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Originally published In Press as doi:10.1074/jbc.M700396200 on April 19, 2007

J. Biol. Chem., Vol. 282, Issue 27, 19938-19947, July 6, 2007
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Signaling by a Non-dissociated Complex of G Protein β{gamma} and {alpha} Subunits Stimulated by a Receptor-independent Activator of G Protein Signaling, AGS8*

Chujun Yuan{ddagger}, Motohiko Sato§1, Stephen M. Lanier§2, and Alan V. Smrcka§3

From the Departments of Pharmacology and Physiology and of {ddagger}Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and the §Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112

Received for publication, January 16, 2007 , and in revised form, April 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gβ{gamma} subunits. Results are presented demonstrating that AGS8 binds to the effector and {alpha} subunit binding "hot spot" on Gβ{gamma} yet does not interfere with G{alpha} subunit binding to Gβ{gamma} or phospholipase C β2 activation. AGS8 stimulates activation of phospholipase C β2 by heterotrimeric G{alpha}β{gamma} and forms a quaternary complex with G{alpha}i1, Gβ1{gamma}2, and phospholipase C β2. AGS8 rescued phospholipase C β binding and regulation by an inactive β subunit with a mutation in the hot spot (β1(W99A){gamma}2) that normally prevents binding and activation of phospholipase C β2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gβ{gamma} interactions with phospholipase C β2. Mutation of an alternate binding site for phospholipase C β2 in the amino-terminal coiled-coil region of Gβ{gamma} prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gβ{gamma} binding proteins, for directing Gβ{gamma} signaling through alternative effector activation sites on Gβ{gamma} in the absence of subunit dissociation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptor signaling systems play key roles in a number of biological processes (1). When bound to specific ligands, G protein-coupled receptors facilitate exchange of GDP for GTP on the G{alpha} subunit, leading to conformational changes in the G{alpha}"switch" regions (2). These changes decrease the affinity of G{alpha} for Gβ{gamma} and are thought to result in subunit dissociation such that G{alpha} and Gβ{gamma} subunits are free to interact with downstream effectors (3). In this model, structural elements in G{alpha} and Gβ{gamma} that bind downstream effectors are masked at the interface between the Gβ{gamma} and G{alpha} subunits and revealed upon subunit dissociation.

Alternatively it has been argued that subunit dissociation is not a required step in the G protein activation cycle, instead, a distinct GTP-bound conformation of the {alpha}β{gamma} complex may represent the activated form of the G protein (4). Considerable experimental evidence has accumulated in favor of this idea. Most recently, cell-based fluorescence resonance energy transfer and bioluminescence energy transfer analyses suggest that G protein heterotrimers may not dissociate upon activation but rather undergo specific conformational rearrangements (5-7). Other evidence includes: A covalently linked G{alpha}-β{gamma} fusion protein is signaling competent in yeast (8); the kinetic coupling model for G protein activation suggests that, in the presence of regulators of G protein signaling proteins, GTP hydrolysis is too rapid to allow subunit dissociation prior to effector activation (9) and the β2-adrenergic receptor can activate adenylyl cyclase without causing nucleotide exchange implying that subunit dissociation is not required for adenylyl cyclase activation by this receptor (10). A potential barrier to acceptance of the model is that it is not clear how conformational rearrangements of the subunits would allow for productive engagement of key signaling surfaces at the G{alpha}-β{gamma} interface with effectors. One proposal is the "clam shell" hypothesis where G{alpha}-β{gamma} contacts are maintained by contacts between the amino-terminal helix of the G{alpha} subunit (G{alpha}-NT)4 with the side of the Gβ subunit β-propeller, while other regions at the G{alpha}-β{gamma} interface are exposed for effector activation.

G protein-coupled receptor-independent mechanisms for G protein activation have been discovered in recent years. For example, activators of G protein signaling (AGS proteins), discovered in a yeast-based screen for receptor-independent activation of the pheromone pathway, are a group of proteins that stimulate G protein signaling (11, 12). The best characterized of the AGS proteins act through interactions with G protein {alpha} subunits to dissociate the G protein heterotrimer either through nucleotide exchange-dependent (Class I AGS proteins) or -independent mechanisms (Class II AGS proteins). Here we explore the mechanism for G protein activation by a recently described AGS protein, AGS8, which binds to G protein β{gamma} subunits (13). AGS8 forms a complex interacting with G{alpha} and Gβ{gamma} simultaneously, occupies the Gβ{gamma}"hot spot," a critical effector binding and signal transfer region on Gβ{gamma} (14-17), yet does not dissociate G{alpha} from Gβ{gamma} subunits. In complexes between G{alpha}, β{gamma}, and AGS8, a signal transfer region on Gβ{gamma} that does not involve the hot spot is critical for signaling to phospholipase C (PLC) β. This introduces a concept for G protein β{gamma} subunit signaling where amino acids outside the G{alpha}-β{gamma} subunit interface drive Gβ{gamma} signaling and provides a model for how non-dissociated G protein complexes can activate downstream targets and a potential modified alternative of the clam shell hypothesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Peptides (SIRK, SIRKALNILGYPDYD; SIGK, SIGKAFKILGYPDYD; and SIRK(L9A) SIRKALNIAGYPDYD) were synthesized by Alpha Diagnostics International, purified by high-performance liquid chromatography to >90% purity, and their identity was confirmed by mass spectrometry analysis. Phosphatidylinositol 4,5-bisphosphate and phosphatidylethanolamine were obtained from Avanti Polar Lipids, nickel-nitrilotriacetic acid-agarose was from Qiagen, GTP{gamma}S was from Sigma, glutathione-Sepharose and anti-GST antibody were from GE Healthcare, TetraLinkTM tetrameric avidin resin was from Promega (Madison, WI), anti-G{alpha}i antibody was from Oncogene.

Purification of β{gamma} and {alpha} Subunits—Gβ1{gamma}2 or biotinylated Gβ1{gamma}2 (bGβ1{gamma}2) subunits were purified from 2 liters of Sf-9 cells triply infected with His6-G{alpha}i1, wild-type, or bGβ1 subunits, and G{gamma}2 subunits essentially as described (18). All of the alanine-substituted Gβ1 subunit mutants used in this study were modified with a biotin acceptor peptide at the amino terminus and were biotinylated. These mutants were expressed with G{gamma}2 in 200 ml of SF9 cells and partially purified exactly as described in a previous study (16). Experiments comparing alanine-substituted bGβ{gamma} activities to wt bGβ{gamma} utilized wt bGβ{gamma} subunits partially purified in parallel with the mutants for direct comparison. Myristoylated G{alpha}i1 was purified from Escherichia coli as previously described (19) and bound GTP{gamma}S (0.5 mol/mol of protein) was determined by an Amido Black protein assay.

Expression and Purification of GST-tagged cAGS8—The coding sequence of cAGS8 (A1359-W1730 (cDNA#1-16)) was fused in-frame to GST in the pGEX-4T vector (GE Healthcare) and expressed and purified from E. coli BL21/DE3 cells as previously described (13) with some modification. Overnight cultures grown in Luria broth containing ampicillin (50 µg/ml) were diluted to an A600 equal to 0.5-0.6 and induced with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside for 3 h at 30 °C. Fusion proteins from a 1-liter culture were purified following the previously described protocol except prior to elution the glutathione-Sepharose column was washed with 5 mM ATP and a denatured E. coli protein extract to remove contaminating GroEL. The resulting eluted protein was 80% pure and dialyzed against 50 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, and protease inhibitors. Protein concentrations were determined by Amido Black protein assay, and purified proteins were snap frozen and stored at -80 °C.

Protein Binding Assays—GST-cAGS8 or GST was incubated with either purified G{alpha}i1, or bGβ1{gamma}2, or various bGβ1 mutants in 200 µl of binding buffer (20 mM HEPES, pH 8.0, 1 mM EDTA, 1.2 mM MgCl2, 0.1% C12E10 (polyoxyethylene-10-lauryl ether), 1mM dithiothreitol, 150 mM NaCl, 10 µM GDP). The binding mixture was rotated at 4 °C overnight. 40 µl of 50% glutathione-Sepharose was added to the binding mixture, and the mixture was further rotated for 1 h. The glutathione matrix was washed with three rounds of centrifugation/washing in binding buffer. Identical conditions were used for avidin-agarose isolations of bGβ1{gamma}2 except avidin-agarose was used. Proteins bound in precipitates were identified by immunoblotting following PAGE. In some cases immunoblots were quantitated by chemiluminescence imaging with a charge-coupled device camera. This has a much larger dynamic range than film and overcomes issues with signal saturation observed with x-ray film.

Gel-filtration Chromatography—Various combinations of GST-cAGS8, G{alpha}i1, Gβ1{gamma}2, and PLCβ2 (100 nM each) were incubated in 500 µl of gel-filtration buffer (20 mM HEPES, pH 8.0, 1 mM EDTA, 1.2 mM MgCl2, 0.1% C12E10, 1 mM dithiothreitol, 150 mM NaCl, 10 µM GDP, and protease inhibitor mixture) at 4 °C for 2 h. Then the mixture was applied to tandem Superdex 75/200 columns (GE Healthcare) pre-equilibrated with gel-filtration buffer and resolved at a flow rate of 0.4 ml/min at 4 °C. 1-ml fractions were collected. An aliquot of each fraction (20 µl) was analyzed by SDS-PAGE on a 12% polyacrylamide gel and visualized by silver staining. No binding of any of the proteins to GST was detected in the gel-filtration assay (data not shown).

GTP{gamma}S Binding Assay—This assay was performed as described previously (20) except without membranes or receptors. Briefly, 120 nM G{alpha}i1 (6 pmol), in the presence or absence of 180 nM GST-cAGS8 and with or without 240 nM Gβ1{gamma}2 subunits, were incubated with 0.4 µM GTP{gamma}S([35S]GTP{gamma}S, ~5000 cpm/pmol specific radioactivity in the assay) in the presence of 2 µM GDP to suppress the basal rate of nucleotide exchange by G{alpha}i1. Bound [35S]GTP{gamma}S detected by binding to nitrocellulose filters.

PLC Assay—PLC assays were performed as described previously (21) and in the figure legends.

Measurement of G{alpha}-β{gamma} Interactions by Flow Cytometry—Equilibrium binding of fluorescein isothiocyanate-labeled myristoylated G{alpha}i1 to bGβ1{gamma}2 subunits was measured using flow cytometry as has been previously described (22-24). Nonspecific binding, determined by the simultaneous addition of 300 pM fluorescein isothiocyanate-labeled myristoylated G{alpha}i1 and 50 nM myristoylated G{alpha}i1 subunits to the bGβ{gamma} bound beads, was 10-20% of the total signal and was subtracted from the mean channel numbers from each experiment unless otherwise indicated.


Figure 1
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  		FIGURE 1.
cAGS8 binds to a domain on the Gβ subunit that overlaps with G{alpha} binding. A, hot spot binding peptides block Gβ1{gamma}2-cAGS8 interactions. 300 nM GST-cAGS8 was incubated with 30 nM purified bGβ1{gamma}2 with or without 20 µM peptides in binding buffer. Proteins bound to GST-cAGS8 were detected with immunoblotting as described under "Experimental Procedures." Samples were also immunoblotted with anti-GST antibodies to validate loading equal amounts of protein (not shown). Data shown are representative of three independent experiments. This Western blot was quantified by chemiluminescence imaging: percent inhibition values relative to control were 20% (L9A), 89% (SIGK), and 89% (SIRK). B, GST pulldown assay showing the effect of various bGβ1 hot spot mutants on bGβ1{gamma}2-cAGS8 interaction. 300 nM GST-cAGS8 protein was incubated with 30 nM wt bGβ1{gamma}2 or with 30 nM various bGβ1 mutants (all partially purified and quantitated in parallel (16)) in binding buffer. The bottom panel is a Western blot of the suspension prior to pulldown assay, demonstrating that equal amounts of each Gβ{gamma} mutant were included in each pulldown assay. Data shown are representative of three independent experiments. C, Western blots from two separate experiments as in B were quantified by chemiluminescence imaging, and the data were pooled and plotted relative to wt Gβ1{gamma}2 binding. Data are mean ± S.E.

 
Trypsin Digestion of G{alpha}i1—3 µg of G{alpha}i1 was mixed with 0.15 µg of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (New England Biolabs Inc.) and incubated in 100 µl of digestion buffer (20 mM Tris, pH 8.0, 0.6 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 70 mM NaCl, 10 µM GDP, 30 µM AlCl3, and 10 mM NaF) at 25 °C for 1 h. The digestion reaction was terminated by adding 5 mM 1-chloro-3-tosylamido-7-amino-2-heptanone. The digestion was analyzed by PAGE and visualization with Coomassie Blue staining. The protein concentration after digestion was determined with an Amido Black protein assay. To confirm that trypsin-treated G{alpha}i1 still bound to cAGS8, binding to GST-cAGS8 was analyzed in a GST pulldown assay as has been described in parallel with undigested G{alpha}i1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AGS8 Binds to a Site on Gβ1{gamma}2 That Overlaps the G{alpha} Binding Site Yet Does Not Affect Binding of G{alpha} to Gβ{gamma}—The carboxyl-terminal domain of AGS8 (amino acids A1359-W1730, cAGS8) binds to β{gamma} and activates Gβ{gamma} signaling in yeast and in mammalian cells transfected with G protein subunits (13). To understand the molecular mechanism for how a protein can bind to Gβ{gamma} and simultaneously propagate Gβ{gamma} signaling, we investigated the molecular nature of the interaction between Gβ{gamma} and cAGS8. To initially identify and characterize a binding site for cAGS8 on Gβ{gamma} we used peptide competition and mutagenic analyses. The peptides SIGK and SIRK bind to a hot spot on Gβ1{gamma}2 that interacts with multiple effectors, including PLCβ2, and corresponds to the G{alpha} subunit switch II binding site (2, 14, 16, 25). SIGK and SIRK compete for binding of both {alpha} subunits and PLCβ2 to Gβ1{gamma}2 (15, 23). These peptides also blocked binding of cAGS8 to Gβ1{gamma}2 indicating the binding sites for the peptides, PLCβ2, G{alpha} subunits and cAGS8 overlap on the surface of Gβ1{gamma}2 (Fig. 1A). A control peptide that does not bind Gβ1{gamma}2, SIRK (L9A) (15), had little effect on cAGS8-Gβ1{gamma}2 interactions.

To identify specific amino acids within the Gβ{gamma} hot spot site required for cAGS8 interactions, GST-cAGS8 binding to a series of alanine-substituted bGβ1{gamma}2 subunit mutants within the hot spot was tested (Fig. 1, B and C; see Fig. 10). Compared with wild-type Gβ1{gamma}2,Gβ1W99A, Gβ1D186A, Gβ1M188A, and Gβ1H311A (Gβ1H311 is outside the hot spot and is a control) mutations did not affect binding to cAGS8, whereas Gβ1K57A, Gβ1Y59A, Gβ1M101A, Gβ1L117A, Gβ1Y145A, and Gβ1N230A mutations all significantly inhibited binding of Gβ1{gamma}2 to cAGS8. These data complement the peptide competition data and clearly demonstrate that cAGS8 interacts with the hot spot on Gβ1{gamma}2.

Many of these amino acids are required for activation of effectors, including PLCβ2, and are directly at the G{alpha}-β{gamma} binding interface (Fig. 10) (3, 26). Thus binding of cAGS8 to this region would be expected to preclude G{alpha} subunit binding and effector activation. We used flow cytometry to analyze G{alpha}-β{gamma} interactions as previously described (22-24). As previously shown, the hot spot-binding peptide, SIGK, competes with G{alpha}i1 for binding to Gβ1{gamma}2 in a concentration-dependent manner (Fig. 2A) consistent with its apparent Kd for Gβ{gamma} of ~1 µM (22); however, cAGS8 did not inhibit G{alpha} binding to Gβ{gamma}, despite apparently binding to the same site as SIGK and G{alpha} subunit switch II. The highest concentration of cAGS8 tested, 1 µM, was higher than concentrations shown to have maximal functional effects on Gβ{gamma} signaling (see Figs. 5B and 9C for examples) and higher than the concentration required for strong binding in the GST pulldown assay and formation of stable complexes by gel filtration.

cAGS8 Binds to G{alpha} Subunits and Gβ{gamma} Simultaneously—SIGK peptide binds to the switch II binding site and competes for {alpha} subunit binding to Gβ{gamma}. How could cAGS8 occupy the same binding site on Gβ{gamma} yet not compete for G{alpha} subunit binding to Gβ{gamma}? We hypothesized that, if cAGS8 bound both Gβ{gamma} and G{alpha} simultaneously, then the complex would not be disrupted. It had previously been shown that G{alpha}i did not bind strongly to cAGS8 (13), but perhaps a weak interaction was not detected that might be significant to formation of this complex. Indeed, G{alpha}i1 bound to GST-cAGS8 in the absence of Gβ{gamma} in a manner independent of the G{alpha}i1 activation state, because inclusion of Formula did not affect the binding (Fig. 2B lanes 3 and 4). As a control to demonstrate that G{alpha}i is activated by Formula in this assay, the G{alpha}i subunit was tested for Formula activation-dependent protection from trypsin digestion. In the absence of Formula treatment there is no detectable binding of G{alpha}i pretreated with trypsin, because the inactive conformation of G{alpha} is not resistant to trypsin digestion and is degraded prior to binding, while a stable proteolytic fragment of G{alpha}i is formed in the presence of trypsin and Formula that binds to GST-cAGS8 (27). This demonstrates that the G{alpha}i1 subunit is activated by Formula treatment. The overall conclusion is that the Formula-dependent activation does not significantly alter binding of G{alpha}i to AGS8. GST-cAGS8 also did not affect the rate of binding of GTP{gamma}S to G{alpha}i1 or G{alpha}i1β1{gamma}2 indicating that AGS8 is not a guanine nucleotide exchange factor (Fig. 2C). These data are consistent with the observation that the cAGS8-mediated activation of G-protein signaling in the yeast-based functional screen was independent of guanine nucleotide exchange (13).


Figure 2
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  		FIGURE 2.
AGS8 does not cause subunit dissociation and bound to G{alpha}i1. A, 300 pM fluorescein isothiocyanate-labeled myristoylated G{alpha}i1 was mixed with 50 pM bGβ1{gamma}2 in the presence of the indicated concentrations of SIGK ({blacktriangleup}) or cAGS8 ({blacksquare}), and the amount of fluorescein isothiocyanate-labeled myristoylated {alpha}i1 bound was assessed under equilibrium conditions by flow cytometry as described under "Experimental Procedures." B, AGS8 bound to G{alpha}i1 in an activation-state independent manner. 300 nM GST-cAGS8 was incubated with 30 nM purified G{alpha}i1 with or without Formula (30 µM AlCl3, 10 mM NaF, 10 mM MgCl2) in GST pulldown buffer, which includes 10 µM GDP. Proteins bound to GST-cAGS8 were pulled down with glutathione-Sepharose and detected with immunoblotting as described under "Experimental Procedures." Data shown are representative of three independent experiments. This Western blot was quantified by chemiluminescence imaging: binding as percentage of input was GST (0%), G{alpha}i1GDP (2.1%), G{alpha}i1 GDP plus Formula (2.4%), G{alpha}i1GDP plus trypsin (0.4%), G{alpha}i1GDP plus Formula plus trypsin (2.1%). C, AGS8 did not cause nucleotide exchange on G{alpha}i1. 120 nM G{alpha}i1 (6 pmol) was mixed with ({square} and bullet) or without ({blacksquare} and {blacktriangledown}) 240 nM bG1{gamma}2, in the presence ({blacktriangledown} and {square}) or absence ({blacksquare} and bullet) of 240 nM AGS8. [35S]GTP{gamma}S binding was assessed as described under "Experimental Procedures." Data are mean ± S.E. from single measurements at each time, pooled from two independent experiments and are representative of at least two other experiments at single 10-min time points.

 
To assess binding of cAGS8 to G protein heterotrimer, 30 nM G{alpha}i1GDP and 30 nM Gβ1{gamma}2 subunit were incubated together with varying concentrations of cAGS8 and compared with binding to either subunit alone. G{alpha}i1 and Gβ1{gamma}2 subunit bound simultaneously to substoichiometric concentrations of GST-cAGS8 (Fig. 3, lane 9, with 10 nM cAGS8 and 30 nM for each subunit), and binding as a function of GST-cAGS8 concentration was more efficient for both subunits together than for either of the subunits alone (Fig. 3A, lanes 6-9 compared with lanes 2-5, Fig. 3B, lanes 6-9 compared with lanes 2-5). The data in Fig. 3 (C and D) are compiled from quantitative analysis of chemiluminescence intensities from three separate binding experiments each. We could not estimate an accurate Kd for complex formation from this analysis, but GST-cAGS8 consistently gave stronger binding signals with the heterotrimer than with either subunit alone. The simplest explanation for this data is that cAGS8 binds to a complex of {alpha} and β{gamma} and that the affinity for this complex is greater than for either subunit alone.

To provide further evidence for formation of the ternary complex we performed gel-filtration chromatography to evaluate the size and composition of potential complexes between G{alpha}i1-GDP, Gβ1{gamma}2, and GST-cAGS8 (Fig. 4). All of the individual proteins and the heterotrimer elute at approximately their predicted molecular masses, including GST-cAGS8, which elutes as an apparent monomer (monomeric molecular mass predicted to be 75 kDa). Incubation of GST-cAGS8 with either G{alpha}i1-GDP or Gβ1{gamma}2 resulted in the appearance of cAGS8 and either G{alpha}i1 or Gβ1{gamma}2 in an earlier fraction (fraction 24) than either of the G protein subunits alone (fraction 27) at a molecular weight indicative of formation of a 1:1 complex between GST-cAGS8 and either G{alpha}i1 or Gβ1{gamma}2. The proportion of cAGS8 associated with a Gβ1{gamma}2 subunit complex appears to be greater than the proportion of cAGS8 with G{alpha}i1 consistent with higher affinity binding of cAGS8 to Gβ1{gamma}2 relative to G{alpha}i1. When cAGS8 was mixed with G{alpha}i1 and Gβ1{gamma}2 together, the three proteins eluted earlier (fraction 23) than the individual cAGS8-G{alpha} or cAGS8-Gβ{gamma} complexes at a position consistent with the predicted molecular weight of a 1:1:1 complex, indicating formation of a stable ternary complex containing GST-cAGS8, G{alpha}i1, and Gβ1{gamma}2.

cAGS8 Binds to a Rearranged G{alpha}β{gamma} Complex—Experiments were conducted to determine how the complex between GST-cAGS8 and the G protein heterotrimer is assembled. There are two interaction sites between G{alpha} and Gβ{gamma}: the G{alpha} switch II interacts with the hot spot on Gβ, and the amino-terminal helix of G{alpha} ({alpha}-NT) interacts with blade 1 of the Gβ propeller (2) (see purple helix, Fig 10A). We hypothesized that cAGS8 binding competes for {alpha} subunit switch II interactions with the hot spot, but interactions of G{alpha}-NT with the Gβ blade region are maintained. In the complex, simultaneous G{alpha} subunit interactions with cAGS8 bound to the hot spot and with Gβ blade 1 result in a bivalent interaction that might be strong enough to maintain a stable ternary complex. To test this idea G{alpha}i1 subunit missing the amino-terminal 21 amino acids (G{alpha}(tt)) of the ~30-amino acid G{alpha}-NT was prepared by limited trypsin digestion. Trypsin treatment of the Formula-activated G{alpha} subunit results in specific cleavage at Arg-21 of G{alpha}i1 (27, 28) and formation of a stable trypsin-resistant fragment, as discussed earlier. This trypsin-digested G{alpha} subunit still binds to cAGS8 (Fig. 2B), but, as has been previously reported, G{alpha}(tt) did not bind Gβ{gamma} (Fig. 4B, top three panels). If interactions between G{alpha}-NT with Gβ{gamma} are required for formation of the ternary complex then it would be expected that G{alpha}(tt) would not form a complex with cAGS8 and Gβ{gamma}. Indeed when GST-cAGS8 was mixed with trypsin-treated G{alpha}i1 and Gβ1{gamma}2, only the GST-cAGS8-Gβ1{gamma}2 or GST-cAGS8-G{alpha}i1(tt) complexes were formed eluting in fraction 24, with no formation of the GST-cAGS8/heterotrimer complex in fraction 23. This clearly demonstrates that cAGS8 is binding to a non-dissociated complex of G{alpha}β{gamma} requiring interaction of {alpha}i1-NT with Gβ{gamma} to maintain the complex.


Figure 3
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  		FIGURE 3.
AGS8 binds to both G{alpha}i1,Gβ1{gamma}2, and the G{alpha}-β{gamma} heterotrimer. A, glutathione-Sepharose isolation of cAGS8 complexed to either G{alpha} alone or G{alpha}β{gamma} heterotrimer. 30 nM G{alpha}i1-GDP or G{alpha}i1-GDP with 30 nM Gβ1{gamma}2 was mixed with the indicated concentration of GST-cAGS8. Proteins bound to GST-cAGS8 were precipitated with glutathione-Sepharose and detected with immunoblotting as described under "Experimental Procedures." Data shown are representative of three independent experiments. B, GST-agarose isolation of GST-cAGS8 complexed to either Gβ{gamma} or G{alpha}β{gamma} heterotrimer. 30 nM Gβ1{gamma}2 or Gβ1{gamma}2 with 30 nM purified G{alpha}i1-GDP were mixed together with the indicated concentration of GST-cAGS8, and the binding was assayed as in A. Data are representative of three independent experiments. C, quantitative evaluation of binding curves of G{alpha}i1 for cAGS8 in the presence ({blacktriangleup}) or absence ({blacksquare}) of Gβ1{gamma}2. The immunoblot in A was evaluated with quantitative chemiluminescence imaging with a charge-coupled device camera to generate binding curves. Data were pooled from experiments performed at least three times. D, quantitative evaluation of binding curves of Gβ{gamma} for cAGS8 in the presence ({blacktriangleup}) or absence ({blacksquare}) of G{alpha}i1. The immunoblot in B was quantitated as described in C. Data were pooled from experiments performed at least three times.

 


Figure 4
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  		FIGURE 4.
Gel-filtration analysis of complex formation between G{alpha}i1, β1{gamma}2, and GST-cAGS8. A, analysis of Gβ1{gamma}2 and {alpha}i1 binding to GST-cAGS8. All proteins were mixed in 500 µl at 100 nM each. B, same as in A with trypsin-treated {alpha}i1 ({alpha}i1(tt)). Excluded blue dextran eluted in fraction 13. Gel-filtration experiments were performed as described under "Experimental Procedures." Each panel is from silver staining of SDS-PAGE separation of proteins in each fraction from the gel-filtration column. Fraction numbers at the top correspond to 1-ml fractions. On the left are the proteins that were mixed prior to gel filtration and on the right are labeling of the individual proteins. GST-cAGS8 elutes at a concentration consistent with a monomer.

 
G{alpha}i1β1{gamma}2-cAGS8 Ternary Complex Is Signaling Competent—AGS8 activates G protein signaling in yeast, and studies in transfected COS7 cells showed that transfection of cAGS8 could stimulate PLC β2 activation in cells transfected with G{alpha}/β{gamma}, suggesting that AGS8 can stimulate Gβ{gamma} signaling from a G protein heterotrimer. Thus the data demonstrating that cAGS8 binds to the heterotrimer raises a central question: How can cAGS8 binding to Gβ{gamma} or G{alpha}β{gamma} cause activation of G protein signaling to allow propagation of signaling by Gβ{gamma}? Based on the data presented so far a simple model where cAGS8 occupies the hot spot and competes with G{alpha} for binding and releasing free Gβ{gamma} cannot be correct. Additionally, either "free" Gβ{gamma} or the cAGS8-{alpha}/β{gamma} complex would have cAGS8 bound at a critical effector binding site and be predicted to be unable to signal to effectors such as PLCβ2, because cAGS8 binding to the Gβ{gamma} hot spot requires amino acids also required for PLCβ2 activation. To determine if cAGS8 could block signaling by Gβ{gamma}, cAGS8 was tested for inhibition of Gβ{gamma}-dependent PLC activation. cAGS8 at concentrations as high as 1 µM did not affect PLCβ2 activation by Gβ1{gamma}2, whereas SIGK peptide, which also binds the hot spot, potently and effectively inhibited Gβ1{gamma}2-dependent activation (Fig. 5A). Thus Gβ1{gamma}2 with cAGS8 bound to a key signaling interface can still regulate a downstream target in vitro.


Figure 5
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  		FIGURE 5.
Gβ{gamma} and G{alpha}β{gamma} activate PLCβ2 in the presence of cAGS8. A, cAGS8 does not affect Gβ1{gamma}2-dependent PLCβ2 activation. 2 ng of PLCβ2 was mixed with phospholipid vesicles containing 50 µM phosphatidylinositol 4,5-bisphosphate, 200 µM phosphatidylethanolamine, and 8000 cpm/assay[3H]phosphatidylinositol 4,5-bisphosphate, 100 nM free Ca2+, and 30 nM β1{gamma}2, in the presence of the indicated concentrations of SIGK peptide ({blacktriangleup}) or cAGS8 ({blacksquare}). The effect of cAGS8 on basal activity of PLCβ2 in the absence of β{gamma} was also studied ({triangleup}). Data were generated from experiments performed at least three times. B, cAGS8-stimulated Gβ{gamma}-dependent PLC activity from an {alpha}β{gamma} heterotrimer. PLC assay conditions are as in A in the presence or absence of 30 nM G{alpha}i1-GDP and the concentrations of cAGS8 as indicated. Data are representative of similar experiments performed at least three times.

 


Figure 6
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  		FIGURE 6.
A quaternary complex is formed between cAGS8, Gβ1{gamma}2, G{alpha}i, and PLCβ2. cAGS8 promotes binding of PLCβ2 to a G{alpha}i1β1{gamma}2 complex. A, avidin-agarose was used to isolate complexes with bGβ1{gamma}2. 30 nM bGβ1{gamma}2 was mixed with either 30 nM G{alpha}i1, and/or 30 nM PLCβ2, and/or 300 nM GST-cAGS8 in binding buffer. Western blots of PLCβ2 from two separate experiments as in B were quantified by chemiluminescence imaging, and the data were pooled and calculated as a percentage of lane 5, PLCβ2 binding to Gβ1{gamma}2 alone: lane 6, Gβ1{gamma}2 plus PLCβ2 plus G{alpha}i1 (8 ± 0.5%); lane 7, Gβ1{gamma}2 plus PLCβ2 plus GSTcAGS8 (122 ± 11%); lane 8, Gβ1{gamma}2 plus PLCβ2 plus G{alpha}i1 plus GSTcAGS8 (115 ± 11%). Data are means ± S.E. B, glutathione-Sepharose was used to isolate complexes with GST-cAGS8. 300 nM GST-cAGS8 was mixed with either 30 nM bGβ1{gamma}2 and/or 30 nM G{alpha}i1, and/or 30 nM PLCβ2 in binding buffer. Complexes were isolated as described under "Experimental Procedures." Data are representative of three independent experiments.

 
To directly test if cAGS8 could activate signaling by a G protein heterotrimer the ability of purified GST-cAGS8 to stimulate PLCβ2 activation in vitro by G{alpha}i1-GDP/Gβ1{gamma}2 was assessed. In the presence of stoichiometric amounts of G{alpha}i1-GDP, Gβ1{gamma}2-dependent PLCβ2 activation is completely inhibited (Fig. 5B). cAGS8 restored the ability of Gβ1{gamma}2 to activate PLCβ2 in the presence of G{alpha}i1-GDP, in a concentration-dependent manner with an EC50 of ~30 nM (Fig. 5B). Because cAGS8 does not dissociate G{alpha} from Gβ{gamma}, the data suggest that the heterotrimeric G protein complex can stimulate PLCβ2 when bound to cAGS8. This is surprising, because both G{alpha}i1 and cAGS8 bind to a region on Gβ{gamma} thought to be required for PLCβ2 activation (14, 16).

Complex Formation between cAGS8, G{alpha}i1-GDP, Gβ{gamma}, and PLCβ2—To directly determine if a complex can be formed between GST-cAGS8, G{alpha}i1-GDP, Gβ{gamma}, and PLCβ2, immobilized avidin was used to precipitate bGβ1{gamma}2 with various potential binding partners. bGβ1{gamma}2 binds PLCβ2 as expected (Fig. 6A, lane 5) and is inhibited in the presence of an equimolar concentration of G{alpha}i1 (Fig. 6A, lane 6). Addition of cAGS8 restored PLCβ2 binding to the level observed in the absence of G{alpha}i1, and G{alpha}i1 remains bound in the complex (Fig. 6A, lane 8). In the absence of G{alpha}i1, cAGS8 did not affect binding of PLCβ2 (lane 7). These data indicate that cAGS8, G{alpha},Gβ{gamma}, and PLCβ2 form a quaternary complex and AGS8-dependent binding of PLCβ2 by the heterotrimer does not involve subunit dissociation. The functional data in Fig. 5 indicate that the AGS8-bound complex is an active signaling complex.

To further confirm the existence of this quaternary complex, we used glutathione-Sepharose to assess binding to GST-cAGS8 (Fig. 6B). PLCβ2 did not bind to glutathione-Sepharose, GST, or GST-cAGS8 (Fig. 6B, lanes 4-6). If G{alpha}i1 subunit was added with PLCβ2, only G{alpha}i1 bound to GST-cAGS8 (Fig. 6B, lane 7). If Gβ1{gamma}2 and PLCβ2 were added, both Gβ1{gamma}2 and its bound PLCβ2 were isolated with GST-cAGS8 (Fig. 6B, lane 8). Addition of G{alpha}i1 did not prevent binding of PLCβ2 to this complex, and binding of G{alpha}i1 was enhanced in the complex compared with binding of G{alpha}i1 alone (Fig. 6B, lane 9). These data further confirm the existence of a quaternary complex between cAGS8, PLCβ2, G{alpha}i1, and Gβ1{gamma}2, with PLC interacting with the heterotrimer only when cAGS8 is present.


Figure 7
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  		FIGURE 7.
Gel-filtration analysis of quaternary complex formation between G{alpha}i1, β1{gamma}2, and GST-cAGS8 and PLCβ2. Experiments were performed as described under "Experimental Procedures" and as in Fig. 4 except 100 nM PLCβ2 was analyzed and included in the mixtures as indicated.

 


Figure 8
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  		FIGURE 8.
cAGS8 rescues the function of inactive Gβ1(W99A){gamma}2. A, cAGS8 rescues binding of Gβ1(W99A){gamma}2 to PLCβ2. 30 nM bGβ1{gamma}2 or bGβ1(W99A){gamma}2 was mixed with either 30 nM PLCβ2, and/or 100 nM GST-cAGS8 in binding buffer. Complexes were isolated with avidin-agarose as described under "Experimental Procedures." Data are representative of three independent experiments. Western blots of PLCβ2 from two separate experiments as in A were quantified by chemiluminescence imaging, and the data were pooled and calculated as a percentage of lane 6, PLCβ2 binding to wt Gβ1{gamma}2 alone: lane 7, Gβ1{gamma}2(W99A) plus PLCβ2 (13 ± 8%); lane 8, wtGβ1{gamma}2 plus PLCβ2 plus GSTcAGS8 (108 ± 9%); lane 9, Gβ1{gamma}2(W99A) plus PLCβ2 plus GSTcAGS8 (96 ± 8%). Data are means ± S.E. B, cAGS8 restored bGβ1(W99A){gamma}2 activation of PLCβ2. PLC assays are as in Fig. 5 with 30 nM bGβ1(W99A){gamma}2 or 30 nM wt bGβ1{gamma}2, in the presence or absence of 100 nM cAGS8, or boiled cAGS8. Data are representative of experiments performed at least three times.

 
Finally we used gel filtration to demonstrate that cAGS8 promotes binding of PLCβ2 to the heterotrimer (Fig. 7). In these experiments PLCβ2 eluted as an apparent monomer. When PLCβ2 was mixed with Gβ1{gamma}2 prior to gel filtration (PLCβ2 plus Gβ1{gamma}2) both PLCβ2 and Gβ{gamma} eluted earlier than either protein alone, indicating complex formation. When PLCβ2 was mixed with the G protein heterotrimer (PLCβ2 plus Gβ1{gamma}2 plus G{alpha}i1) a small proportion of the PLCβ2 eluted at a higher molecular weight, but only Gβ{gamma} was associated with this complex indicating that PLC formed a stable complex with the free Gβ1{gamma}2 in the mixture but not with the heterotrimer. In contrast, when GST-cAGS8 was added to the G protein heterotrimer (cAGS8 plus PLCβ2 plus Gβ1{gamma}2 plus G{alpha}i1), all of the proteins eluted at a much higher molecular weight than any of the individual proteins or complexes demonstrating formation of a stable complex between GST-cAGS8, G{alpha}i1,Gβ1{gamma}2, and PLCβ2. This demonstrates conclusively that bound cAGS8 promotes association of PLCβ2 with the cAGS8-G{alpha}/β{gamma} complex despite AGS8 binding to a key signaling surface on Gβ{gamma}.

cAGS8 Rescues the Loss of Function of Gβ1(W99A){gamma}2 Mutant—The currently accepted model for activation of Gβ{gamma} signaling to downstream effectors is that separation of Gβ{gamma} from G{alpha} uncovers the hot spot on Gβ{gamma} allowing for the binding of effectors to this region (3). In the presence of cAGS8, or both cAGS8 and G{alpha} subunit, this surface would be expected to be occupied and the resulting complexes would be unable to signal downstream. Nevertheless, we demonstrated that both the cAGS8-Gβ{gamma} complex and the cAGS8-G{alpha}β{gamma} complex can bind and activate PLCβ2. To understand this we hypothesized that, in these complexes, the hot spot on Gβ{gamma} is no longer utilized for PLCβ2 activation, and a new activation site is exposed in the cAGS8-Gβ{gamma} complexes that can activate PLCβ2. To test this idea a mutant that disables the hot spot for PLCβ2 activation, but not cAGS8 binding, was analyzed. It had been previously reported that Gβ1(W99A){gamma}2 is defective for PLCβ2 activation (14), but Gβ1(W99A){gamma}2 still bound efficiently to cAGS8 (Fig. 1C, see Fig 10A, blue amino acid). If a new binding/signal transfer surface for PLC activation was created in the presence of cAGS8, then binding of cAGS8 might rescue PLCβ2 binding and activation by Gβ1(W99A){gamma}2. First binding of PLCβ2 to bGβ1{gamma}2 and bGβ1(W99A){gamma}2 was tested (Fig. 8A). Mutation of Gβ1W99 to Ala significantly inhibited binding of PLCβ2, indicating that this amino acid, previously shown to be important for activation of PLCβ2, is also important for binding to PLCβ2 (Fig. 8A, lanes 6 and 7). Addition of cAGS8 rescued binding of PLCβ2 to bGβ1(W99A){gamma}2 to levels approaching binding to wt bGβ1{gamma}2 (Fig. 8A, compare lanes 6, 7, and 9); whereas the binding of PLCβ2 to wtGβ1{gamma}2 did not change in the absence or presence of cAGS8 (Fig. 8A, compare lanes 6 and 8).

Next it was determined if cAGS8 could restore the ability of Gβ1(W99A){gamma}2 to activate PLCβ2 (Fig. 8B). Wild-type Gβ1{gamma}2 stimulated PLCβ2 12-fold, whereas mutation of βW99 to Ala greatly impaired this activation. cAGS8 restored the capacity of Gβ1(W99A){gamma}2 to stimulate PLCβ2 to near wt Gβ1{gamma}2 levels. cAGS8 itself did not stimulate PLCβ2, as described earlier and cAGS8 did not affect wt Gβ1{gamma}2-dependent activation of PLCβ2. Together these data indicate that cAGS8 unmasks and/or creates a new binding and signal transfer site for PLCβ2 that does not involve the hot spot on Gβ{gamma}.


Figure 9
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  		FIGURE 9.
The amino-terminal coiled-coil region of Gβ{gamma} is an alternate site required for PLCβ2 binding and activation in the presence of cAGS8. A, AGS8 inhibits binding of Gβ1(23-27){gamma}2 to PLCβ2. 30 nM bGβ1{gamma}2 or bGβ1(23-27){gamma}2 mutant was mixed with 30 nM PLCβ2, and/or 100 nM GST-AGS8 in binding buffer. Pulldown assay with avidin-agarose was performed as described under "Experimental Procedures." Data are representative of three independent experiments. Western blots of PLCβ2 from two separate experiments as in A were quantified by chemiluminescence imaging, and the data were pooled and calculated as a percentage of lane 7, PLCβ2 binding to wt Gβ1{gamma}2 alone: lane 8, Gβ1(23-27){gamma}2 plus PLCβ2 (97 ± 2%); lane 9, wtGβ1{gamma}2 plus PLCβ2 plus GSTcAGS8 (98 ± 4%); lane 10, Gβ1(23-27){gamma}2 plus PLCβ2 plus GSTcAGS8 (12 ± 1%). Data are means ± S.E. B, Gβ1(23-27){gamma}2 does not form a complex with PLCβ2 in the presence of cAGS8. Gel-filtration analysis was performed as described under "Experimental Procedures" and in legends for Figs. 4 and 7 except bGβ1(23-27){gamma}2 was analyzed. C, AGS8 inhibited Gβ1(23-27){gamma}2 activation of PLCβ2. PLC assays were performed as described in Fig. 5 with 1 ng of purified PLCβ2/assay, 30 nM Gβ1(23-27){gamma}2 ({blacktriangleup}), or 30 nM wt Gβ1{gamma}2 ({blacksquare}), in the presence of varying concentrations of AGS8. In this experiment basal PLC activity was 5.4 mmol/mg/min and with wt bGβ1{gamma}2-stimulated activity was 25 µmol/mg/min, and bGβ1(23-27){gamma}2-stimulated activity was 42 µmol/mg/min (as previously reported, bGβ1(23-27){gamma}2 stimulates PLC activity more effectively than wt bGβ1{gamma}2 (29)). Data are representative of experiments performed at least three times.

 
cAGS8 May Unmask a New Binding Surface at the Amino Terminus of β{gamma}—The data support a model where AGS binds to the heterotrimer with simultaneous interactions with Gβ{gamma} at the hot spot and the G{alpha} subunit and that the hot spot is no longer used for signal transfer to PLCβ2 activation, because it is occupied with cAGS8. To accommodate this model we propose that another site for effector interactions is unmasked on Gβ{gamma} that signals to downstream effectors. We recently presented evidence that PLCβ2 can bind to two distinct regions on Gβ, at the amino-terminal coiled-coil domain, and at the hot spot (29). In this earlier work it was proposed that association of PLCβ2 with the Gβ{gamma} coiled-coil region inhibited PLC activation.

To test whether the amino-terminal region on the Gβ subunit might participate in activation of PLCβ2 by the cAGS8-Gβ{gamma} complex, binding of PLCβ2toa β subunit mutant that no longer interacts with PLCβ2 at the amino terminus (bGβ1(23-27){gamma}2) (29) was tested in the presence or absence of cAGS8 (Fig. 9A, see Fig 10A, yellow amino acids). Addition of cAGS8 greatly inhibited the binding of PLCβ2 to the Gβ1(23-27){gamma}2 (mutant, but not wild-type Gβ1{gamma}2 (Fig. 9A, compare lanes 7, 9, and 10). The Gβ1(23-27){gamma}2 mutation did not significantly alter interactions with PLCβ2 in the absence of cAGS8 (Fig. 9A, compare lane 7 and 8). This data were confirmed by gel-filtration analysis where, in contrast to data in Fig. 7 for the wild-type heterotrimer, cAGS8 did not promote binding of PLCβ2 to a heterotrimer containing bGβ1(23-27){gamma}2 (compare Figs. 7 and 9B). Free bGβ1(23-27){gamma}2 associates with PLCβ2, presumably through the hot spot, indicating that this mutant Gβ subunit protein is viable.

This indicates that the amino-terminal coiled-coil binding region on Gβ binds PLCβ2 when the hot spot is occupied by cAGS8. We next tested whether activation of PLCβ2 by cAGS8-Gβ{gamma} complex was affected by the amino-terminal mutation. We predicted that, if this alternate binding site for PLCβ2 was disabled, the Gβ{gamma} would no longer be able to activate PLCβ2 with cAGS8 bound to the hot spot. cAGS8 did not significantly inhibit wild-type Gβ1{gamma}2-stimulated PLCβ2 activity (see also Fig. 5A). However, cAGS8 inhibited Gβ1(23-27){gamma}2-stimulated PLCβ2 activity with an IC50 of ~15 nM, indicating that the amino-terminal interaction region was in fact a second site required for stimulation of PLCβ2 activity by the cAGS8-Gβ{gamma} complex. This also suggests that the apparently inhibiting activity of the amino terminus toward PLCβ2 participates in activation of PLCβ2 upon cAGS8 binding.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we describe a mechanism for activation of G protein signaling that does not involve nucleotide exchange or subunit dissociation. Multiple assays of direct binding demonstrate that cAGS8 binds to the G protein heterotrimer and promotes binding of PLCβ2 to Gβ{gamma} subunits to form a quaternary signaling complex. Gel-filtration analysis demonstrates that the quaternary complex is stable, indicating the proteins in the complex interact with relatively high affinity. In addition, AGS8 modulated G protein-dependent PLC regulation with EC50 or IC50 values of ~30 nM (Figs. 5B and 9C). A key experiment, supporting the idea that subunit dissociation is not needed for signaling in the quaternary complex, demonstrated that cAGS8 could rescue the function of a mutant Gβ{gamma} (Gβ1W99A) with the hot spot at the G{alpha}/β{gamma} interface disabled for PLCβ2 binding and activation. This experiment rules out the possibility that undetected free Gβ{gamma} might be generated upon AGS8 binding that somehow transiently reveals the hot spot for PLCβ2 binding and activation. Clearly cAGS8 cannot be acting simply by dissociating G{alpha} from Gβ{gamma} to expose the hot spot on Gβ{gamma}.


Figure 10
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  		FIGURE 10.
Model for cAGS8-promoted Gβ{gamma} signaling from G protein heterotrimers. A, three-dimensional representation of the structure of Gβ{gamma} showing relevant binding surfaces and mutants. In dark gray is the β subunit with bright green and red spaces showing amino acids important for cAGS8 binding. In red are amino acids important for both cAGS8 and PLCβ2 interactions. Trp-99, whose mutation to alanine disables PLC activation but not cAGS8 binding, is in blue. The orange ribbon helix representation is the {alpha} subunit switch II, and the purple ribbon helix is the {alpha} subunit amino-terminal helix. The gamma subunit is represented by the red helical ribbon. In yellow are amino acids mutated in the Gβ1(23-27){gamma}2 mutant. B, in the classic model for G protein activation, subunit dissociation after activation by G protein-coupled receptors leads to exposure of the hot spot required for binding and activation of PLCβ2. During activation by cAGS8, cAGS8 binds to the hot spot and {alpha} remains associated through a bivalent interaction with the blade regions on β{gamma} and bound cAGS8. This creates a hot spot-independent signaling surface that can bind and activate effectors.

 
It was initially surprising that cAGS8 binding to the hot spot on Gβ{gamma} did not result in subunit dissociation, whereas the peptide SIGK that binds to an overlapping site promotes subunit dissociation (16, 22). These observations can be reconciled by the observation that AGS8, but not SIGK, also exhibits some affinity for G{alpha} subunits. In this scenario, when AGS8 binds the hot spot, G{alpha} loses contact with the hot spot but gains new interactions with AGS8 sufficient to maintain the ternary complex in cooperation with interactions between the G{alpha}-NT and Gβ{gamma}. Alternatively, a model could be suggested based on a recent structural model of GRK2 simultaneously bound to both Gβ{gamma} and G{alpha}q. Here G{alpha} subunits are dissociated from the Gβ{gamma} subunits but remain bound to two different sites of GRK2 (30). In support of the first model, the amino-terminal helix of G{alpha}i1 is required for maintenance of the quaternary complex but not AGS8 binding. Thus we propose a modified version of the clam shell hypothesis, where the activator (AGS8) rather than the effector binds to a rearranged complex of G{alpha} and Gβ{gamma} that is maintained by contacts between the amino-terminal helix of G{alpha} with Gβ{gamma} (see Fig. 10B).

Because GβW99A disables the hot spot for PLCβ2 binding and activation, cAGS8 itself did not bind to or activate PLCβ2, and cAGS8 occupied the region on the Gβ subunit required for PLCβ2 binding. The only way that cAGS8 could rescue the binding and activation of PLCβ2 by GβW99A would be by creating a new binding site for PLCβ2 in the cAGS8-Gβ{gamma} complex. Site-directed mutagenesis has shown that there are contact surfaces outside of the hot spot that are important for effector recognition (29, 31).

A binding site for PLCβ2 in the amino-terminal coiled-coil region of the β{gamma} subunits has previously been identified by chemical cross-linking and site-directed mutagenesis (29, 32). In those studies it was demonstrated that disabling this binding surface by site-directed mutagenesis resulted in increased Gβ{gamma}-dependent activation of PLCβ2 leading to the conclusion that this binding site for PLC was inhibitory while the binding site in the hot spot was stimulatory. Here it is demonstrated that the Gβ{gamma} coiled-coil region is required for binding and activation of PLCβ2 when AGS8 is bound. This suggests that this binding site is converted from an inhibitory to a stimulatory site in the presence of cAGS8. It is also possible that PLCβ2 binding and activation in the complex requires both binding to the Gβ{gamma} amino terminus and cAGS8, although no binding or activation of PLCβ2 by cAGS8 alone was detected. These data suggest a signaling mechanism for non-dissociated G protein complexes that uses binding sites outside the G{alpha}/β{gamma} interface for effector activation.

Interestingly, the signal transfer surfaces of the Gβ{gamma} subunit in yeast, Saccharomyces cerevisiae, have not been shown to involve amino acids at the G{alpha}/β{gamma} interface but rather have been mapped to a region in the amino-terminal coiled-coil at a position that is nearly identical to the region we described for PLC binding (33-35). In contrast to the hot spot region where G{gamma} subunits cannot directly contribute to binding, the more variable G{gamma} subunits could conceivable directly contribute to this contact interface. Thus some signaling specificity could be supplied by binding effectors to this region.

Mechanisms for activation of G protein signaling by group II AGS proteins are distinct from receptor-mediated mechanisms for G protein activation but involve an apparently straightforward subunit dissociation-based mechanism where the GPR motif of the AGS protein binds G{alpha}-switch II and either promotes subunit dissociation or competes for Gβ{gamma} subunit binding, leading to accumulation of free Gβ{gamma} (22, 36, 37). Here we describe a model for Gβ{gamma} activation that does not involve revealing the hot spot on Gβ{gamma} for PLC binding but rather involves alternative utilization of binding sites outside the G{alpha}-β{gamma} interface upon binding of the activator AGS8. This mechanism has significant implications for mechanisms of signaling by non-dissociated G protein complexes that probably extends beyond AGS8 regulation.

It is not clear from our studies how AGS8 is regulated. One possible mechanism is transcriptional up-regulation of AGS8 as was observed in cardiac myocytes subjected to ischemic stress (13). Another possibility is that AGS8 is regulated post-translationally by an upstream stimulus. It is important to emphasize that this study examined only a carboxyl-terminal portion of the full-length AGS8 protein. We elucidated how this G protein-binding domain of AGS8 can regulate heterotrimeric G protein function, but this carboxyl terminus may in turn be regulated by intramolecular interactions in full-length AGS8. Thus, it is important that we understand how this domain operates in the context of the full-length AGS8 protein. Full-length AGS8 has been difficult to express and purify. It has been expressed in COS7 cells and does not inhibit Gβ{gamma}-dependent PLCβ2 activation (13), consistent with the data presented here. We are currently exploring various strategies to address the properties of full-length AGS8 as we move forward with these studies.

The subject of G protein subunit dissociation has been the subject of significant interest driven in part by the advent of cellular resonance energy transfer methods that allow alterations in protein interactions to be monitored in intact cells. Based primarily on the observation that, in some cases receptor activation leads to increased resonance energy transfer between G{alpha} and Gβ or G{gamma} subunits, it has been proposed that G protein subunits undergo conformational rearrangement rather than dissociation (5, 7). Recent fluorescence recovery after photobleaching studies, on the other hand, favors a subunit dissociation-based model for at least some G protein isoforms (38). Analysis of G protein subunit interactions with GIRK channels suggests that G{alpha} and Gβ{gamma} subunits are prebound to the channel and that conformational rearrangements lead to channel activation (39). These experiments suggest that conformational alterations of assembled G protein heterotrimer-effector complexes occur in cells that are presumably sufficient to activate effector functions, but the nature of these conformations are difficult to discern from this type of analysis. Recent determination of the structure of GRK2 bound to G protein {alpha}q and Gβ{gamma} subunits simultaneously provides atomic level detail for how a G protein trimer can interact with a target to form a quaternary complex, but in this complex the G protein subunits are dissociated from each other with each subunit separately interacting with different domains of GRK2 (30). The model presented here, although based on in vitro data, provides molecular details for one potential model for how non-dissociated G protein trimers may regulate effector targets that would be difficult to approach in intact cells.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants GM060286 and GM053536 (to A. V. S.) and NS24821 and MH55391 (to S. M. L.) and by an American Heart Association predoctoral fellowship grant (to C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Cardiovascular Research Institute, Yokohama City University, School of Medicine, 3-9 Fukuura, Kanazawa-Ku, Yokohama 236-0004, Japan. Back

2 Supported by the David R. Bethune/Lederle Laboratories Professorship in Pharmacology and the Research Scholar Award from Yamanouchi Pharmaceutical Company (now named Astellas Pharma Inc.). Present address: Dept. of Pharmacology, Colcock Hall, 2nd. Floor, P. O. Box 250002, Medical University of South Carolina, 179 Ashley Ave., Charleston, SC 29425. Back

3 To whom correspondence should be addressed: Dept. of Pharmacology & Physiology and Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-0892; Fax: 585-273-2652; E-mail: Alan_Smrcka{at}urmc.rochester.edu.

4 The abbreviations used are: {alpha}-NT, G{alpha} subunit animo-terminal helix; bβ{gamma}, biotinylated β{gamma}; AGS, activator of G protein signaling; GRK2, G protein-coupled receptor kinase 2; GST, glutathione S-transferase; PLC, phospholipase C; GTP{gamma}S, guanosine 5'-O-(thiotriphosphate); b, biotinylated; wt, wild type; tt, trypsin-treated. Back


   ACKNOWLEDGMENTS
 
We thank Tabetha Bonacci for preparation of the G protein β subunit mutants and Dr. Elliott M. Ross for comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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