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


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 (Gα, Gβ, and Gγ) have revealed the determinants involved in heterotrimer assembly and effector protein engagement as well as the mechanism of nucleotide hydrolysis by the Ga 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 tetrafluoride. Recent identification 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.



As their name suggests, G protein-coupled receptors link ligand binding to intracellular changes functionally via their engagement of three distinct proteins (Gα, Gβ, Gγ) known collectively as the G-protein heterotrimer (Fig. 1). The Ga subunit of the trimer is the bona fide 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 Gβ and Gγ) and guanosine 5'-triphosphate (GTP) when in the activated state. An activating ligand binds its respective GPCR and changes the conformation of the receptor’s transmembrane regions and intracytosolic loops to activate the receptor’s guanine nucleotide exchange factor (GEF) activity on the Gα-GDP/Gβγ complex. Receptor-catalyzed exchange of GTP for the bound GDP within Gα leads to release of bound Gβγ, and subsequent engagement of α-effector and βγ-effector proteins by GTP-bound Ga and the now-freed Gβγ heterodimer.

Four general classes of Ga subunits have been defined based on their functional couplings (in the GTP-bound state) to various α-effector proteins. Gs-subfamily Gα 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 Gαs/Gβγ heterotrimer activation) by forskolin, a plant-derived diterpene. Conversely, G1-subfamily Gα subunits generally are inhibitory to adenylyl cyclases. Gq-subfamily Gα subunits are potent activators of phospholipase-Cβ 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 Gβγ subunits released by activated Gαβγ heterotrimers are also known to stimulate the activity of phospholipase-Cβ 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 Reference 2). G12/13-subfamily Gα subunits activate the small G-protein RhoA through stimulation of RhoA-specific GEFs such as p115-RhoGEF, LARG, and PDZ-RhoGEF.

Gα 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) specific to Ga subunits: namely, the “regulator of G-protein signaling” (RGS) proteins (3). α-Effectors can also exhibit GAP activity [e.g., phospholipase-Cβ activity in accelerating GTP hydrolysis by Gαq (4)]. GTP hydrolysis controls the timing of signal duration, as the loss of the third phosphate reverts the Gα back to the GDP-bound state with characteristic high affinity for Gβγ and low affinity for a-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 Gα subunit of the G-protein heterotrimer.



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 α-effector and βγ-effector proteins. Pi, inorganic phosphate.



X-ray diffraction crystallography has been the main technique employed to establish the structures of GDP-bound G-proteins (both as isolated Gα subunits and Gαβγ heterotrimeric complexes; Table 1), as well as activated states of Gα induced either by the binding of the nonhydrolyzable GTP analog guanosine 5'-(γ-thio)triphosphate (GTPγS) or by the addition of aluminum tetrafluoride (AlF4-) to GDP-bound Ga subunits. These efforts have unveiled the precise secondary and tertiary structures of both Gα and Gβγ (Figs. 2 and 3) (5), how the heterotrimeric complex is formed by these two binding partners, the conformational changes that are induced within Ga by the exchange of GDP for GTP (Fig. 2b), and the mechanisms of both intrinsic (Fig. 4) and RGS protein-accelerated GTP hydrolysis (6-8).


Table 1. Important heterotrimeric G-protein subunit structures obtained by X-ray crystallography


Structure(s) of G-protein subunit(s)

Database identifier*

Inactive conformation of isolated Gα subunits (bound to GDP)

Inactive heterotrimeric G-protein complexes of Gα-GDP/Gβγ

Active conformation of Gα subunits (bound to GTPγS)

Transition-state conformations of Gα subunits (bound to GDP∙AlF4-)

Activated Gαs bound to adenylyl cyclase and activated Gα13 bound to p115-RhoGEF; basis for Gα/effector interactions

i1 bound to GDP∙AlF4- and RGS4; basis for RGS protein GTPase-accelerating activity

i1 bound to GDP and RGS14 GoLoco motif; basis for GoLoco-mediated GDI activity

i1 bound to GEF peptides derived from phage-display (KB-752) and the dopamine D2-receptor (D2N); roles of β3/α2 loop and β6 strand in Gα GDP release


1GP2; 1GOT







*Structural data is accessible via


The Ga subunit, in its inactive state, binds GDP within a nucleotide-binding pocket circumscribed by residues derived from both of its constituent domains: a Ras-like domain (resembling the structural fold of small G-proteins) and an all α-helical domain unique to the “large” Gα family, comprising a structurally distinct six-helix bundle (Fig. 2a). An extended N-terminal α-helix is modified by covalent attachment of the fatty acids myristate and/or palmitate, which facilitates membrane targeting as well as assembly with Gβγ subunits (9). Exchange of GDP for GTP is catalyzed in a poorly understood process by an activated GPCR acting on the Gα∙GDP/Gβγ heterotrimer (10) and causes a structural rearrangement within three “switch” regions (I-III) of Ga (Fig. 2b) that results from nucleotide-pocket residues interacting with the γ-phosphate of the newly-bound GTP (8, 11). The particular conformations of these three switch regions are critical to the protein-protein interactions that Gα makes with its nucleotide-selective binding partners such as Gβγ, effectors, RGS proteins, and GoLoco motifs (6-8, 12). Structures of complexes between activated Gα subunits and several different a-effectors (e.g., Gas/adenylyl cyclase, Ga13/p115-RhoGEF) have highlighted a universal site for effector engagement within GTP-bound Gα: a conserved hydrophobic cleft (Fig. 2a) formed by the α2 (switch II) and α3 helices (reviewed in Reference 13).



Figure 2. Overall structural fold of the heterotrimeric G-protein Ga subunit in its inactive, GDP-bound form (a) and details of structural differences between GDP- and GTP-bound states (b). (a) The Ga subunit is composed of a Ras-like domain (blue) and an all α-helical domain (green), between which is found the guanine nucleotide binding pocket (GDP in purple). The three conformationally flexible switch regions (SI, SII, and SIII) are highlighted in cyan. The "hotspot" region of the Ras-like domain, circumscribed by the switch II (α2) and α3 helices and defined on the basis of interactions with the Gα-binding peptides KB-752, KB-1753, and the GoLoco motif-derivative R6A-1, is highlighted with a transparent yellow oval. Ga 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.



Gβ and Gγ subunits form tightly associated heterodimers (Fig. 3). GP begins with an extended N-terminal α-helix and is composed mainly of a β-propeller fold formed by seven individual segments of a ~40-amino acid sequence known as the WD-40 repeat. Gγ is an extended stretch of two a-helices joined by an intervening loop. Assuming no significant tertiary structure on its own, the N-terminus of Gγ participates in a coiled-coil interaction with the N-terminal α-helix of GP (Fig. 3); much of the remainder of Gγ binds along the outer edge of the Gβ toroid (14, 15). Gγ is prenylated posttranslationally on a cysteine residue that is four amino acids from the C-terminus. Most Gγ subunits receive a 20-carbon geranylgeranyl group at this position (Fig. 3), whereas Gγ1, Gγ8, and Gγ11 alternatively receive a 15-carbon farnesyl group (9). This lipid modification aids in the resultant membrane localization of the Gβγ heterodimer that is important to receptor coupling.

Gβγ and GDP-bound Gα form the G-protein heterotrimer via two principal sites of interaction: 1) extensive burial of the β3/α2 loop and α2 helix (switch II) of Gα within six of the seven WD repeats of GP and 2) contact between the side of the first β-propeller blade of GP and the extended N-terminal helix of Ga (6, 8). These extensive interactions form the basis for competition for Gβγ binding between Gα-GDP and βγ-effectors. Structures of Gβγ bound to the βγ-effector GRK2, the regulatory protein phosducin, and SIGK (a peptide capable of disrupting effector activation) have shown that the effector-binding site on Gβγ overlaps significantly with the region responsible for binding switch II of Gα near the central pore of the Gβ toroid (5, 16, 17).



Figure 3. Overall structural fold of the Gβγ heterodimer. The Gβ subunit is colored to highlight the seven WD40 repeats that comprise the β-propeller (or "torus") fold: WD1, green; WD2, purple; WD3, cyan; WD4, orange; WD5, gray; WD6, wheat; WD7; blue. Residues within Gβ that contact the Gβ1γ2-binding peptide SIGK, which constitutes the Gpy "hotspot" as defined by Bonacci et al. (5), are highlighted in light green. The cysteine residue within Gγ (red) that is subjected to posttranslational geranylgeranylation is highlighted in sticks configuration. The relative positioning of the N-terminal α-helix of the Gα subunit (when in the Gα∙GDP/Gβγ heterotrimeric complex) is also highlighted. Coordinates are from PDB record 10MW.


Mechanism of Guanine Nucleotide Hydrolysis

The mechanism of GTP hydrolysis by Gα, as well as RGS protein-mediated acceleration of this hydrolysis, has been discerned from x-ray diffraction crystallographic structures of the Gα transition state-mimetic form (Gα 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 Ga bound to guanosine 5'-(βγ-imido)triphosphate (GppNHp) or GDP plus inorganic phosphate (19, 20). The GTP hydrolysis reaction is mediated by three conserved Ga amino acids (Fig. 4; residues numbered as found in Gaβ). Glutamine-204 in switch II coordinates the critical nucleophilic water molecule responsible for hydrolysis of the γ-phosphate, 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 Ga are both necessary and sufficient for GTP hydrolysis to GDP and inorganic phosphate; thus, the mechanism of action of RGS proteins, the GTPase-accelerating proteins (GAPs) specific for Ga 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 Ga stabilizes the transition state conformation, which lowers the activation energy required for the hydrolysis reaction (7, 22).




Figure 4. Residues within Gα 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 Gαi1; coordinates are from PDB record 1GFI). Magnesium ion is highlighted in yellow. The planar anion aluminum tetrafluoride, which mimics the y-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 Gα

As mentioned previously, the planar anion aluminum tetrafluoride is used experimentally to activate Gα subunits; by mimicking the γ-phosphate leaving group, AlF4- binding to Gα∙GDP causes a rearrangement of the switch regions away from their high-affinity Gβγ-binding state. This action of aluminum tetrafluoride was discovered first by the serendipitous activation of purified G-protein preparations via sodium fluoride (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 Gα subunits, and the mechanism of action that has been identified 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 Gas, 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 Ga subunits (25); this posttranslational ADP-ribosylation occurs only in the context of the intact G-protein heterotrimer (Gαi∙GDP/Gβγ) 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 Gαqactivation by phosphorylating serine-47, a residue located in the highly conserved diphosphate-binding loop of Gα (Fig. 4), which impairs GTP binding (26). Although it is not a toxin, YM-254890, a cyclic depsipeptide identified from Chromobacterium sp. QS3666 culture broth that blocks ADP-induced platelet aggregation, is also an inhibitor of signaling from Gαq/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 specifically Gαq and not Gα11. Although the differential responsiveness of these two highly related Ga subunits has been mapped to two histidine residues present only in the all-helical domain of Gαq (28), the particular mechanism of Gαq activation by PMT has not yet been determined.


G protein-binding peptides reveal mechanistic insights and ''hotspots'' for chemical intervention

Using various random-peptide screening strategies, several groups have recently identified 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 affinity for Gi-subclass Gα subunits in their activated form (either GTPγS- or GDP∙AlF4--bound); KB-1753 can block α-effector and RGS protein binding to these two activated Ga states. Its structural determination by X-ray crystallography (12) was instrumental in highlighting a universal site of effector/Gα-GTP engagement (as described above). A second peptide derived from the same screen, KB-752, has selective binding affinity for Gi-subclass Gα subunits in their inactive (GDP-bound) form. On binding, it enhances the rate of spontaneous GDP release; structural determination of a KB-752/Gα∙GDP complex by X-ray crystallography (29) provided strong support for a prevailing model of receptor-catalyzed nucleotide exchange in which the β3/α2 loop of Ga, normally an occlusive barrier to GDP release, is remodeled by receptor-mediated tilting of the Gβγ dimer (10).

The 36-amino acid GoLoco motif, a naturally occurring, Gα∙GDP-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 Gβγ-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 identified a family of “R6A” peptides (including a minimal 9-amino-acid sequence called R6A-1) that interact with GDP-bound Ga subunits in a manner competitive to Gβγ binding (30). Whether the R6A peptides retain the GDI activity of their parental GoLoco motif sequence remains controversial (32). However, the finding that all three peptides (KB-1753, KB-752, R6A-1) interact with their Ga binding partners via engagement of switch II (13, 29, 32) identifies this particular region of Ga as a “hotspot” potentially amenable to targeting by small molecules for future chemical modulation of Ga function. That peptides can define binding hotspots on G-protein targets has already been exploited successfully by Bonacci et al. (5) for the Gβγ heterodimer. Using bacteriophage display, this group identified four distinct groups of Gβγ-binding peptides that, despite divergent sequences, were found to bind the same site on Gβγ—a site identified previously in mutagenesis studies as critical to βγ-effector interactions. A crystal structure of Gβ1γ2 bound to one of these peptides (SIGK) was determined by this group, which revelaed a partial a-helical conformation of the SIGK peptide reminiscent of that adopted by switch II of Ga within the GaPy heterotrimer.


Small molecules that modulate the function of G-protein subunits and their regulators

Bonacci et al. (5) identified several Gβ1γ2-binding compounds using the Gβ1γ2/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 Gβγ hotspot, which includes inhibition of Gβ1γ2-mediated PLCP2 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 μ-opioid GPCR. In a screen for compounds that can inhibit cholera toxin-stimulated (but not forskolin-stimulated) cAMP accumulation in intact cells, Prevost et al. (33) identified 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 Gy 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 P-adrenergic receptor signaling in isolated cardiac myocytes by reducing Gγ isoprenylation and thus decreasing the amount of functional Gαs/Gβγ heterotrimers at the cell membrane (34).

Another promising venue for developing chemical modulators of G-protein function is the Gα/RGS protein interface. With a chemical genetics approach that employs the egg-laying behavior of the nematode Caenorhabditis elegans, Fitzgerald et al. (35) identified two Ga subunits (GOA-1/Gαo, EGL-30/Gαq) 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 Gaq-GTP/RGS protein pair in an unproductive complex, similar to the action of brefeldin A on the ARF1-GDP/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 flow-cytometry-based protein interaction assay to identify methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (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 action—namely, covalent modification of surface-exposed cysteine-free thiols (Fig. 5b), which include cysteine-132 present in the Ga-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 Ga/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 identification of peptide-binding “hotspots” on G-protein subunits, akin to recent Gβ1γ2/SIGK peptide and dopamine receptor loop/Gaαi1 crystal structures (5, 39), should facilitate the rational design or discovery of direct chemical modulators of heterotrimeric G-protein action.



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).


1. Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat. Rev. Drug Discov. 2006; 5:993-996.

2. Clapham DE, Neer EJ. G protein beta gamma subunits. Annu. Rev. Pharmacol. Toxicol. 1997; 37:167-203.

3. Neubig RR, Siderovski DP. Regulators of G-protein signalling as new central nervous system drug targets. Nat. Rev. Drug Discov. 2002; 1:187-197.

4. Berstein G, Blank JL, Jhon DY, Exton JH, Rhee SG, Ross EM. Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell 1992; 70:411-418.

5. Bonacci TM, Mathews JL, Yuan C, Lehmann DM, Malik S, Wu D, Font JL, Bidlack JM, Smrcka AV. Differential targeting of Gbetagamma-subunit signaling with small molecules. Science 2006; 312:443-446.

6. Bohm A, Gaudet R, Sigler PB. Structural aspects of heterotrimeric G-protein signaling. Curr. Opin. Biotechnol. 1997; 8:480-487.

7. Tesmer JJ, Berman DM, Gilman AG, Sprang SR. Structure of RGS4 bound to AlF4-activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 1997; 89:251-261.

8. Wall MA, Posner BA, Sprang SR. Structural basis of activity and subunit recognition in G protein heterotrimers. Structure 1998; 6:1169-1183.

9. Wedegaertner PB, Wilson PT, Bourne HR. Lipid modifications of trimeric G proteins. J. Biol. Chem. 1995; 270:503-506.

10. Johnston CA, Siderovski DP. Receptor-mediated activation of heterotrimeric G-proteins: current structural insights. Mol. Pharmacol. 2007; 72:219-230.

11. Lambright DG, Noel JP, Hamm HE, Sigler PB. Structural determinants for activation of the alpha-subunit of a heterotrimeric G protein. Nature 1994; 369:621-628.

12. Willard FS, Kimple RJ, Siderovski DP. Return of the GDI: the GoLoco motif in cell division. Annu. Rev. Biochem. 2004; 73:925-951.

13. Johnston CA, Lobanova ES, Shavkunov AS, Low J, Ramer JK, Blaesius R, Fredericks Z, Willard FS, Kuhlman B, Arshavsky VY, Siderovski DP. Minimal determinants for binding activated G alpha from the structure of a G alpha(i1)-peptide dimer. Biochemistry 2006; 45:11390-11400.

14. Sondek J, Bohm A, Lambright DG, Hamm HE, Sigler PB. Crystal structure of a G-protein beta gamma dimer at 2.1 A resolution. Nature 1996;379:369-374.

15. Wall MA, Coleman DE, Lee E, Iniguez-Lluhi JA, Posner BA, Gilman AG, Sprang SR. The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell 1995; 83:1047-1058.

16. Gaudet R, Bohm A, Sigler PB. Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin. Cell 1996; 87:577-588.

17. Lodowski DT, Pitcher JA, Capel WD, Lefkowitz RJ, Tesmer JJ. Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma. Science 2003; 300:1256-1262.

18. Coleman DE, Berghuis AM, Lee E, Linder ME, Gilman AG, Sprang SR. Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science 1994; 265:1405-1412.

19. Coleman DE, Sprang SR. Structure of Gialpha1. GppNHp, autoinhibition in a galpha protein-substrate complex. J. Biol. Chem. 1999; 274:16669-16672.

20. Raw AS, Coleman DE, Gilman AG, Sprang SR. Structural and biochemical characterization of the GTPgammaS-, GDP. Pi-, and GDP-bound forms of a GTPase-deficient Gly42 → Val mutant of Gialpha1. Biochemistry 1997; 36:15660-15669.

21. Ahmadian MR, Stege P, Scheffzek K, Wittinghofer A. Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat. Struct. Biol. 1997; 4:686- 689.

22. Berman DM, Kozasa T, Gilman AG. The GTPase-activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J. Biol. Chem. 1996; 271:27209-27212.

23. Sternweis PC, Gilman AG. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc. Natl. Acad. Sci. U.S.A. 1982; 79:4888-4891.

24. Cassel D, Selinger Z. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. U.S.A. 1977; 74:3307-3311.

25. Burns DL. Subunit structure and enzymic activity of pertussis toxin. Microbiol. Sci. 1988; 5:285-287.

26. Navarro L, Koller A, Nordfelth R, Wolf-Watz H, Taylor S, Dixon JE. Identification of a molecular target for the Yersinia protein kinase A. Mol. Cell 2007; 26:465-477.

27. Takasaki J, Saito T, Taniguchi M, Kawasaki T, Moritani Y, Hayashi K, Kobori M. A novel Galphaq/11-selective inhibitor. J. Biol. Chem. 2004; 279:47438-47445.

28. Orth JH, Lang S, Aktories K. Action of Pasteurella multocida toxin depends on the helical domain of Galphaq. J. Biol. Chem. 2004; 279:34150-34155.

29. Johnston CA, Willard FS, Jezyk MR, Fredericks Z, Bodor ET, Jones MB, Blaesius R, Watts VJ, Harden TK, Sondek J, Ramer JK, Siderovski DP. Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange. Structure 2005; 13:1069-1080.

30. Ja WW, Roberts RW. G-protein-directed ligand discovery with peptide combinatorial libraries. Trends Biochem. Sci. 2005; 30:318-324.

31. Webb CK, McCudden CR, Willard FS, Kimple RJ, Siderovski DP, Oxford GS. D2 dopamine receptor activation of potassium channels is selectively decoupled by Galpha-specific GoLoco motif peptides. J. Neurochem. 2005; 92:1408-1418.

32. Willard FS, Siderovski DP. The R6A-1 peptide binds to switch II of Galphai1 but is not a GDP-dissociation inhibitor. Biochem. Biophys. Res. Commun. 2006; 339:1107-1112.

33. Prevost GP, Lonchampt MO, Holbeck S, Attoub S, Zaharevitz D, Alley M, Wright J, Brezak MC, Coulomb H, Savola A, et al. Anticancer activity of BIM-46174, a new inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex. Cancer Res. 2006; 66:9227-9234.

34. Muhlhauser U, Zolk O, Rau T, Munzel F, Wieland T, Es- chenhagen T. Atorvastatin desensitizes beta-adrenergic signaling in cardiac myocytes via reduced isoprenylation of G-protein gamma-subunits. FASEB. J. 2006; 20:785-787.

35. Fitzgerald K, Tertyshnikova S, Moore L, Bjerke L, Burley B, Cao J, Carroll P, Choy R, Doberstein S, Dubaquie Y, et al. Chemical genetics reveals an RGS/G-protein role in the action of a compound. PLoS. Genet. 2006; 2:e57.

36. Mossessova E, Corpina RA, Goldberg J. Crystal structure of ARF1*Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism. Mol. Cell 2003; 12:1403-1411.

37. Lan KL, Sarvazyan NA, Taussig R, Mackenzie RG, DiBello PR, Dohlman HG, Neubig RR. A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins. J. Biol. Chem. 1998; 273:12794-12797.

38. Kimple AJ, Willard FS, Giguere PM, Johnston CA, Mocanu V, Siderovski DP. The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face. Biochim. Biophys. Acta 2007; 1774:1213-1220.

39. Johnston CA, Siderovski DP. Structural basis for nucleotide exchange on G alpha i subunits and receptor coupling specificity. Proc. Natl. Acad. Sci. U.S.A. 2007; 104:2001-2006.


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: 551-577.

Siderovski DP, Willard FS. The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits. Int. J. Biol. Sci. 2005; 1:51-66.

Sprang SR. G protein mechanisms: insights from structural analysis. Ann. Rev. Biochem. 1997; 66:639-678.