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J Biol Chem, Vol. 274, Issue 43, 31087-31093, October 22, 1999

Regulators of G Protein Signaling 6 and 7
PURIFICATION OF COMPLEXES WITH G5 AND ASSESSMENT OF THEIR EFFECTS ON G PROTEIN-MEDIATED SIGNALING PATHWAYS^*

Bruce A. Posner, Alfred G. Gilman^§, and Bruce A. Harris^¶

From the  Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and ^¶ Hoechst Marion Roussel, Inc., Bridgewater, New Jersey 08807

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein  subunits, and certain biochemical properties of RGS6/5 and RGS7/5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the G5 subunit (but not Gs 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/5 in that they act as GTPase-activating proteins specifically toward G_o. Unlike conventional G complexes, RGS6/5 and RGS7/5 do not form heterotrimeric complexes with either G_o-GDP or G_q-GDP. Neither RGS6/5 nor RGS7/5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-1 or -2. However, the RGS/5 complexes inhibited ₁₂-mediated activation of phospholipase C-2. RGS/5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G_i and G_o by binding to phospholipase C and stimulating the GTPase activity of G_o.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Initial studies of proteins that belong to the regulators of G protein signaling (RGS)¹ family demonstrated that they are GTPase-activating proteins (GAPs) that accelerate hydrolysis of GTP bound to the  subunits of certain heterotrimeric G proteins (1-3). As such, RGS proteins can function as negative regulators of G protein-mediated signal transduction by speeding deactivation of the active form of G subunits, thereby promoting formation of inactive G protein heterotrimers (G_GDP). It is now clear that some proteins that contain an RGS domain have more complex functions. For example, p115 RhoGEF, which contains an RGS domain near its amino terminus, is an effector for G protein action, catalyzing guanine nucleotide exchange on the monomeric G protein Rho more efficiently when stimulated by G₁₃-GTP. The capacity of G₁₃ to activate p115 RhoGEF is dependent on the RGS domain of p115, which also accelerates hydrolysis of GTP by G₁₃ (4, 5). p115 RhoGEF thus resembles phospholipase C, a well characterized effector for G protein action that also deactivates its regulator (G_q) by acting as a GAP (6-8).

A subfamily of RGS proteins has been identified in which each member possesses so-called DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein subunit-like) domains in addition to an RGS domain (9, 10). Members of this group include mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11), a Drosophila RGS protein (dRGS7), and EGL-10, an RGS protein found in Caenorhabditis elegans (10-12) Functionally, the GGL domain was shown to specify binding of RGS11 or a fragment of RGS7 to the G protein 5 subunit (10). It has also been shown that both RGS7 and RGS9 can be isolated from brain and retina as a complex with G5 (13-15)² and that the distribution of mRNA for the GGL-containing RGS proteins and G5 overlap in these tissues (10, 11, 16-20). In addition, Snow and co-workers (10) demonstrated that the RGS11/5 complex accelerates hydrolysis of GTP bound to the  subunit of the G protein G_o. The functional significance of the DEP domains of these proteins remains unknown. The mammalian members of this subfamily of RGS proteins are found predominantly in the central nervous system (10, 11, 16, 17), and to date, their role in G protein-mediated signal transduction is unknown.

The superficial resemblance of the complex formed by G5 and a GGL-containing RGS protein to a G protein subunit complex suggests potential targets for modulation of G protein signaling. G protein subunits regulate the activity of a diverse array of effectors (adenylyl cyclases, ion channels, and phospholipase C- isoforms, among others), play a significant role in interactions of G proteins and G protein-coupled receptor kinases with receptors, and facilitate signal transfer from G protein-coupled receptors to mitogen-activated protein kinase cascades (21). However, relatively little is known about the signaling properties of G5. Of the five known G subunits, G5 is the clear outlier. Although Gs 1-4 are 80-90% identical to each other, G5 is only about 50% homologous to its relatives (18). Functionally, the recombinant 52 dimer appears capable of stimulating the phospholipase activity of PLC-2 (18, 22, 23) and coupling G_q with ET_B or m1 muscarinic receptors in crude reconstituted systems (23). However, this complex was unable to stimulate either type II adenylyl cyclase in vitro (23) or the mitogen-activated protein kinase pathway in COS cells (22).

We describe herein the cloning of RGS6 and explore the biochemical properties of RGS6 and RGS7, the most closely related member of this group of RGS proteins. We demonstrate that both RGS6 and RGS7 associate strongly and specifically with G5. These RGS/5 complexes have been purified following expression directed by recombinant baculoviruses and tested for interactions with certain recombinant G protein  subunits and known effectors for G protein action.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³⁵S]GTPS and [-³²P]GTP were obtained from NEN Life Science Products. Spodoptera frugiperda (Sf9) cells were maintained and recombinant baculoviruses were amplified as described previously (24). Baculoviruses encoding amino-terminally hexahistidine-tagged G1 and 5 have been described previously (10, 25). Viruses encoding hexahistidine-tagged G3 and 4 were prepared similarly. G_q, G_z, G₁₂, and p115 RhoGEF were generously provided by Dr. Tohru Kozasa (University of Texas Southwestern Medical Center); PLC-1 and PLC-2 by Dr. Paul Sternweis (University of Texas Southwestern Medical Center); antisera against RGS6 (U1895) and RGS7 (U1480) by Dr. Andrejs Krumins (University of Texas Southwestern Medical Center); antiserum against G5 (SGS-1) by Dr. William Simonds (National Institutes of Health); an antiserum that reacts with G1-4 subunits (B600) by Dr. Susanne Mumby (University of Texas Southwestern Medical Center); and a recombinant baculovirus encoding RGS7 by Dr. Sheu-Fen Lee (University of Texas Southwestern Medical Center). Synthetic oligonucleotides were obtained from commercial sources or prepared by the Pharmacology Core Facility of the University of Texas Southwestern Medical Center.

Cloning of Human RGS6 cDNA-- A partial rat RGS6 cDNA sequence (GenBank: RNU32436), identified by homology to the C. elegans EGL-10 protein, was used with the BLAST algorithm to identify a human clone (GenBank: HUMORFE) containing sequences homologous to the consensus RGS domain found in all members of the RGS protein family. Further BLAST alignments with a human expressed sequence tag data base identified g874443 as identical to HUMORFE except with 11 additional nucleotides and an EcoRI site at its 5' end. This incomplete RGS6 cDNA clone was obtained from the Lawrence Livermore National Laboratory collection of expressed sequence tags through Genome Systems (St. Louis, MO), and sequences derived from its 5' region were used to prepare oligonucleotide primers for 5'-RACE (rapid amplification of cDNA ends) cloning of the missing DNA sequences.

Marathon-Ready^TM (CLONTECH Laboratories, Palo Alto, CA) cDNA from human brain was used for 5'-RACE. Oligonucleotide primer B413 (5'-CCACATCCTGTAGGGGTTGTTTC-3') derived from RGS6 sequence downstream of the EcoRI site at nucleotide position 1117 (Fig. 1) was used with oligonucleotide primer AP1 (supplied by CLONTECH) to produce double-stranded cDNA according to a polymerase chain reaction protocol provided by the supplier. Following removal of oligonucleotide primers by gel filtration, the enriched cDNA was used to prepare a specific 1153-base pair human RGS6 5'-RACE product using primers B413 and B415 (5'-TCTAGACTGCAGGATGGCTCAAGGATCCGGGGATC-3'). The latter primer was derived from a human RGS6 cDNA sequence obtained from GenBank with the accession number AF073920. The human RGS6 5'-RACE cDNA was joined to the g874443 partial RGS6 cDNA at their common EcoRI restriction site at nucleotide position 1117 by a sequential polymerase chain reaction protocol. The amplified cDNA from this procedure (1730 base pairs) was ultimately cloned into baculovirus transfer vector pFastBac HTb for preparation of a human RGS6 protein with a hexahistidine tag near its amino terminus.

Construction of a Truncated RGS6 cDNA-- A modified RGS6 cDNA lacking the sequences coding for the consensus RGS box domain was constructed using the polymerase chain reaction, and the amplified product (997 base pairs) was cloned into pFastBac HTb. The truncated RGS6 protein (RGS6R, 41.4 kDa) includes amino acid residues 1-324 of human RGS6 in addition to 25 residues of amino-terminal sequences containing the hexahistidine tag and 8 residues of carboxyl-terminal sequence derived from the pFastBac HTb vector.

Interaction of RGS/G5 Complexes with G-GDP Proteins-- The ability of purified, myristoylated G_o and G_q to form heterotrimeric complexes with 12 and RGS6 or 7/G5 complexes was assessed as follows. To 100 µl of buffer A (20 mM Na Hepes (pH 8), 20 mM -mercaptoethanol, 2 mM MgSO₄, 2 µM GDP, 100 mM NaCl, and 0.005% C₁₂E₁₀) was added 10 pmol of myristoylated G_o or 10 pmol of G_q and 25 pmol of hexahistidine-tagged RGS6/5, RGS6R/5, RGS7/5, or 12. Following a 30-min incubation on ice, 25 µl of Ni-NTA resin (Qiagen) equilibrated in buffer A was added and incubated for an additional 30 min. The Ni-NTA resin was collected by centrifugation and washed three times with 150 µl of buffer B (buffer A supplemented with 400 mM NaCl and 10 mM imidazole). Proteins bound to the Ni-NTA resin were eluted with 50 µl of buffer C (buffer A supplemented with 150 mM imidazole). The identity of proteins eluted from Ni-NTA resin was determined by immunoblotting after separation through 11% sodium dodecyl sulfate-polyacrylamide gels.

Protein Purification-- The hexahistadine-tagged (His₆) RGS6/5 and RGS7/(His₆)5 complexes were synthesized in Sf9 cells using a recombinant baculovirus expression system. Sf9 cells (6 liters) were grown in suspension to a density of 1.4 × 10⁶ cells/ml in complete medium (IPL-41 medium (Life Technologies, Inc.) supplemented with 1% heat-inactivated fetal calf serum, 1% Pluronic F68, and 10 µg/ml gentamicin; Ref. 24) and then infected with 1 plaque-forming unit of each virus per cell. The cells were harvested 48 h later by centrifugation, suspended to a density of 2.5 × 10⁷ cells/ml in lysis buffer (50 mM NaHepes (pH 8), 10 mM -mercaptoethanol, 50 mM NaCl, and protease inhibitors (0.02 mg/ml phenylmethylsulfonyl fluoride, 0.03 mg/ml leupeptin, 0.02 mg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, 0.02 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone, and 0.03 mg/ml lima bean trypsin inhibitor)) and subjected to nitrogen cavitation as described previously (26). The cell lysates were clarified by centrifugation at 100,000 × g for 30 min at 4 °C and applied to Ni-NTA resin (5 ml; Qiagen, Chatsworth CA) equilibrated with buffer D (20 mM NaHepes (pH 8), 10 mM -mercaptoethanol, 10% glycerol, and protease inhibitors). The resin was washed with 80 ml of buffer E (buffer D plus 400 mM NaCl), 80 ml of buffer F (buffer D plus 150 mM NaCl and 5 mM imidazole), and 50 ml of buffer G (buffer D plus 50 mM NaCl), and the RGS/5 complex was then eluted with buffer H (buffer D plus 100 mM NaCl and 150 mM imidazole) in the case of (His₆)RGS6/5 and (His₆)RGS6R/5 or buffer I (buffer D plus 50 mM NaCl and 50 mM EDTA) in the case of RGS7/(His₆)5. The Ni-NTA pool was diluted 5-fold into Mono Q buffer (50 mM Tris-HCl (pH 8), 2 mM dithiothreitol, protease inhibitors) and applied to a Mono Q 10/10 column (Amersham Pharmacia Biotech) at a flow rate of 1 ml/min. Protein was eluted from the column using a linear gradient of increasing ionic strength (0-400 mM NaCl in Mono Q buffer). The RGS/5 complexes eluted from the column between 180 and 200 mM NaCl. The purified complexes were exchanged into storage buffer (50 mM NaHepes (pH 8), 5 mM dithiothreitol, and 10% glycerol), flash-frozen in liquid nitrogen, and stored at 80 °C.

Interaction of RGS6 and RGS7 with G Subunits-- Cultures of Sf9 cells (50 ml, 1.0 × 10⁶ cells/ml) were infected with RGS6 or RGS7 baculoviruses in addition to baculoviruses encoding one of each of the five G subunits. In these experiments, the G proteins were synthesized with a His₆ tag at their amino termini; the RGS proteins were not tagged. Infections were carried out at a multiplicity of infection of 1 for each virus. Infected cells were harvested by centrifugation, suspended to a density of 2.5 × 10⁷ cells/ml in lysis buffer, subjected to nitrogen cavitation, and clarified by centrifugation as described above. Supernatant fractions were applied to Ni-NTA resin in small columns. The resin was washed with 10 column volumes each of buffer D, buffer E, and buffer J (buffer A plus 150 mM NaCl and 20 mM imidazole). Each sample was eluted with buffer H. Column fractions were analyzed by immunoblotting using the following antibodies: U1895 (anti-RGS6 diluted 1:10,000), U1480 (anti-RGS7 diluted 1:4000), SGS-1 (anti-5 diluted 1:2000), and B600 (anti-G1-4 diluted 1:10,000). Particulate fractions of the cells were washed once with lysis buffer and then resuspended in lysis buffer, flash-frozen in liquid nitrogen, and stored at 80 °C.

Hydrolysis of Bound GTP-- Hydrolysis of G-bound [-³²P]GTP in solution was monitored at 4 °C essentially as described by Berman et al. (27). To prepare GTP-bound substrate with G_i1, G_i2, G_i3, and G_z, G_a proteins (10-500 nM) were incubated with 10-30 µM [-³²P]GTP (700-25000 cpm/pmol), 50 mM NaHepes (pH 8), 5 mM EDTA, 3 mM dithiothreitol, and 0.05% C₁₂E₁₀ for 30 min at 30 °C prior to passage through a gel filtration column at 4 °C to remove excess [-³²P]GTP and [³²P]P_i (G-50 Sephadex; Amersham Pharmacia Biotech). G_s-GTP and G_o-GTP were prepared similarly except that they were incubated at 20 °C for 20 min. Assays were initiated by addition of excess unlabeled GTP, MgSO₄, and RGS protein (purified protein or crude Sf9 cell membranes) to G substrate at 4 °C. The final concentrations of components in the assay were 50 mM NaHepes (pH 8.0), 5 mM EDTA, 200 µM GTP, 3 mM dithiothreitol, 10 mM MgSO₄, and 0.05% C₁₂E₁₀. Reactions were quenched with neutral charcoal (Norit A) at appropriate time intervals. Assays with R183C G_q and G₁₂ were performed as described previously (5, 28).

Miscellaneous Procedures-- G subunits were purified from Escherichia coli using the methods of Lee et al. (29). Hexahistidine-tagged RGS4 was purified from E. coli as described by Berman et al. (27). R183C G_q was prepared from Sf9 cells as described previously (28), as was G_o-GTPS (30). Measurements of phospholipase C activity and adenylyl cyclase activity were conducted according to published protocols (31, 32).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of Human RGS6 cDNA-- A full-length coding sequence for RGS6 was obtained by joining a human expressed sequence tag clone (g874443) encoding all of the 3' region to a primer-extended and polymerase chain reaction-amplified cDNA encoding the complete 5' region of RGS6 (Fig. 1). The full-length clone contains 2946 nucleotides and has an open reading frame encoding a protein of 472 amino acid residues with a deduced molecular weight of 54,422. 

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Cloning of human RGS6 cDNA and comparison with murine RGS7. A, clone g874443 (open rectangle) containing human 3' RGS 6 cDNA sequences was fused to a 1143-base pair polymerase chain reaction product obtained from sequential amplification of human brain cDNA with primers B413 and AP1 followed by B413 and B415 (gray rectangle). The complete RGS6 open reading frame was cloned into pFastBac1 and pFastBac Htb for expression in Sf9 cells. B, the deduced amino acid sequence of human RGS6 (GenBank accession no. AF156932, upper sequence) is compared with that of murine RGS7 (lower sequence). Areas of sequence identity are boxed. Open bar, DEP domain; gray bar, GGL domain; black bar, RGS domain. Amino acids conserved among other RGS proteins that contact G protein  subunits are indicated with an asterisk.

There are three regions of particular interest in the RGS6 protein. The DEP domain comprises amino acid residues 42-121. It is a highly conserved hydrophobic domain found in RGS6, 7, 9, and 11, in addition to a wide variety of other signaling proteins (9). The second region of interest is the GGL domain, defined by Snow et al. (10) as a domain that contains the required elements for interaction with G5. Within the full-length RGS6 protein, the GGL domain is found between amino acid residues 261 and 309 (Fig. 1). The third recognizable motif, the RGS domain, is found between amino acid residues 325 and 451 of RGS6. A series of critical amino acid residues in the RGS domain that contact G was identified from the crystal structure of the complex formed between RGS4 and G_i1 (33). Although there is a substantial tolerance for amino acid substitutions within the consensus RGS domain of the family, there is an expected conservation of those residues that are critical for the direct interaction with the switch domains of the G subunit (at least for those RGS proteins that interact with members of the G_i subfamily). Within RGS6, those critical residues are: Glu³⁵⁷-Phe-Ser³⁵⁹, Glu³⁶¹-Asn³⁶², Asn⁴⁰¹, Asp⁴⁰³, Leu⁴³², Asp⁴³⁶-Ser⁴³⁷ and Arg⁴⁴⁰. Based on the conservation of these critical residues, we predicted that RGS6 would function as a GTPase-activating protein.

Association of RGS6 or RGS7 with G Protein  Subunits-- In a previous study with RGS11 (10), Snow and co-workers demonstrated that full-length RGS11 or a fragment of RGS7 formed specific and high affinity complexes with G5. This association required the GGL domain present in RGS11 and RGS7; this domain can also be recognized in RGS6 and RGS9. Although several studies have now shown that RGS7 and RGS9 form heterodimers with G5 (10, 13-15), the specificity of this interaction with G5 has yet to be generalized for the entire subfamily of RGS proteins.

We have addressed this question for full-length RGS6 and RGS7 with a recombinant baculovirus expression system. In these experiments, Sf9 cells were infected with an RGS virus either alone or in combination with one of five viruses encoding known G subunits (Fig. 2). When expressed alone or with an RGS protein, significant amounts of G5 were found in the supernatant (cytosolic fraction) produced by high speed centrifugation of cell lysates (Fig. 2 and data not shown). With longer exposure times, it was possible to detect the presence of the other  subunits in the cytosolic fraction (Fig. 2). When expressed alone or with Gs 1-4, RGS6 and RGS7 are found predominantly in the particulate fraction. However, concurrent expression of RGS6 or RGS7 with G5 alters this distribution dramatically, and a significant amount of the RGS protein is found in the cytosolic fraction (Fig. 2).

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Specificity of interaction of RGS6 and RGS7 with G subunits. Sf9 cells were infected with recombinant baculoviruses encoding RGS6 or RGS7 and hexahistidine-tagged G subunits 1-5. Cells were then processed as described under "Experimental Procedures," and immunoblots were analyzed for the presence of RGS6 or RGS7 and Gs 1-5. Upper panel, RGS7. The membrane fraction (M, 3 µg), the cytosolic fraction (C, 20 µg), and the eluted material from the Ni-NTA resin (B, 250 ng) are shown for each experiment. The exposure time for the main immunoblot was 30 s. Longer exposures were necessary to detect Gs 1-4 in the cytosolic fraction and eluted material from the Ni-NTA resin. The lower series of panels represent a region of the blot shown above where RGS7 and Gs 1-4 would be present if detected. The time (in minutes) for each exposure is shown to the left of each panel. Bottom panel, RGS6. The membrane fraction (M, 2 µg), the cytosolic fraction (C, 15 µg), and the eluted material from the Ni-NTA resin (B, 1.5%) are shown for each experiment. The exposure time of the immunoblot shown was 30 s; a longer exposure (5 min) was necessary to detect G4 in the cytosolic fraction and eluted material from the Ni-NTA resin.

Although attempts to purify an RGS/G complex from the cytosol were unsuccessful in experiments with Gs 1-4, both RGS6 and RGS7 co-purified with G5 reproducibly (see below). Gel filtration of crude cytosolic fractions also revealed that only G5 formed a heterodimer with these RGS proteins (data not shown). In summary, these data demonstrate strong and specific association of the mammalian GGL-containing RGS family members with G5.

Purification of the RGS6/5 and RGS7/5 Complexes-- The distribution of the RGS6/5 and RGS7/5 complexes between the soluble and particulate fractions is similar to that observed for G5 in brain homogenates (18). Both complexes can be purified to homogeneity from the soluble fraction (Fig. 3) and remain intact when passed through anion and cation exchange resins. Gel filtration analyses indicate a 1:1 complex of RGS7 with G5 (data not shown). Two protein bands are visualized with the RGS7 antibody and migrate electrophoretically near the expected position for the protein (Fig. 3, left panel). The upper band in these preparations disappears after treatment with phosphoprotein phosphatases (data not shown). RGS6 migrates as a single protein species (Fig. 3, right panel).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Purification of the RGS6/5 and RGS7/5 complexes from Sf9 cell cytosol. Sf9 cells were infected with recombinant baculoviruses and processed as described under "Experimental Procedures." The crude cytosolic fraction (A, 15 µg), the eluate from the Ni-NTA resin (B, 3 µg), and the concentrated pool from the Mono Q column (C, 3 µg) were subjected to SDS-polyacrylamide gel electrophoresis and subsequently stained with Coomassie Brilliant Blue. Left panel, RGS7/(His6)5; right panel, (His6)RGS6/5 and (His6)RGS6R/5.

Efforts to purify these RGS/5 complexes from the particulate fraction have not been successful. This may be due to several factors, including an instability of the complex in the presence of detergent and/or the presence of a large amount of poorly folded or aggregated recombinant protein in the particulate fraction.

Stimulation of G GTPase Activity by RGS6/5 and RGS7/5-- Although GAP activity that is largely specific for G_o-GTP was observed previously with the RGS11/5 complex (10), Levay et al. (14) claim that the nucleotide-dependent interaction of RGS7 with G_o is abolished in the presence of G5. Despite this claim, GAP activity of purified RGS6/5 or RGS7/5 complexes was detected readily. Of interest, both of these complexes showed the same remarkable specificity for G_o observed previously with RGS11/5 (Fig. 4). The GTPase activity of G_o was clearly enhanced by both RGS6/5 and RGS7/5, but the activities of G_i1, G_i2 (data not shown), G_i3 (data not shown), G_z, G_s, G_q, and G₁₂ were not.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   The RGS6/5 and RGS7/5 complexes specifically stimulate the GTPase activity of Go in solution. Each G protein  subunit (A, myristoylated Go, 200 nM; B, myristoylated Gi1, 200 nM; C, Gq(R183C), 4 nM; D, G12, 80 nM; E, Gz, 10 nM; F, Gs, 140 nM) was bound to GTP and assayed as substrates for the potential GAP activity of RGS6/5 (, 200 nM), RGS7/5 (, 200 nM), or (as positive controls) RGS4 (, 40 nM in panels A, B, E, and F; 150 nM in panel C) or p115 RhoGEF (, 200 nM, panel D) as described under "Experimental Procedures." The rate of hydrolysis of GTP in the absence of added RGS protein is also indicated ().

Substrate specificity was not altered when crude membranes containing RGS6/5 or RGS7/5 were tested for GAP activity (data not shown). Interestingly, RGS6 and RGS7 in crude membrane fractions exhibited modest GAP activity toward G_o-GTP in the absence of recombinant G5 (data not shown). Although we cannot rule out the presence of a S. frugiperda ortholog of G5 in these experiments, it is certainly possible (or likely) that association with G5 is not a prerequisite for the GAP activity of these proteins. The GAP activity of membrane-associated RGS7 was consistently greater in the presence of recombinant G5 but was not augmented by coexpression of RGS7 with other G subunits. However, there may be several explanations for this difference, including activation of RGS7 by G5 and/or greater stability or proper folding of RGS7 protein in the presence of G5.

Interactions of RGS6/5 or RGS7/5 with G-GDP-- We have also assessed the capacity of RGS6 or 7/5 complexes to form heterotrimers with G_q or myristoylated G_o proteins (Fig. 5). Purified (His₆)RGS6/5, RGS7/(His₆)5, or a truncated RGS6 protein lacking the RGS domain ((His₆)RGS6R/5) were incubated on ice for 30 min with GDP and purified G proteins. We then attempted to detect heterotrimeric complexes by adsorption to and elution from Ni-NTA resin. Both G_q and myristoylated G_o readily formed stable heterotrimers with 1/(His₆)2. By contrast, heterotrimeric complexes were not detected when RGS6, RGS7, or RGS6R/5 complexes were tested. In at least this sense, these proteins do not appear to function as G protein subunit-like complexes.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Failure of (His6)RGS6/5, (His6)RGS6R/5, and RGS7/(His6)5 to associate with G-GDP. Purified RGS/5 complexes or (as a positive control) 12 and myristoylated Go (top panel) or Gq (bottom panel) were incubated in the presence of 2 µM GDP and then bound to Ni-NTA resin. After washing, bound proteins were eluted from the resin with 150 mM imidazole, resolved through 11% polyacrylamide gels, and identified by immunoblotting as described under "Experimental Procedures." The migration positions of G1, RGS6, RGS6R, RGS7, myristoylated Go, and Gq are indicated. L, sample loaded onto Ni-NTA; F, fraction not bound to Ni-NTA; W, wash; and E, fraction eluted from Ni-NTA resin.

Interactions with Adenylyl Cyclase and Phospholipase C--- Although previous studies have demonstrated several roles for complexes in G protein-mediated signaling (21), the G5 subunit appears to be functionally restricted when compared with Gs 1-4 (18, 22, 23). Although the 52 dimer can stimulate PLC-2, it does not activate type II adenylyl cyclase. 5/2 also associates preferentially with members of the G_q subfamily of G proteins in vitro and may be selectively released by G_q-linked receptors. Nevertheless, G5 forms heterodimers with 3, 4, 5, and 7 subunits, as well as the GGL-containing RGS proteins (10, 18). Thus, the role of G5 in signaling may be quite extensive.

We have considered the potential of the RGS6/5 and RGS7/5 complexes to interact with three isoforms of adenylyl cyclase and two isoforms of PLC-. Several dimers inhibit the G_s-stimulated activity of type I adenylyl cyclase and activate (conditionally with G_s) type II adenylyl cyclase (34); subunits do not appear to interact with type V adenylyl cyclase. Interestingly, neither RGS6/5 nor RGS7/5 was able to modulate the activity of any of these adenylyl cyclases in the presence or absence of activated G_s (Fig. 6). In addition, neither complex was able to interfere with the capacity of 12 to activate type II adenylyl cyclase (data not shown). We considered the possibility that the RGS domain might influence interactions of the complex with effectors. However, neither deletion of the RGS domain of RGS6 nor inclusion of activated G_o in these assays failed to alter the earlier results.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Interaction of 12, RGS6/5, and RGS7/5 with three isoforms of adenylyl cyclase. Assays with the type I, type II, and type V isoforms of adenylyl cyclase were performed as described previously (32). The presence or absence of 12 (100 nM), RGS6/5 (200 nM), RGS7/5 (200 nM), and Go-GTPS (150 nM) is indicated in the figure. The concentration of Gs-GTPS (100 nM) was constant in each assay. RGS6/5 and RGS7/5 also failed to modulate cyclase activity when Gs was omitted from the assay (data not shown).

G protein subunits are also capable of stimulating the activity of selected isoforms of PLC-. PLC-2 can be stimulated up to 20-fold by subunits, while the activity of PLC-1 is relatively insensitive to the dimer (32, 35). However, both RGS/5 complexes failed to influence the activity of PLC-1 or PLC-2 in the presence or absence of activated G_o (Fig. 7, A and B); removal of the RGS domain did not alter these results (Fig. 7B). We did observe a moderate, concentration-dependent inhibition by RGS/5 complexes of the ability of 12 to activate PLC-2 (Fig. 7C). At the highest concentration of RGS/5 tested, -stimulated phospholipase activity was reduced roughly 25% and 40% by RGS6/5 and RGS7/5, respectively. The inclusion of activated G_o did not influence these results (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Interaction of RGS6/5 and RGS7/5 with PLC-1 and PLC-2. The RGS6/5 and RGS7/5 complexes were tested for their ability to activate PLC-1 (A) and PLC-2 (B). The final concentrations of PLC-1 and PLC-2 were 0.13 and 0.2 nM, respectively, while those of Gq-GDP-AlF4 and 12 were both 100 nM. The presence or absence of Go-GTPS (150 nM) or Go-GDP (150 nM) is indicated in the figure. Competition assays (C) were performed with RGS6/5 () or RGS7/5 () in the presence of 12 (100 nM) and PLC-2. The effect of the RGS/5 complexes on PLC-2 activity in the absence of 12 is indicated by the closed symbols.

	DISCUSSION

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We describe herein the cloning of RGS6, the specificity of interaction of RGS6 and RGS7 with the G protein 5 subunit, and biochemical properties of the RGS6/5 and RGS7/5 complexes. Sequence alignments of RGS6 with known RGS proteins reveal high sequence homology to a subfamily of RGS proteins that includes RGS7, dRGS7, RGS9, RGS11, and EGL-10. The members of this subfamily each contain a DEP domain near the amino terminus, a GGL domain roughly in the middle of the protein, and an RGS domain near the carboxyl terminus. The mRNA transcripts of the mammalian members of this subfamily are found primarily in the central nervous system, including the retina (10, 11, 16, 17, 19, 36). Among the subfamily members, RGS7 is most similar in sequence to RGS6. It thus seemed likely that RGS6 and RGS7 would share other important properties. Interestingly, their distribution in the central nervous system is somewhat distinct, which may suggest subtle differences in their roles in G protein-mediated signaling (17).

Both RGS6 and RGS7 can be expressed in Sf9 cells and purified to homogeneity from cytosolic extracts as complexes with G5. Like RGS11 (10), the GAP activities of the RGS6/5 and RGS7/5 complexes in solution (in vitro) appear specific for G_o. Previous studies conducted with the RGS homology domain of RGS7 fused to glutathione S-transferase suggested a broader substrate specificity (G_o, G_i1, G_i3, and G_z) (20, 37, 38). Interaction of full-length RGS7 or RGS6 with these  subunits may be precluded by the presence of the GGL domain, the DEP domain, other elements of the protein, and/or G5.

The GAP activity of RGS6/5 and RGS7/5 is a property of the complexes themselves. We have not been able to dissociate the RGS7/5 complex by incubation with G_o-GDP-AlF₄. For example, RGS7/G_oa-GDP-AlF₄ complexes were not observed when a mixture of RGS7/5 and an 8-fold molar excess of G_o-GDP-AlF₄ were gel-filtered. These observations contradict a previous assertion that G5 prevents the interaction of RGS7 and other GGL-containing RGS proteins with G_o (14). This assertion was based on binding assays that failed to reveal complexes of G_o with RGS7/5; GAP assays were not performed. In addition, the authors were incorrect in their statement that Snow et al. (10) tested only fragments of RGS11 (associated with G5) for GAP activity. Compared with RGS proteins such as RGS4 and GAIP (39), the affinity of the RGS7/5 complex for G_o-GDP-AlF₄ is modest,³ and interaction between these proteins would likely go undetected in assays designed to measure binding rather than catalytic activity. It has also been suggested that RGS7 may be important in Gq-mediated signaling (38). To date, we have failed to detect GAP activity for these RGS/5 complexes with G_q in single turnover assays (Fig. 4) or in steady-state GTPase assays in which the m1 muscarinic cholinergic receptor and heterotrimeric G_q were reconstituted in phospholipid vesicles (data not shown).

The specific and high affinity association of the GGL domains of RGS6, RGS7, and RGS11 with G5 appears to be a general property of this subfamily of RGS proteins. RGS9 has been isolated from rod outer segment membranes as a complex with G5L, a long splice variant of G5 found primarily in retina (18). In addition, RGS9 can also form a complex with G5 when the two genes are coexpressed in Sf9 cells using a recombinant baculovirus system⁴; however, the specificity of the RGS9 GGL domain for G5 has not yet been tested. There is an apparent ortholog of G5 in C. elegans (Q206636), but its association with EGL-10 has not been described (14).

Many of the experiments that we have performed to date have been guided by the hypothesis that RGS/5 complexes may play roles analogous to more conventional G protein subunits. Many of these experiments have proven negative, particularly including those designed to detect interactions between RGS/5 complexes and GDP-bound G protein  subunits. Similarly, these RGS/5 complexes by themselves appear unable to modulate the activities of at least certain effectors for G proteins. However, both complexes are capable of inhibiting stimulation of PLC-2 by ₁₂ (Fig. 7C). Although the magnitude of the inhibition appears modest, the affinity of the interaction may be sufficient to target the RGS domain of these proteins to PLC-2. Deactivation of G_o in the vicinity of PLC-2 by the RGS domain would ensure rapid sequestration of and termination of phosphoinositide-mediated signaling from the relevant subset of receptors. Importantly, these RGS/5 complexes do not inhibit ₁₂ stimulation of type II adenylyl cyclase, and they do not display significant GAP activity (at least in vitro) for the three isoforms of G_i. This constellation of effects raises the possibility of a role of RGS6/5 and RGS7/5 as enforcers of selectivity in signaling initiated from receptors that can activate G_i and G_o.

	ACKNOWLEDGEMENTS

We thank Pam Sternweis and Dr. Tohru Kozasa for assistance with the phospholipase C assays and Michelle Clark for excellent technical assistance. We are grateful to Drs. Suchetana Mukhopadhyay and Elliott Ross for performing the steady state GTPase assays in which the m1 muscarinic cholinergic receptor and heterotrimeric G_q were reconstituted in phospholipid vesicles. We thank Drs. Tohru Kozasa, Andrejs Krumins, Sheu-Fen Lee, and Clive Slaughter for helpful comments and suggestions.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM34497 and the Raymond and Ellen Willie Distinguished Chair of Molecular Neuropharmacology (to A. G. G.).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.

^§ To whom correspondence should be addressed.

² A. Krumins and A. G. Gilman, unpublished observations.

³ B. A. Posner and A. G. Gilman, unpublished observations.

⁴ T. K. Harden and A. Krumins, personal communication.

	ABBREVIATIONS

The abbreviations used are: RGS, regulators of G protein signaling; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; DEP, disheveled, EGL-10, and pleckstrin; GGL, G protein  subunit-like; C₁₂E_10, dodecyl poly(ethylene oxide) (n~10), GTPS, guanosine 5'-O-thiotriphosphate; PLC, phospholipase C; RACE, rapid amplification of cDNA ends; Ni-NTA, nickel-nitrilotriacetic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES