RGS6, RGS7, RGS9, and RGS11 Stimulate GTPase Activity of Gi Family G-proteins with Differential Selectivity and Maximal Activity*

Shelley B. HooksDagger §, Gary L. WaldoDagger , James CorbittDagger , Erik T. BodorDagger , Andrejs M. Krumins||, and T. Kendall HardenDagger

From the Dagger  Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599 and the || Department of Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas 75390

Received for publication, November 7, 2002, and in revised form, December 23, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha -subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma -like (GGL) domain that mediates dimeric interaction with Gbeta 5. Gbeta 5/R7 dimers stimulated steady state GTPase activity of Galpha -subunits of the Gi family, but not of Galpha q or Galpha 11, when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta 5/R7 proteins revealed differences in potencies and efficacies toward Galpha -subunits of the Gi family. Although all four Gbeta 5/R7 proteins exhibited similar potencies toward Galpha o, Gbeta 5/RGS9 and Gbeta 5/RGS11 were more potent GAPs of Galpha i1, Galpha i2, and Galpha i3 than were Gbeta 5/RGS6 and Gbeta 5/RGS7. The maximal GAP activity exhibited by Gbeta 5/RGS11 was 2- to 4-fold higher than that of Gbeta 5/RGS7 and Gbeta 5/RGS9, with Gbeta 5/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta 5/RGS7 and Gbeta 5/RGS9 inhibited Gbeta 5/RGS11-stimulated GTPase activity of Galpha o. Therefore, R7 family RGS proteins are Gi family-selective GAPs with potentially important differences in activities.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Heterotrimeric guanine nucleotide-binding proteins (G-proteins) act as molecular switches in multiple GPCR1 signaling pathways via regulation of specific effector molecules such as phospholipase C and adenylyl cyclase. The biological activity of G-protein alpha -subunits is determined by the identity of the bound guanine nucleotide (GTP or GDP), which in turn is governed by the relative rates of guanine nucleotide exchange and hydrolysis of GTP by the intrinsic GTPase activity of Galpha -subunits. These opposing reactions are stimulated by agonist-occupied GPCR and GTPase-activating proteins (GAPs).

Although some effector proteins exhibit GAP activity (1-3), the primary regulators of GTPase activity of Galpha -subunits are a diverse family of regulator of G-protein signaling (RGS) proteins that act as GAPs for heterotrimeric G-protein alpha -subunits (4-7). This family is defined by a conserved RGS domain, which markedly increases the rate of GTP hydrolysis by Galpha -subunits and terminates effector activation by both Galpha - and Gbeta gamma -subunits. More than 30 RGS proteins have been identified and organized into subfamilies based on sequence similarity and domain structure. These families vary in size and complexity, from the R4 family whose structure is largely limited to the RGS domain to the R12 and RhoGEF families whose members are large multifunctional proteins containing several domains (for reviews see Refs. 8-10).

The R7 RGS family is a unique multidomain family, which consists of RGS proteins containing a novel G-gamma -like (GGL) domain homologous to the Ggamma -subunit of heterotrimeric G-proteins (11). This domain, found in the mammalian proteins RGS6, -7, -9, and -11 and the Caenorhabditis elegans proteins EAT16 and EGL10 (7, 12), confers specific binding to Gbeta 5-subunits but not to Gbeta 1-4 (11, 13). Heterodimeric association with Gbeta 5 appears necessary for stability and biological activity of R7 proteins (14-16). R7 proteins also contain a conserved N-terminal DEP (dishevelled, EGL10, pleckstrin homology) domain of unknown function (17).

RGS proteins may modify GPCR signaling through selective increases in GTP hydrolysis by a subset of G-proteins. For example, RZ family members specifically accelerate GTPase activity of Galpha z (18), and the recently characterized sorting nexin 13 (RGS-PX1) has been reported to increase the rate of GTP hydrolysis by Galpha s but not by Galpha i (19). The G-protein selectivity of the R7 family of RGS proteins has not been clearly defined. In single turnover GTPase assays, Gbeta 5/RGS6 and Gbeta 5/RGS7 increased GTPase activity of Galpha o (20) and Gbeta 5/RGS11 increased GTPase activity of Galpha o and, to a much lesser degree, that of Galpha i1, Galpha i2, and Galpha i3 (11). However, the R7 RGS proteins did not affect the single turnover GTPase rates of other Galpha -subunits, including Galpha q (R183C), Galpha s, and Galpha 12. In contrast, when expressed in cultured cell lines, RGS7 inhibited Galpha q-promoted Ca2+ responses downstream of M3 receptors (14) and 5-HT2c receptors (21, 22) and inhibited Galpha i-regulated K+ channel activity in a Gbeta 5-dependent manner (23). Therefore, assays of soluble Galpha -subunits suggest Galpha o selectivity, while intact cell signaling studies implicate R7 proteins in regulation of Gq as well as Gi pathways.

In face of the uncertainty of the actions of R7 RGS proteins, we purified the four members of the mammalian R7 family. The specificities of action of Gbeta 5/R7 heterodimers were determined in steady state GTPase assays of Gi and Gq family Galpha -subunits reconstituted with GPCR in phospholipid vesicles. Gbeta 5/RGS6, -7, -9, and -11 increased the GTPase activity of Galpha o, Galpha i1, Galpha i2, and Galpha i3 but not Galpha q or Galpha 11. Notable differences in maximal GAP activities were observed among R7 family proteins, and the maximal activity of the most efficacious RGS protein (Gbeta 5/RGS11) was inhibited by Gbeta 5/RGS7 and Gbeta 5/RGS9.

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INTRODUCTION
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Protein Purification-- The generation of baculoviruses for Gbeta 5, RGS6, RGS7, and RGS11 have been described previously (11, 20). A baculovirus for RGS9-1 in pFastbac Htb was prepared similarly. Cultures of Sf9 insect cells (1.4 × 106 cells/ml) were co-infected with virus encoding the RGS6, RGS7, or RGS9 gene (m.o.i. of 1) and a hexahistidine-tagged Gbeta 5 (short isoform) (m.o.i. of 0.5). Forty-eight hours postinfection, 4 liters of cells were collected by centrifugation. Due to lower expression under these conditions, Sf9 cells were infected with the RGS11:Gbeta 5 viruses at a 10:1 ratio (m.o.i. of 0.5 and 0.05), and cell lysates were obtained 65 h postinfection. Cells were resuspended and lysed in 600 ml of Buffer A (20 mM KPO4, pH 8, 150 mM NaCl, 2 mM MgCl2, 5 mM beta -mercaptoethanol, protease inhibitors (500 nM aprotinin, 10 µM leupeptin, 200 µM phenylmethylsulfonyl fluoride, 1 nM pepstatin, 10 µM L-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone)) with 20 strokes of a Dounce homogenizer at 4 °C. The lysate was cleared by low speed centrifugation, and the supernatant was centrifuged at 100,000 × g for 30 min. The soluble protein fraction was loaded on a 3- to 5-ml column of Ni-NTA-agarose resin (Qiagen, Germany) over 3 h. The column was washed with 15 ml of Buffer B (20 mM KPO4, pH 8, 400 mM NaCl, 2 mM MgCl2, 5 mM beta -mercaptoethanol, and protease inhibitors) followed by 5 ml of Buffer C (20 mM KPO4, pH 8, 25 mM NaCl, 2 mM MgCl2, 5 mM beta -mercaptoethanol, and protease inhibitors). The Gbeta 5/R7 dimers eluted with 50-150 mM imidazole in Buffer C. This eluate was further purified using 1 ml of HighTrap FPLC columns of either Q-Sepharose (Gbeta 5/RGS6 and Gbeta 5/RGS7) or S-Sepharose (Gbeta 5/RGS9 and Gbeta 5/RGS11) (Amersham Biosciences). The Ni-NTA eluate was diluted 5:1 in starting buffer for ion exchange chromatography (buffer for Q-Sepharose: 50 mM Tris, pH 8, 2 mM dithiothreitol, protease inhibitors; buffer for S-Sepharose: 50 mM Hepes, pH 8, 1 mM EDTA, 2 mM dithiothreitol, 10% glycerol, protease inhibitors), loaded onto the FPLC column, and eluted in the same buffer with a 0-400 mM NaCl gradient over 30-column volumes. Each dimer eluted at ~200 mM NaCl. Fractions were collected and concentrated using a Centricon centrifugal filter device (Millipore, Bedford, CT). The concentration of purified Gbeta 5/R7 dimers was determined by Coomassie staining purified product and a standard curve of protein standards resolved by SDS-PAGE. Yield was ~1 mg of Gbeta 5/R7 dimer per 4 liters except for Gbeta 5/RGS11, whose purification yielded ~250 µg per 4 liters. Galpha - and Gbeta gamma -subunits (24) and muscarinic receptors (25) were purified after expression from baculoviruses in Sf9 insect cells as described.

Vesicle Reconstitution and Characterization-- Detergent/phospholipid mixed micelles were prepared by drying 110 µg of phosphatidylethanolamine, 70 µg of phosphatidylserine, and 8 nmol of cholesteryl hemisuccinate under argon and resuspending in detergent buffer (0.4% deoxycholate, 20 mM Hepes, 1 mM EDTA, 100 mM NaCl). Fifty µl of this preparation was combined with 15 pmol of muscarinic receptor, 50 pmol of Galpha , and 150 pmol of Gbeta 1gamma 2 in Buffer D (20 mM Hepes, 100 mM NaCl, 1 mM EDTA, 2 mM MgCl2) and immediately loaded onto a G-50-Sepharose column equilibrated with Buffer D. The eluate was collected in 200-µl fractions. Fractions were assayed for the presence of muscarinic receptor by incubation of 5 µl per fraction with 20 nM [3H]quinuclidinyl benzilate (~200,000 cpm) in a volume of 100 µl for 90 min at 30 °C and filtration over GF/F filters (Whatman). Peak fractions also were assayed for Galpha incorporation by incubation of 5 µl of the vesicle preparation with 1 µM 35S-labeled GTPgamma S (500,000 cpm) in the presence or absence of 0.1% C12E10 detergent (total volume = 100 µl) at 30 °C for 90 min. Samples labeled in the absence of C12E10 were filtered over GF/F filters, which collect vesicles but not free protein, to quantitate incorporation of Galpha -subunits into vesicles, and C12E10-containing samples were filtered over nitrocellulose to quantitate total Galpha .

Steady State GTPase Assays-- One microliter (for Galpha i/o-containing vesicles) or 5 µl (for Galpha q/11-containing vesicles) of the vesicle preparations was equilibrated on ice in Buffer D in the presence or absence of 100 µM carbachol and various concentrations of RGS protein. [gamma -32P]GTP (2 µM; ~400,000 cpm) was added to each 25 µl of reaction. The reaction was transferred to a 30 °C water bath for 15 min (for Galpha i/o-containing vesicles) or 30 min (for Galpha q/11-containing vesicles) and quenched on ice with 975 ml of cold 5% activated charcoal in 20 mM NaH2PO4. The charcoal was pelleted by centrifugation, and a portion of the supernatant was added to scintillant for 32Pi quantitation.

    RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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The specificity of RGS proteins for G-protein substrates determines in part their physiological effects on signaling. Previous in vitro studies with Galpha -subunits in solution have illustrated specificity of R7-RGS proteins for Galpha o, whereas in vivo observations have suggested broader activities. To more specifically address the selectivity of individual Gbeta 5/R7 heterodimers for Galpha -subunits, we purified Gbeta 5/RGS6, Gbeta 5/RGS7, Gbeta 5/RGS9, and Gbeta 5/RGS11 to near homogeneity and directly measured their selectivity in steady state GTPase assays with proteoliposomes reconstituted with M1 or M2 muscarinic receptors and various heterotrimeric G proteins of the Gq and Gi families, respectively.

Gbeta 5/R7 Protein Purification-- Full-length RGS6, -7, -9, and -11 were co-expressed with hexahistidine-tagged Gbeta 5 in Sf9 insect cells using the baculovirus expression system. Dimers were purified from the soluble fraction using Ni-NTA-agarose and ion exchange chromatography. Twenty-five to seventy-five percent of the total cellular immunoreactive R7 protein was recovered in the soluble fraction, 25-75% of the soluble protein was recovered following Ni-NTA chromatography, and nearly 100% of the Ni-NTA eluate was recovered following the final ion exchange purification. The results of a typical purification (Gbeta 5/RGS7) are shown in Fig. 1A. Purified Gbeta 5/RGS6, Gbeta 5/RGS9, and Gbeta 5/RGS11 dimers are illustrated in Fig. 1B.


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Fig. 1.   Gbeta 5/R7 purification. A, the purification of Gbeta 5/RGS7 is shown on a Coomassie Blue-stained SDS-PAGE. The soluble fraction of Sf9 cells infected with RGS7 and hexahistidine-tagged Gbeta 5 was loaded onto a Ni-NTA column. The column was consecutively washed with 400 mM NaCl (lane 1), 25 mM NaCl (lane 2), 50 mM imidazole (lane 3), 150 mM imidazole (lane 4), and 300 mM imidazole (lane 5). The 50 mM imidazole elution was further purified on a HighTrap Q anionic exchange column and eluted with a gradient of 0-400 mM NaCl. Lanes 6-13 show fractions eluting at ~175-225 mM NaCl. Fractions shown in lanes 9-11 were pooled and concentrated. B, the final purified products of all four Gbeta 5/R7 dimers are shown: Gbeta 5/RGS6 (lane 1), Gbeta 5/RGS7 (lane 2), Gbeta 5/RGS9 (lane 3), and Gbeta 5/RGS11 (lane 4).

Vesicle Reconstitution-- G-protein alpha -subunits (Galpha o, Galpha i1, Galpha i2, Galpha i3, Galpha q, Galpha 11) were reconstituted in phospholipid vesicles with Gbeta 1gamma 2 and either M1 (Gq family G-proteins) or M2 (Gi family G-proteins) muscarinic receptors under conditions similar to those described by Ross and coworkers (26, 27). Recovery of Galpha -subunits and M1 or M2 muscarinic receptors in the various vesicle preparations was quantitated as described under "Experimental Procedures." Essentially 100% of added Galpha o, Galpha i1, Galpha i2, or Galpha i3 was incorporated into vesicles, and receptor recovery in the proteoliposomes was ~50%. We also prepared and resolved Galpha o-containing vesicles using the higher exclusion limit Sephacryl S-300 gel filtration resin, which separates vesicles from free Galpha o, and observed nearly all of the Galpha o immunoreactivity co-migrating with vesicles in the void volume (data not shown). The four varieties of M2·Galpha i/o vesicles contained similar Galpha protein levels (~100 fmol/µl) and receptor: Galpha ratios (1:6) (data not shown). Quantitation of Galpha q and Galpha 11 is difficult due to the low rates of guanine nucleotide turnover by these Galpha -subunits. Therefore, calculations of GTPase activity reported below were made assuming that incorporation of Galpha q/11 into vesicles was equal to that of Gi family Galpha -subunits.

Steady State GTPase Assays-- The GAP activity of Gbeta 5/R7 proteins toward Galpha -subunits of the Gi and Gq families was assessed in steady state GTPase assays, which measure multiple rounds of GTP hydrolysis and, as such, reflect both guanine nucleotide exchange and GTPase activity. RGS4, an effective GAP against Gq and Gi family Galpha -subunits (28), was used as a reference RGS protein in all experiments. Only minor increases were observed in the rate of GTP hydrolysis in the presence of either agonist (100 µM carbachol) or GAP (200 nM RGS4) alone in proteoliposomes formed by reconstitution of M2 muscarinic receptor, Galpha o, and Gbeta 1gamma 2 (Fig. 2). In contrast, the combined presence of carbachol and RGS4 resulted in a markedly synergistic increase in GTPase activity, and the rate of hydrolysis of GTP was linear for at least 15 min.


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Fig. 2.   Steady state GTPase activity of reconstituted Galpha -subunits. The time course of GTP hydrolysis by Galpha o reconstituted in phospholipid vesicles with the M2 muscarinic receptor and Gbeta 1gamma 2 under basal conditions (open circle ), in the presence of 100 µM carbachol (black-triangle), 200 nM RGS4 (black-down-triangle ), or carbachol plus RGS4 (black-diamond ) is shown.

In the presence of a maximally effective concentration of RGS protein, guanine nucleotide exchange is rate-limiting, and therefore stimulation of GTPase activity by carbachol was observed with a concentration dependence of agonist that approximated its occupancy curve for binding to the M2 muscarinic receptor (data not shown). Similarly, in the presence of a maximally effective concentration of carbachol, guanine nucleotide exchange was no longer rate-limiting, and marked concentration-dependent stimulation of GTPase was observed with RGS4 (data not shown) and Gbeta 5/R7 RGS proteins (see below).

Gbeta 5/R7 Proteins Stimulate Steady State GTPase Activity of Gi Family Galpha -subunits-- To compare the capacity of Gbeta 5/R7 proteins to accelerate GTPase rates of Gi family Galpha -subunits, steady state GTPase activities were determined in the presence and absence of 100 µM carbachol and in the presence and absence of 1 µM RGS protein (either RGS4 or each Gbeta 5/R7 dimer). RGS4 markedly increased GTPase activity for Galpha o, Galpha i1, Galpha i2, and Galpha i3 in the presence of 100 µM carbachol (Fig. 3). Each of the Gbeta 5/R7 dimers also stimulated to varying degrees GTP hydrolysis by Galpha o, Galpha i1, Galpha i2, and Galpha i3 in the presence of agonist (Fig. 3 and Table I). The rate observed with Gbeta 5/RGS11 was as high or higher than the rate with RGS4 with all four Gi family Galpha -subunits, while the GTPase rates in the presence of 1 µM Gbeta 5/RGS6, Gbeta 5/RGS7, and Gbeta 5/RGS9 were significantly lower. Vesicles containing Galpha o achieved the highest maximal GTPase rates irrespective of the RGS protein. However, the basal rate of GTP hydrolysis by Galpha o in the absence of RGS protein was also higher than that observed in Galpha I-containing vesicles. Thus, the fold increase in activity (GTPase rate in the presence of RGS and agonist divided by the GTPase rate with agonist alone) of Galpha i3 was as high or higher than that of Galpha o in response to Gbeta 5/RGS6, -7, -9, and -11 stimulation. Further, the effects of R7 proteins on GTPase activity of Galpha o-subunits reconstituted with purified P2Y12 receptors was also determined (in the presence of the agonist 2-methylthio ADP). Similar to the results observed with M2 receptor-coupled G-proteins, each of the Gbeta 5/RGS11 dimers stimulated steady state GTPase activity of Galpha o, and Gbeta 5/RGS11 stimulated much higher GTPase rates than the other R7 proteins (data not shown).


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Fig. 3.   Gbeta 5/R7 proteins stimulate GTPase activity of Gi family Galpha -subunits. The effects of 100 µM carbachol and 1 µM of the indicated RGS protein on the steady state GTPase activity of Galpha o, Galpha i1, Galpha i2, and Galpha i3 reconstituted in phospholipid vesicles with the M2 muscarinic receptor and Gbeta 1gamma 2 were determined as described under "Experimental Procedures." Open bars, no agonist. Shaded bars, 100 µM carbachol. Results shown are representative of at least three separate experiments using three independent vesicle preparations.


                              
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Table I
GTPase rates of Gi family Galpha -subunits in the presence of RGS4 and Gbeta 5/R7 proteins
GTPase rates (min-1) attained in the presence of 100 µM carbachol and 1 µM RGS protein are shown with mean ± S.D.

Gbeta 5/R7 Proteins Do Not Stimulate Steady State GTPase Activity of Gq Family Galpha -subunits-- Regulation of GTPase activities of Galpha q and Galpha 11 was examined in vesicles reconstituted with M1 muscarinic receptor and heterotrimeric G-proteins (Fig. 4). RGS4 increased steady state GTPase activity of Galpha q and Galpha 11 in the presence of agonist by nearly 5-fold to final GTPase rates of ~200 fmol of GTP/min/pmol of Galpha . Consistent with previous observations of guanine nucleotide exchange/GTPase kinetics of Gq (29), these rates are lower than those observed for Gi family alpha -subunits. In contrast to the activity of RGS4, none of the Gbeta 5/R7 dimers significantly increased steady state GTPase activity of Galpha q or Galpha 11 in the presence of carbachol (Fig. 4A). Likewise, Gbeta 5/R7 dimers did not stimulate GTPase activity of Galpha q- or Galpha 11-subunits reconstituted with purified P2Y1 receptors (in the presence of the agonist 2-methylthio ADP) (data not shown). Further, 1 µM Gbeta 5/R7 dimers had no effect on the GTPase activity of M1 receptor-coupled Galpha q and Galpha 11 stimulated by RGS4 and carbachol (Fig. 4B). The partial inhibition observed with Gbeta 5/RGS11 was nonspecific as demonstrated by equivalent inhibitory activity observed with boiled Gbeta 5/RGS11. Therefore, under the conditions of these assays, R7 proteins neither stimulate GTPase activity of Galpha q or Galpha 11 nor affect GTPase activity stimulated by agonist and RGS4.


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Fig. 4.   Gbeta 5/R7 proteins do not stimulate GTPase activity of Gq family Galpha -subunits. A, the effects of 100 µM carbachol and 1 µM of the indicated RGS protein on the steady state GTPase activity of Galpha q and Galpha 11 reconstituted in phospholipid vesicles with the M1 muscarinic receptor and Gbeta 1gamma 2 were determined as described under "Experimental Procedures." Open bars, no agonist. Shaded bars, 100 µM carbachol. Results shown are representative of three separate experiments using three independent vesicle preparations. B, the effects of 1 µM Gbeta 5/RGS proteins on agonist-stimulated steady state GTPase activity of M1 receptor-coupled Galpha q and Galpha 11 were determined in the presence of 100 nM RGS4. The effects of heat inactivated Gbeta 5/RGS11 were also determined. The results are presented as a percentage of the agonist-stimulated activity.

Gbeta 5/R7 Proteins Exhibit Differences in Maximal Activity and Potency toward Gi Family Galpha -subunits-- To more fully elucidate any selectivity of Gbeta 5/R7 proteins as GAPs for Gi family subunits, full concentration effect curves of each Gbeta 5/R7 protein were generated in the presence of a maximally effective concentration of carbachol. Consistent with the data in Fig. 3, maximally effective concentrations of Gbeta 5/RGS11 produced larger effects than Gbeta 5/RGS7, Gbeta 5/RGS6, and Gbeta 5/RGS9 on the GTPase activity of each Galpha -subunit (Fig. 5 and data not shown), and the highest maximal rate observed with each RGS protein was observed with Galpha o as substrate (not shown). Each of the Gbeta 5/RGS dimers produced a near maximal effect at a concentration of 1 µM, and therefore each activation curve was normalized to 100% of maximal activity for comparison of EC50 values (Fig. 6). All four Gbeta 5/R7 dimers exhibited similar potency for Galpha o (EC50 = 16-47 nM), while Gbeta 5/RGS9 and Gbeta 5/RGS11 were more potent (EC50 = 25-80 nM) than Gbeta 5/RGS6 and Gbeta 5/RGS7 (EC50 = 150-350 nM) for Galpha i1, Galpha i2, and Galpha i3 (Table II).


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Fig. 5.   Gbeta 5/R7 proteins stimulate different maximal GTPase activity. Steady state GTPase rates were determined for M2·Galpha o·Gbeta 1gamma 2 vesicles in the presence of 100 µM carbachol and various concentrations of R7 proteins to generate concentration effect curves. Values observed at each RGS concentration in the absence of vesicles have been subtracted from the data. Data are plotted as GTPase rates (min-1). Symbols are Gbeta 5/RGS11 (black-diamond ), Gbeta 5/RGS9 (), Gbeta 5/RGS7 (black-down-triangle ), and Gbeta 5/RGS6 (black-square). Results are representative of at least three independent determinations using three separate vesicle preparations.


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Fig. 6.   Gbeta 5/R7 proteins exhibit differences in potency toward Gi family Galpha -subunits. Steady state GTPase rates were determined for M2·Galpha i/o·Gbeta 1gamma 2 vesicles in the presence of 100 µM carbachol and various concentrations of R7 proteins to generate concentration effect curves. Data are normalized to 100% activity for comparison of EC50 values. Symbols are Gbeta 5/RGS11 (black-diamond ), Gbeta 5/RGS9 (), Gbeta 5/RGS7 (black-down-triangle ), and Gbeta 5/RGS6 (black-square). Results are representative of at least three independent determinations using three separate vesicle preparations.


                              
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Table II
EC50 values Gbeta 5/R7 proteins for stimulation of GTPase activity of Gi family Galpha -subunits
The mean ± S.D. of EC50 values (nM) are shown from three independent determinations of the concentration effect relationship of the R7 proteins as GAPs of Gi family Galpha -subunits using three different vesicle preparations.

Gbeta 5/RGS7 and Gbeta 5/RGS9 Inhibit Gbeta 5/RGS11-stimulated Galpha o GTPase Activity-- Marked differences in the maximal GTPase rate of Galpha -subunits were observed across the Gbeta 5/R7 protein family. For example, Gbeta 5/RGS11-stimulated GTPase activity of Galpha o was twice that achieved in the presence of Gbeta 5/RGS9 or Gbeta 5/RGS7. These results suggest that Gbeta 5/RGS7 and Gbeta 5/RGS9 interaction with G-proteins results in a less active Galpha conformation with respect to GTPase activity than that promoted by Gbeta 5/RGS11 interaction. To test this hypothesis, steady state GTPase activity of M2·Galpha o·beta 1gamma 2 vesicles was measured in the presence of 100 nM Gbeta 5/RGS11 or 1 µM Gbeta 5/RGS7 alone or with 100 nM Gbeta 5/RGS11 plus 1 µM Gbeta 5/RGS7. As illustrated in Fig. 7A, GTPase activity in the presence of Gbeta 5/RGS11 was nearly twice that observed with a 10-fold higher concentration of Gbeta 5/RGS7. However, the combined presence of 1 µM Gbeta 5/RGS7 and 100 nM Gbeta 5/RGS11 resulted in activity only slightly greater than that of Gbeta 5/RGS7 alone, suggesting that Gbeta 5/RGS7 competitively antagonizes the action of the more efficacious Gbeta 5/RGS11. Heat-inactivated Gbeta 5/RGS7 neither stimulated GTPase activity nor inhibited the stimulatory effect of Gbeta 5/RGS11 on GTPase activity (Fig. 7A), demonstrating that both effects of Gbeta 5/RGS7 are dependent on protein activity. To further characterize the interaction of RGS proteins and Galpha o, the concentration dependence of the inhibitory effect of Gbeta 5/RGS7 and Gbeta 5/RGS9 was determined by varying the concentrations of these proteins in the presence of carbachol and 100 nM Gbeta 5/RGS11. Both Gbeta 5/RGS7 and Gbeta 5/RGS9 significantly inhibited Gbeta 5/RGS11-stimulated GTPase activity of M2·Galpha o vesicles with IC50 values of 100-200 nM (Fig. 7B). These data indicate that while Gbeta 5/RGS7 and Gbeta 5/RGS9 are less efficacious activators of GTPase activity, they interact with a similar region of Galpha o as does Gbeta 5/RGS11.


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Fig. 7.   Gbeta 5/RGS7 and Gbeta 5/RGS9 inhibit Gbeta 5/RGS11-stimulated GTPase activity of Galpha o. A, steady state GTPase activity of M2·Galpha o·Gbeta 1gamma 2 vesicles was determined in the presence of 100 µM carbachol alone or in the presence of Gbeta 5/R7 proteins. The effects of 100 nM Gbeta 5/RGS11, 1 µM Gbeta 5/RGS7, and heat-inactivated Gbeta 5/RGS7 (1 µM) were assayed separately and in combination. Results are representative of two independent experiments with two RGS11 preparations. B, Gbeta 5/R11-stimulated steady state GTPase activity of Galpha o was determined at various concentrations of added Gbeta 5/RGS7 and Gbeta 5/RGS9.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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The results of this study demonstrate Galpha i/o specificity of Gbeta 5/R7 proteins and found no evidence of regulation of Galpha q GTPase activity by these proteins. Further, we demonstrated that differences exist in the potencies and relative efficacies of the Gbeta 5/R7 proteins for their Galpha i/o substrates. Finally, we illustrated that Gbeta 5/RGS7 and Gbeta 5/RGS9, which are less effective promoters of maximal GTPase activity than is Gbeta 5/RGS11, inhibit Gbeta 5/RGS11-stimulated GTPase activity of Galpha o.

The drug selectivity of G-protein-coupled receptors has been widely exploited therapeutically to manipulate specific cellular processes (30-34). Similarly, selectivities of G-proteins for effector activation and potential selectivities of RGS proteins for deactivation of G-proteins may provide equally rich targets for pharmacological modulation of G-protein-regulated signaling (35, 36). Whereas the role of receptor activity and selectivity in regulating various classes of G-proteins has been studied extensively in the past decades, the roles of proteins exhibiting Galpha GAP activity and selectivities within this class of proteins remain undefined.

Members of the Gbeta 5/R7 family previously were reported to be specific for Galpha o in single turnover assays of soluble Galpha -subunits (11, 20). These assays may not accurately represent physiological interactions between G-proteins and RGS proteins for several reasons including the lack of a lipid bilayer with which G-proteins and RGS proteins may associate, the lack of a GPCR, which may form a complex with RGS proteins (37-39) and facilitate interaction with Galpha -subunits, and the necessity of using GTPase-deficient mutants of Gq family G-proteins given their low rates of exchange. These limitations may explain discrepancies between selectivities for G-protein alpha -subunits observed in single turnover versus cell-based assays. Indeed, RGS2 behaves as a Galpha q-specific GAP in single turnover assays, but exhibits GAP activity toward Galpha i as well as Galpha q in steady state GTPase assays of proteoliposomes reconstituted with GPCR and heterotrimeric G-proteins (28).

In this study, we examined the GAP activity of Gbeta 5/R7 proteins using steady state GTPase assays of receptor-coupled G-proteins reconstituted in phospholipid bilayers. Because these assays measure multiple rounds of hydrolysis in the presence of receptor-stimulated guanine nucleotide exchange, wild type Galpha q or Galpha 11 may be used, and the contributions made by agonist, receptor, Gbeta gamma -subunits, and the phospholipid bilayer to GTPase activity are likely more representative of a cellular environment. Our results differ from those from single turnover assays (11, 20) with respect to the selectivity within the Galpha i/o family since all four Galpha -subunits of this family are substrates for Gbeta 5/R7 proteins.

Our results also differ from published reports that indirectly suggest that Gbeta 5/R7 proteins stimulate GTPase activity of Galpha q or Galpha 11 (14, 21, 22) in that we did not observe stimulation of Galpha q or Galpha 11 GTPase activity in response to R7 proteins. A trivial explanation for our observation is that R7 proteins inhibit agonist promoted exchange and thereby mask GAP activity in steady state GTPase assays. However, the lack of an effect of R7 proteins on the steady state GTPase activity achieved in the presence of carbachol and RGS4 demonstrates that Gbeta 5/R7 heterodimers do not significantly affect agonist-promoted exchange of guanine nucleotides under the conditions of these assays (Fig. 4B). We also observed minimal to no effects of Gbeta 5/R7 proteins on agonist-stimulated guanine nucleotide exchange measured directly in GTPgamma S binding assays (not shown). Therefore, the inability of Gbeta 5/R7 proteins to stimulate steady state GTPase activity of Galpha q and Galpha 11 indicates that they do not function as Gq family GAPs under the conditions of our assay. In the absence of GAP activity, the reported effects of RGS7 on Gq family G-protein signaling could reflect direct inhibition of phospholipase enzymes, as observed by Posner et al. (20). Although we have observed some inhibition of receptor-stimulated inositol phosphate accumulation in cells cotransfected with R7 RGS proteins and Gbeta 5, this inhibition is less pronounced and requires expression to much higher levels than does the marked inhibition of phospholipase C response observed in cells overexpressing RGS2 or RGS4 (data not shown). Thus, the reported effects of R7 proteins on cellular Gq pathways may reflect either loss of GAP selectivity due to protein overexpression or a more complex interaction of Gbeta 5/R7 dimers with the G-protein signaling cycle.

Gbeta 5/R7 proteins exhibited differences in the potency and efficacy of their GAP activity against the Galpha i/o family. Gbeta 5/RGS6 and Gbeta 5/RGS7 each exhibited 10-fold lower potency for Galpha i alpha -subunits than for Galpha o. In contrast, Gbeta 5/RGS9 and Gbeta 5/RGS11 exhibited similar potency for all four Gi family Galpha -subunits. This pattern mirrors the grouping of R7 proteins by sequence similarity; that is, RGS6 and RGS7 have higher sequence identity to each other than to RGS9 and RGS11 and vice versa (13). The R7 proteins group differently with respect to their apparent efficacies for stimulation of GTPase activity. Gbeta 5/RGS11 exhibited the highest maximal effect, while the maximal effects of Gbeta 5/RGS7 and Gbeta 5/RGS9 were much less and Gbeta 5/RGS6 exhibited an intermediate maximal effect. These differences in activity inversely correlate with the expression of R7 transcripts in rat brain where RGS11 is expressed at much lower levels than RGS7 and -9, and again RGS6 is intermediate (42). We speculate that expression of the robustly active RGS11 may be regulated differently than the less active proteins.

Galpha i1, Galpha i2, and Galpha i3 share high sequence homology, and these signaling proteins are essentially interchangeable in many signaling processes. However, although these proteins often are expressed in the same cell, they may not be entirely functionally redundant. For example, selectivity of coupling of certain G protein-coupled receptors among these three Galpha i proteins has been illustrated (43-46), and several reports suggest selective coupling of receptors to ion channels through specific Galpha i-subunits (for review see Ref. 47). Our observation of selectivity of action of beta 5/RGS proteins among Galpha i1, Galpha i2, Galpha i3, and Galpha i/o likely has physiologically important ramifications. For example, this family of RGS proteins is highly expressed in the central nervous system as are Gi family Galpha -subunits. Given the broadly different patterns of expression of RGS6, RGS7, RGS9, and RGS11 in the brain (42), we hypothesize that otherwise functionally redundant Galpha i-subunits may exhibit cell-specific differences in signaling activities as a consequence of the presence of different RGS proteins.

The differences observed in the maximal GAP activity of R7 family RGS proteins may reflect differences in their interactions with Galpha . Unlike GAP proteins for the Ras superfamily GTPases, which contribute a catalytic arginine required for GTPase activity, RGS proteins are considered to enhance GTPase activity solely by stabilizing Galpha switch regions (48). Presumably, R7 RGS proteins bind Galpha -subunits through interactions similar to those observed in the RGS4/Galpha i (48) and the RGS9/Galpha t/i1 crystal structures (49), in which the base of a 4-helix bundle (alpha 4, alpha 5, alpha 6, alpha 7) of the RGS domain directly contacts portions of the three switch regions of Galpha . Martemyanov and Arshavsky recently reported that mutation of RGS9 residues in the base of Galpha -interacting helices alpha 5 and alpha 6 (L353E/R360P) resulted in markedly higher maximal GAP activity toward Galpha transducin than observed in wild type RGS9, consistent with a role for this region in determining maximal GAP activity (50). It will be equally important to determine if regions outside the RGS box play important roles in defining selectivities of beta 5-RGS proteins among Galpha -subunits as suggested in studies of Galpha selectivity of C. elegans R7 family members (40, 41).

In summary, we have demonstrated that R7 family RGS proteins selectively stimulate GTPase activity of Gi family Galpha -subunits. We have shown differences in potency and efficacy of Gbeta 5/R7 dimers as GAPs among the Gi family Galpha -subunits. Further, lower efficacy GAPs were shown to inhibit GTPase activity achieved in the presence of a more efficacious GAP, indicating that RGS proteins apparently interacting with the same activating surface of a Galpha -subunit promote different maximal rates of catalysis by the Galpha GTPase.

    ACKNOWLEDGEMENTS

The authors would like to thank Dr. Alfred Gilman for input and support and Drs. David Siderovski and John Sondek for thoughtful suggestions during preparation of the manuscript.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants GM29536, GM65533, and GM34497.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by postdoctoral National Research Service Award GM66561 from the United States Public Health Service. To whom correspondence should be addressed: Department of Pharmacology, Campus Box 7365, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5356; Fax: 919-966-5640; E-mail: shelleyb@med.unc.edu.

Supported by a postdoctoral fellowship from the Pharma Foundation.

Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M211382200

    ABBREVIATIONS

The abbreviations used are: GPCR, G-protein coupled receptor; RGS, regulator of G-protein signaling; GAP, GTPase activating protein; GGL, G-gamma like; m.o.i., multiplicity of infection; Ni-NTA, nickel-nitrilotriacetic acid; C12E10, polyoxyethylene 10-lauryl ether.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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