Large G-Proteins

David P. Siderovski, Adam J. Kimple and Francis S. Willard, University of North Carolina at Chapel Hill, North Carolina
doi: 10.1002/9780470048672.wecb219

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Background Structures Mechanism of Guanine Nucleotide Hydrolysis Toxin, Peptide, and Chemical Modulators

Large G-proteins, also known as heterotrimeric G-proteins, cycle through inactive (GDP-bound) and active (GTP-bound) states, thereby coupling the activation of cell-surface receptors to the modulation of various second messengers and intracellular effector proteins. High-resolution structures of the constituent subunits of the heterotrimer (G, G, and G) have revealed the determinants involved in heterotrimer assembly and effector protein engagement as well as the mechanism of nucleotide hydrolysis by the G subunit and its acceleration by regulator of G-protein signaling (RGS) proteins. Mechanistic details have also been gleaned from studies of G-protein modulation by pathogen toxins and chemicals such as aluminum tetrauoride. Recent identication of peptide-binding hotspots on G-protein subunits should facilitate discovery and design of new chemical entities that can modulate nucleotide cycling and receptor/effector coupling at the level of the G-protein heterotrimer.

A wide variety of extracellular signaling molecules, such as hormones, neurotransmitters, growth factors, tastants, and odorants, communicate their information via binding and activating cell membrane-bound receptors and thus eliciting changes in various intracellular processes. The largest class of such receptors, the superfamily of seven-transmembrane, G protein-coupled receptors (GPCRs), has been an attractive target historically for discovery of small molecule therapeutics. GPCRs remain the largest single fraction of the druggable proteome, with GPCR-targeted drugs continuing to have annual sales in the tens of billions of dollars worldwide (1). Thus considerable effort has been made over the past few decades to establish a complete mechanistic understanding of how GPCRs communicate extracellular signals into the cell, in the hopes of elucidating the mechanism of action of existing therapeutics as well as facilitating new modalities of GPCR-directed drug discovery/design (beyond the receptor/ligand binding interface per se ). The precise structural determinants of GPCR-mediated G-protein activation on binding activating ligand remain enigmatic; however, the mechanisms by which heterotrimeric G-proteins couple receptor activation to modulation of intracellular processes are now known in great detail.

Background
As their name suggests, G protein-coupled receptors link ligand binding to intracellular changes functionally via their engagement of three distinct proteins (G, G, G) known collectively as the G-protein heterotrimer (Fig. 1). The G subunit of the

trimer is the bona de G-protein that binds guanine nucleotides in one of two states: guanosine 5 -diphosphate (GDP) when in the inactive, heterotrimeric state (i.e., complexed with G and G) and guanosine 5 -triphosphate (GTP) when in the activated state. An activating ligand binds its respective GPCR and changes the conformation of the receptors transmembrane regions and intracytosolic loops to activate the receptors guanine nucleotide exchange factor (GEF) activity on the GGDP/G complex. Receptor-catalyzed exchange of GTP for the bound GDP within G leads to release of bound G, and subsequent engagement of -effector and -effector proteins by GTP-bound G and the now-freed G heterodimer. Four general classes of G subunits have been dened based on their functional couplings (in the GTP-bound state) to various -effector proteins. Gs -subfamily G subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3 -5 -cyclic adenosine monophosphate (cAMP); cellular adenylyl cyclase activity can be stimulated directly (i.e., in the absence of Gs /G heterotrimer activation) by forskolin, a plant-derived diterpene. Conversely, Gi -subfamily G subunits generally are inhibitory to adenylyl cyclases. Gq -subfamily G subunits are potent activators of phospholipase-C enzymes that catalyze breakdown of the cell membrane lipid constituent phosphatidylinositol-4,5-bisphosphate into the second messengers diacylglycerol and inositol triphosphate, which leads to transient increases in intracellular calcium content. The G subunits released by activated G heterotrimers are also known to stimulate the activity of phospholipase-C enzymes, as well as to modulate adenylyl cyclase activity, to activate phosphatidylinositol-3 kinase and inward-rectifying potassium channels, and to inhibit calcium channel current (reviewed in 1

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Large G-Proteins

activating ligand

G protein-coupled receptor

O N O OH P O

a GDP
NH

g b

O O P O

N O

NH2

O HO P O

GDP
OH OH O N NH N

Pi

GEF GTP
NH2

RGS GAP

O OH P O

O O P O

O O P O

N O

OH OH

a GTP

g b

a-effectors

bg-effectors

Figure 1 Cycle of guanine nucleotide exchange and GTP hydrolysis by heterotrimeric G-proteins that serves to couple receptor activation to the modulation of various -effector and effector proteins. Pi , inorganic phosphate.

Reference 2). G12/13 -subfamily G subunits activate the small G-protein RhoA through stimulation of RhoA-specic GEFs such as p115-RhoGEF, LARG, and PDZ-RhoGEF. G subunits from all four subfamilies have the intrinsic ability to hydrolyze bound GTP to GDP and inorganic phosphate (Fig. 1). This intrinsic GTPase activity can be accelerated dramatically by the GTPase-accelerating proteins (GAPs) specic to G subunits: namely, the regulator of G-protein signaling (RGS) proteins (3). Effectors can also exhibit GAP activity [e.g., phospholipase-C activity in accelerating GTP hydrolysis by Gq (4)]. GTP hydrolysis controls the timing of signal duration, as the loss of the third phosphate reverts the G back to the GDP-bound state with characteristic high afnity for G and low afnity for effectors. Hence, the root mechanism for coupling receptor activation to the modulation of intracellular processes is a cycle of GTP binding and GTP hydrolysis transacted by the G subunit of the G-protein heterotrimer. 2

Structures
X-ray diffraction crystallography has been the main technique employed to establish the structures of GDP-bound G-proteins (both as isolated G subunits and G heterotrimeric complexes; Table 1), as well as activated states of G induced either by the binding of the nonhydrolyzable GTP analog guanosine 5 -(-thio)triphosphate (GTPS) or by the addition of aluminum tetrauoride (AlF4 ) to GDP-bound G subunits. These efforts have unveiled the precise secondary and tertiary structures of both G and G (Figs. 2 and 3) (5), how the heterotrimeric complex is formed by these two binding partners, the conformational changes that are induced within G by the exchange of GDP for GTP (Fig. 2b), and the mechanisms of both intrinsic (Fig. 4) and RGS protein-accelerated GTP hydrolysis (68).

G
The G subunit, in its inactive state, binds GDP within a nucleotide-binding pocket circumscribed by residues derived

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Large G-Proteins

Table 1 Important heterotrimeric G-protein subunit structures obtained by X-ray crystallography Database identier 1GDD; 1TAG 1GP2; 1GOT 1GIA; 1TND 1GFI; 1TAD

Structure(s) of G-protein subunit(s) Inactive conformation of isolated G subunits (bound to GDP) Inactive heterotrimeric G-protein complexes of GGDP/G Active conformation of G subunits (bound to GTPS) Transition-state conformations of G subunits (bound to GDPAlF4 ) Activated Gs bound to adenylyl cyclase and activated G13 bound to p115-RhoGEF; basis for G/effector interactions Gi1 bound to GDPAlF4 and RGS4; basis for RGS protein GTPase-accelerating activity Gi1 bound to GDP and RGS14 GoLoco motif; basis for GoLoco-mediated GDI activity Gi1 bound to GEF peptides derived from phage-display (KB-752) and the dopamine D2-receptor (D2N); roles of 3/2 loop and 6 strand in G GDP release
*

1AZS; 1SHZ

1AGR

1KJY

2HLB

Structural data is accessible via http://www.wwpdb.org/ .

from both of its constituent domains: a Ras-like domain (resembling the structural fold of small G-proteins) and an all -helical domain unique to the large G family, comprising a structurally distinct six-helix bundle (Fig. 2a). An extended N-terminal -helix is modied by covalent attachment of the fatty acids myristate and/or palmitate, which facilitates membrane targeting as well as assembly with G subunits (9). Exchange of GDP for GTP is catalyzed in a poorly understood process by an activated GPCR acting on the GGDP/G heterotrimer (10) and causes a structural rearrangement within three switch regions (IIII) of G (Fig. 2b) that results from nucleotide-pocket residues interacting with the -phosphate of the newly-bound GTP (8, 11). The particular conformations of these three switch regions are critical to the proteinprotein interactions that G makes with its nucleotide-selective binding partners such as G, effectors, RGS proteins, and GoLoco motifs (68, 12). Structures of complexes between activated G subunits and several different -effectors (e.g., Gs /adenylyl cyclase, G13 /p115-RhoGEF) have highlighted a universal site for effector engagement within GTP-bound G: a conserved hydrophobic cleft (Fig. 2a) formed by the 2 (switch II) and 3 helices (reviewed in Reference 13).

G
G and G subunits form tightly associated heterodimers (Fig. 3). G begins with an extended N-terminal -helix and is composed mainly of a -propeller fold formed by seven individual segments of a 40-amino acid sequence known as

the WD-40 repeat. G is an extended stretch of two -helices joined by an intervening loop. Assuming no signicant tertiary structure on its own, the N-terminus of G participates in a coiledcoil interaction with the N-terminal -helix of G (Fig. 3); much of the remainder of G binds along the outer edge of the G toroid (14, 15). G is prenylated posttranslationally on a cysteine residue that is four amino acids from the C-terminus. Most G subunits receive a 20-carbon geranylgeranyl group at this position (Fig. 3), whereas G1, G8, and G11 alternatively receive a 15-carbon farnesyl group (9). This lipid modication aids in the resultant membrane localization of the G heterodimer that is important to receptor coupling. G and GDP-bound G form the G-protein heterotrimer via two principal sites of interaction: 1) extensive burial of the 3/2 loop and 2 helix (switch II) of G within six of the seven WD repeats of G and 2) contact between the side of the rst -propeller blade of G and the extended N-terminal helix of G (6, 8). These extensive interactions form the basis for competition for G binding between GGDP and -effectors. Structures of G bound to the -effector GRK2, the regulatory protein phosducin, and SIGK (a peptide capable of disrupting effector activation) have shown that the effector-binding site on G overlaps signicantly with the region responsible for binding switch II of G near the central pore of the G toroid (5, 16, 17). 3

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Large G-Proteins

Ras domain

a3
l ca eli in h - ma do
GDP

SIII

b3

SII

SI N

(a)

G a GDP G GTP

Gly-203

Thr-181

(b)
Figure 2 Overall structural fold of the heterotrimeric G-protein G subunit in its inactive, GDP-bound form (a) and details of structural differences between GDP- and GTP-bound states (b). (a) The G subunit is composed of a Ras-like domain (blue) and an all -helical domain (green), between which is found the guanine nucleotide binding pocket (GDP in purple). The three conformationally exible switch regions (SI, SII, and SIII) are highlighted in cyan. The hotspot region of the Ras-like domain, circumscribed by the switch II (2) and 3 helices and dened on the basis of interactions with the G-binding peptides KB-752, KB-1753, and the GoLoco motif-derivative R6A-1, is highlighted with a transparent yellow oval. G structural coordinates are derived from PDB record 1GP2 (see Table 1). (b) The additional (third) phosphate (orange and red ) of bound GTP establishes contacts with residues threonine-181 and glycine-203 of switches I and II, respectively, which leads to changes in all three switch regions (green) versus their conformation in the GDP-bound state (cyan). Magnesium ion is highlighted in yellow . Coordinates are from PDB records 1GP2 and 1GIA.

Mechanism of Guanine Nucleotide Hydrolysis

The mechanism of GTP hydrolysis by G, as well as RGS protein-mediated acceleration of this hydrolysis, has been discerned from x-ray diffraction crystallographic structures of the G transition state-mimetic form (G bound to GDP and AlF4 ), both in isolation and bound to the archetypal RGS protein RGS4 (7, 18), as well as hydrolysis reaction intermediates including G bound to guanosine 5 -(-imido)triphosphate (GppNHp) or GDP plus inorganic phosphate (19, 20). The GTP hydrolysis reaction is mediated by three conserved G amino acids (Fig. 4; residues numbered as found in Gi1 ). Glutamine-204 in switch II coordinates the critical nucleophilic water molecule responsible for hydrolysis of the -phosphate, 4

whereas arginine-178 and threonine-181 (both from switch I) help to stabilize the leaving group (as mimicked by the planar anion AlF4 ), with the latter coordinating a bound Mg2+ ion (18). These three residues within G are both necessary and sufcient for GTP hydrolysis to GDP and inorganic phosphate; thus, the mechanism of action of RGS proteins, the GTPase-accelerating proteins (GAPs) specic for G subunits, necessarily differs from those of the GAPs for small G-proteins that introduce an additional catalytic residue in trans (e.g., Reference 21). Instead, RGS protein binding to G stabilizes the transition state conformation, which lowers the activation energy required for the hydrolysis reaction (7, 22).

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Large G-Proteins

Geranylgeranyl group N-end a-helix of Ga

N Gg N

Figure 3 Overall structural fold of the G heterodimer. The G subunit is colored to highlight the seven WD40 repeats that comprise the -propeller (or torus) fold: WD1, green; WD2, purple; WD3, cyan; WD4, orange; WD5, gray ; WD6, wheat ; WD7; blue. Residues within G that contact the G1 2 -binding peptide SIGK, which constitutes the G hotspot as dened by Bonacci et al. (5), are highlighted in light green. The cysteine residue within G (red ) that is subjected to posttranslational geranylgeranylation is highlighted in sticks conguration. The relative positioning of the N-terminal -helix of the G subunit (when in the GGDP/G heterotrimeric complex) is also highlighted. Coordinates are from PDB record 1OMW.

a3

AlF4
Q2 R1 78

04

H2O T181 S47

SII

Figure 4 Residues within G that are critical to the GTP hydrolysis mechanism include arginine-178 and threonine-181 from switch I and glutamine-204 from switch II (colored as in Fig. 2 and numbered as in Gi1 ; coordinates are from PDB record 1GFI). Magnesium ion is highlighted in yellow . The planar anion aluminum tetrauoride, which mimics the -phosphate leaving group in the hydrolysis reaction, is depicted in metallic red . Note the position of serine-47, the target of phosphorylation by the Y. pestis protein kinase YpkA.

Toxin, Peptide, and Chemical Modulators

Chemicals and pathogen toxins that modify the activation state of G
As mentioned previously, the planar anion aluminum tetrauoride is used experimentally to activate G subunits; by mimicking the -phosphate leaving group, AlF4 binding to GGDP causes a rearrangement of the switch regions away from their high-afnity G-binding state. This action of aluminum

tetrauoride was discovered rst by the serendipitous activation of puried G-protein preparations via sodium uoride (used as a protease inhibitor) combining with aluminum present as a contaminant of laboratory water and glassware (23). Other naturally occuring agents have also been discovered to either activate or inactivate G subunits, and the mechanism of action that has been identied for some agents serve to highlight key amino acids involved in guanine nucleotide cycling and/or other regulatory roles. Severe diarrhea and whooping cough, characteristic of infection by Vibrio cholerae and Bordetella pertussis , respectively, are caused by direct effects on G-protein activity catalyzed by pathogen-produced exotoxins. Cholera toxin catalyzes the ADP-ribosylation of arginine-178 within Gs , which cripples its GTP hydrolysis mechanism and leads to a constitutively active, GTP-bound state (24). In contrast, the S1 subunit of pertussis toxin catalyzes the ADP-ribosylation of a C-terminal cysteine found only within Gi-subclass G subunits (25); this posttranslational ADP-ribosylation occurs only in the context of the intact G-protein heterotrimer (Gi GDP/G) and leads to inhibition of receptor/heterotrimer signal transduction (i.e., the heterotrimer becomes uncoupled from receptor-mediated activation). The causative agent of the Black Plague, Yersinia pestis , harbors several essential virulence determinants that it injects into host cells. One protein, the serine/threonine kinase YpkA, inhibits Gq activation by phosphorylating serine-47, a residue located in the highly conserved diphosphate-binding loop of G (Fig. 4), which impairs GTP binding (26). Although it is not a toxin, YM-254890, a cyclic depsipeptide identied from Chromobacterium sp. QS3666 culture broth that blocks ADP-induced platelet aggregation, is also an inhibitor of signaling from Gq/11 -containing heterotrimers, albeit with an unresolved mechanism of action potentially related to effects on receptor-catalyzed nucleotide exchange (27). Conversely, Pasteurella multocida , a common cause of animal infections, produces a toxin (PMT) that activates specically Gq and not G11 . Although the differential responsiveness of these two 5

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Large G-Proteins

highly related G subunits has been mapped to two histidine residues present only in the all-helical domain of Gq (28), the particular mechanism of Gq activation by PMT has not yet been determined.

crystal structure of G1 2 bound to one of these peptides (SIGK) was determined by this group, which revelaed a partial -helical conformation of the SIGK peptide reminiscent of that adopted by switch II of G within the G heterotrimer.

G protein-binding peptides reveal mechanistic insights and hotspots for chemical intervention
Using various random-peptide screening strategies, several groups have recently identied linear polypeptide sequences that bind to heterotrimeric G-protein subunits in unique and informative ways (5, 13, 29, 30). One example is KB-1753, a 19-amino acid peptide derived from bacteriophage screening (29) with selective binding afnity for Gi-subclass G subunits in their activated form (either GTPS- or GDPAlF4 -bound); KB-1753 can block -effector and RGS protein binding to these two activated G states. Its structural determination by X-ray crystallography (12) was instrumental in highlighting a universal site of effector/GGTP engagement (as described above). A second peptide derived from the same screen, KB-752, has selective binding afnity for Gi-subclass G subunits in their inactive (GDP-bound) form. On binding, it enhances the rate of spontaneous GDP release; structural determination of a KB-752/GGDP complex by X-ray crystallography (29) provided strong support for a prevailing model of receptor-catalyzed nucleotide exchange in which the 3/2 loop of G, normally an occlusive barrier to GDP release, is remodeled by receptor-mediated tilting of the G dimer (10). The 36-amino acid GoLoco motif, a naturally occurring, GGDP-binding peptide sequence found in several G-protein regulators such as RGS12 and RGS14, exhibits the opposite biochemical activity to that of KB-752: namely, guanine nucleotide dissociation inhibitor (GDI) activity (i.e., reducing the rate of spontaneous GDP release) (12). Whole-cell electrophysiologic studies of GPCR coupling to ion channel modulation have established that GoLoco motif-derived peptides are useful tools to uncouple heterotrimeric G-protein signaling, but they have no intrinsic ability to activate directly G-dependent signaling per se (31). By the use of mRNA display, Ja and Roberts (30) screened a set of semi-randomly permutated peptide sequences based on the GoLoco motif and identied a family of R6A peptides (including a minimal 9-amino-acid sequence called R6A-1) that interact with GDP-bound G subunits in a manner competitive to G binding (30). Whether the R6A peptides retain the GDI activity of their parental GoLoco motif sequence remains controversial (32). However, the nding that all three peptides (KB-1753, KB-752, R6A-1) interact with their G binding partners via engagement of switch II (13, 29, 32) identies this particular region of G as a hotspot potentially amenable to targeting by small molecules for future chemical modulation of G function. That peptides can dene binding hotspots on G-protein targets has already been exploited successfully by Bonacci et al. (5) for the G heterodimer. Using bacteriophage display, this group identied four distinct groups of G-binding peptides that, despite divergent sequences, were found to bind the same site on Ga site identied previously in mutagenesis studies as critical to -effector interactions. A 6

Small molecules that modulate the function of G-protein subunits and their regulators
Bonacci et al. (5) identied several G1 2 -binding compounds using the G1 2 /SIGK interface obtained by X-ray crystallography as the basis for computational docking screens of virtual compound libraries (5). One lead compound from this virtual screening, (1S,2S)-2-(3,4,5-trihydroxy-6-oxoxanthen-9-yl) cyclohexane-1-carboxylic acid (or M119; Fig. 5a), possesses several in vitro and in vivo activities consistent with steric blockade of the G hotspot, which includes inhibition of G1 2 -mediated PLC2 activation and chemoattractant-induced calcium signaling in immune cells, as well as sensitization of mice to the antinociceptive effects of morphine, an activator of the -opioid GPCR. In a screen for compounds that can inhibit cholera toxin-stimulated (but not forskolin-stimulated) cAMP accumulation in intact cells, Pr evost et al. (33) identied an imidazo-pyrazine derivative, 2-amino-1-(2-cyclohexyl-8(cyclohexylmethyl)-6,8-dihydro-5H-imidazo(2,1-c)pyrazin-7-yl) -3-sulfanylpropan-1-one (or BIM-46174) as a pan-inhibitor of heterotrimeric G-protein signaling that emanates from Gs-, Gi-, and Gq-coupled GPCRs as well as Frizzled receptors (33). As this screen was conducted using a library of cysteine-related compounds designed originally as farnesyltransferase inhibitors, one likely mechanism of action of BIM-46174 is alteration of the G isoprenylation that is critical to proper heterotrimer membrane targeting and receptor coupling. Statin drugs are used clinically to reduce serum cholesterol; this class of compounds also exerts pleotropic, cholesterol-independent effects, which include the reduced production of isoprenoids because statins inhibit the rate-limiting enzyme (HMG-CoA reductase) in the mevalonate pathway. At least one statin, atorvastatin, has been found to affect -adrenergic receptor signaling in isolated cardiac myocytes by reducing G isoprenylation and thus decreasing the amount of functional Gs /G heterotrimers at the cell membrane (34). Another promising venue for developing chemical modulators of G-protein function is the G/RGS protein interface. With a chemical genetics approach that employs the egg-laying behavior of the nematode Caenorhabditis elegans , Fitzgerald et al. (35) identied two G subunits (GOA-1/Go , EGL-30/Gq ) and an RGS protein (EAT-16) as the molecular targets for two related inhibitors of rat bladder muscle tone and spontaneous contractions (BMS-192364, BMS-195270; Fig. 5a). A model of trapping a Gq GTP/RGS protein pair in an unproductive complex, similar to the action of brefeldin A on the ARF1GDP/Sec7 complex (36), has been proposed for the mechanism of action of BMS-192364 and BMS-195270 (35); however, no in vitro biochemistry has yet been published to support this model directly. Neubig et al. (37) used a ow-cytometry-based protein interaction assay to identify methyl-N -[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulnimi-

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Large G-Proteins

M119 OH O HS HO OH O OH O

BIM-46174

O N NH2 N N

H3C H N O N BMS-192364 N OH CH3 BMS-195270 N N H N O OH

Cl (a) Cl H2N CH CH2 SH O S N S O Cysteine O C OH H2N

Cl

O CH CH2 S C OH

O S Disulfidebridged adduct

N+ O O O

N+ O

CCG-4986 (b)
Figure 5 Structures of the G-protein subunit modulating chemicals M119, BIM-46174, BMS-192364, and BIM-195270 (a) and the proposed mechanism of action of the reactive RGS4 inhibitor CCG-4986 (b).

doate (CCG-4986) as a selective inhibitor of RGS4 (and not the related RGS protein RGS8). However, it has been shown subsequently that the selective nature of CCG-4986 lies in its mechanism of actionnamely, covalent modication of surface-exposed cysteine-free thiols (Fig. 5b), which include cysteine-132 present in the G-interacting surface of RGS4, but not RGS8 (38). Placing this cysteine within RGS8 by site-directed mutagenesis engenders sensitivity to CCG-4986 inhibition. Along with earlier evidence that subtle changes in the G/RGS protein interface can lead to loss of RGS protein-mediated GAP activity (37), the precedence established by the early RGS protein inhibitors BMS-192364, BMS-195270, and CCG-4986 supports the notion that these particular G-protein regulators will be valuable drug discovery

targets in the future (3). In addition, continued identication of peptide-binding hotspots on G-protein subunits, akin to recent G1 2 /SIGK peptide and dopamine receptor loop/Gi1 crystal structures (5, 39), should facilitate the rational design or discovery of direct chemical modulators of heterotrimeric G-protein action.

References
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WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

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Further Reading
McCudden CR, Hains MD, Kimple RJ, Siderovski DP, Willard FS. G-protein signaling: back to the future. Cell. Mol. Life Sci. 2005;62: 551577. Siderovski DP, Willard FS. The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits. Int. J. Biol. Sci. 2005;1:5166. Sprang SR. G protein mechanisms: insights from structural analysis. Ann. Rev. Biochem. 1997;66:639678.

WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.