicated heat map. Also shown is a ribbon diagram of GαsAH in a ‘closed’ position (grey) similar to that observed in the crystal structure of the GDP- bound Gαi heterotrimer. The location of the GDP binding site is shown as spheres. b, Surface rendering of a rotated back 90° to show the cytoplasmic side of the ‘open’ conformation of nucleotide-free Gαs.

From Chung et al., Nature 2011 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

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, strands Receptor binding & 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)

Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt  blocks receptor interaction

Pasteurella toxin attacks Gq, (mechanism unknown)  sensitizes to receptor agonist action Schematic AC structure

The structure of the core catalytic domains of adenylate cyclase complexed with forskolin and Gsα–GTPγS. Ribbon diagram of the structure of a recombinant heterodimer of AC5 C1 (C1, blue) and AC2 C2 (C2, yellow) bound to Gsα– GTPγS and forskolin (red ball-and-stick)2. The view is into the ventral cleft. On the right the structure has been rotated toward the viewer 90° around a horizontal axis to illustrate the dorsal and ventral surfaces of the cyclase catalytic core1.. On the left the likely position of the cyclase catalytic site (ATP, green hexagon) in the ventral cleft opposite the site of forskolin binding (red star) is shown.The hypothesized position of Giα* relative to the cyclase complex is indicated. On the right the most probable orientation of the complex in relation to the plasma membrane is shownIn this model, substrate would have ready access to ventral cleft of the catalytic core because of its cytoplasmic orientation. The fatty acid groups that are known to modify the N-termini of Gsα and Giα and known to be involved in their membrane targeting are shown as squiggly lines. From Simonds, TiPS 1999 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

via co- (aminoterminal myristoylation, e.g. G i) and post-translational modification (palmitoylation on cystein Membrane anchoring  close to the N-terminus)

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 G) Deactivation RGS proteins exert GAP function on G proteins 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 Gs (G t)  persistent activation due to impaired GTP-hydrolysis

Pertussis toxin of a cysteine 4 amino acids from C-terminus in Go, Gi, Gt  blocks receptor interaction

Pasteurella toxin attacks Gq, (mechanism unknown)  sensitizes to receptor agonist action Deletion of rod-specific RGS9 prolongs the photoresponse

RGS…negative regulators of G- protein mediated signaling

•Recognize G-GTP /G- transition

•Bind to switch 2 and switch 1 region

•Accelerate hydrolysis

•G protein specificity by recognition of -helical domain From Chen et al., Nature 2000 RGS 17 Gi RGS 19 RGS 20 Gz Gi/o Gq

, 21

Gi/o Gt

Gi/o

Rho-GEF G 12/13 GRK Heterogeneity in RGS-domain interactions with the Gα all-helical domain switch 2

switch 1

Soundararajan M. et.al. PNAS 2008;105:6457-6462

Structure •tightly associated, rigid dimer – no coformational change upon activation  (35 kDa) •7 WD40 repeats (1 repeat = 4 antiparallel -strands) form a 7-bladed propeller (7-10 kDa) core •extended -helix of G forms coiled-coil with -subunit membrane •via carboxyterminal isoprenoid lipid modification on G (C20 = anchoring geranoyl-geranyl; C15= farnesyl in Gt) • plasma membrane association needs G (formation of heterotrimer)

Receptor •-dimers required for receptor mediated activation of G-subunits recognition •G1 identified in transducin (Gt) only

Effector Effector regulation site on G overlaps with G switch II binding site regulation Deactivation •by GDP-liganded -subunits Regulation •5 genes for G, 12 for G •G: Folding assistance by CCT1; G chaperone = phosducin like protein •G chaperone = DRiP78 •phosducin a 33 kDa protein, binds and scavenges free -dimers •G5 complexes with R7 family RGS proteins

Activation •Guanine nucleotide exchange on -subunit through receptor action triggers complex dissociation •AGS proteins (AGS-1 and-3 by accelerating nucleotide exchange on heterotrimer in a receptor-like

manner; AGS-8 by dissociating G and G ) Structure of the G protein beta-gamma dimer Structure of the heterotrimeric G-protein

From Sprang and Gilman, Annu. Rev. Pharmacol. 1997 Biochemical Properties of G Protein -Dimers

Structure •tightly associated, rigid dimer – no coformational change upon activation  (35 kDa) •7 WD40 repeats (1 repeat = 4 antiparallel -strands) form a 7-bladed propeller core (7-10 kDa) •extended -helix forms coiled-coil with -subunit membrane •via carboxyterminal isoprenoid lipid modification on G (C20 = geranoyl-geranyl; C15= farnesyl in Gt) anchoring • plasma membrae association needs G

Receptor •-dimers required for receptor mediated activation of G-subunits recognition •G1 identified in transducin (Gt) only

Activation •Guanine nucleotide exchange on -subunit through receptor action triggers complex dissociation •AGS-3 binds to G-GDP (switch 2) and displaces G •AGS-8 binds to G and dissociates the heterotrimer when GDP bound Physiological G effectors (direct)

• inwardly rectifying K+ channel (GIRK1/GIRK2,GIRK1/GIRK4) • GPCR kinase 2 and 3 •PLC 1, 2 and 3 • Adenylyl cyclase (activation), II, IV, VII • Adenylyl cyclase (inhibition), I, III, V, VI How to test for G effects • N type Ca2+ channels  • P/Q type Ca2+ channels • Gallein/M119, inhibitor of G activation of PLC, PI3K, of GRK2 • Phosphoinositide 3 kinase  and Rac-GEF (pREX-1) • SNAP-25 • Inhibition of subunit dissociation •P-Rex1 RacGEF pertussis toxin • Quenching: Overexpression of GRK2 (-ark) c-tail or of transducin (Gαt) • Reconstitute effect in cell free membranes using purified G

Binding of M201 (M119 derivative) to G “hot spot”. M201 (NSC201400) is depicted in yellow. G is in blue with some of the key amino acids in the hot spot From: Lin and Smrcka, 2011 A goal of the „Signal transduction“ programme, you should be able to offer an opinion on the following issues

• What are biochemical similarities and differences between G-protein α-subunits and small GTPases? • Which are the principles of activation and deactivation of GTPases? • Classification of small GTPases • Classification of heterotrimeric G proteins • Structure of heterotrimeric G proteins • Interaction of receptors with G proteins (physical coordinates and conceptual principles) • Experimental tools to investigate G protein mediated signaling • Typical effectors regulated by G-protein -subunits and -dimers • Structure of G -subunit and consequences of heterotrimer formation