Mapping the Galpha 13 Binding Interface of the rgRGS Domain of p115RhoGEF*

Zhe ChenDagger §, William D. Singer§, Clark D. Wells||, Stephen R. SprangDagger **DaggerDagger, and Paul C. Sternweis§§

From the Departments of Dagger  Biochemistry and  Pharmacology and the ** Howard Hughes Medical Institute, the University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, December 12, 2002

    ABSTRACT
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Structural requirements for function of the Rho GEF (guanine nucleotide exchange factor) regulator of G protein signaling (rgRGS) domains of p115RhoGEF and homologous exchange factors differ from those of the classical RGS domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS domain was undertaken to determine its functional interface with the Galpha 13 subunit. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Galpha 13 and the interactions of RGS4 and RGS9 with their Galpha substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Lack of sequence conservation within the N terminus may also explain the lack of GTPase-activating protein (GAP) activity in a subset of the rgRGS domains. For all mutations, loss of functional GAP activity is paralleled by decreases in binding to Galpha 13. The same mutations, when placed in the context of the p115RhoGEF molecule, produce deficiencies in GAP activity as observed with the rgRGS domain alone but show no attenuation of the regulation of Rho exchange activity by Galpha 13. This suggests that the rgRGS domain may serve a structural or allosteric role in the regulation of the nucleotide exchange activity of p115RhoGEF on Rho by Galpha 13.

    INTRODUCTION
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INTRODUCTION
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The intrinsic guanosine triphosphatase (GTPase) activity of the alpha  subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins) can be stimulated by members of the regulators of G protein signaling (RGS)1 family (1-3). The catalytic activity of these GTPase-activating proteins (GAPs) resides in an ~120-residue alpha -helical domain that corresponds to a consensus "RGS box" defined by amino acid sequence similarity. RGS box sequences show considerable diversity and can be subdivided into distinct homologous clusters (4), but all share conserved features recognizable in the three-dimensional structures of representative family members: RGS4 (5), GAIP (6) and RGS9 (7). p115RhoGEF, a guanine nucleotide exchange factor (GEF) for the monomeric G protein RhoA (8, 9), is the prototype of a family that includes LARG (10), Lsc (11), PDZRhoGEF (12), and GTRAP48 (13). These proteins contain regions in their N-terminal halves with low sequence identity to RGS domains (14). In p115RhoGEF, this region extends from residue 42 to 163. Recombinant p115RhoGEF and fusion proteins containing the first 246 (14) or 252 (15) N-terminal residues of p115RhoGEF had specific GAP activity toward the alpha  subunits of the heterotrimeric G proteins, G12 and G13, but not toward members of the Gs, Gi, or Gq subfamily of Galpha proteins. In contrast to other RGS proteins, p115RhoGEF requires elements outside of the RGS box to function as a GAP. A construct that extends about 75 residues beyond the C terminus of the RGS box is the smallest fragment of the holoprotein which could be overexpressed as a soluble protein in Escherichia coli, although as little as 50 additional C-terminal residues allowed expression of a functional domain in eukaryotic cells. In addition to an extended C terminus, 25 residues that precede the RGS box were also required for full GAP activity (15). We refer to the N- and C-terminally extended RGS box segment of p115RhoGEF and its homologs as the rgRGS (RhoGEF RGS) domain. Two members of the p115RhoGEF family, GTRAP48 (13, 15) and PDZRhoGEF,2 bind to Galpha 13 but have little or no GAP activity; these form a distinct sequence subset with respect to both the RGS box domain and the N-terminal segment.

Structural (5, 7) and mutagenesis (16-19) studies have defined three distinct regions of the RGS domain which interact with Galpha and convey GAP activity. These correspond to two surface polypeptide turns that join helical segments, together with the surface of the C-terminal alpha  helix of the RGS domain. These RGS elements directly contact the catalytic site of Galpha , principally, the switch I and switch II segments that undergo conformational rearrangement upon hydrolysis of GTP (20). Together, these two segments in Galpha subunits contain residues that participate directly in the catalytic mechanism of GTP hydrolysis or bind the magnesium ion cofactor (21, 22). The complex formed by GDP, Mg2+, and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, which promotes an activated state of Galpha (23), also mimics the pentacoordinate transition state for GTP hydrolysis (21, 22). RGS proteins have been shown to bind more strongly to the GDP·Mg2+·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> complexes of Galpha subunits than to those formed with GTP analogs (24). This, together with structural evidence, indicates that RGS proteins accelerate GTP hydrolysis by stabilizing a transition state-like conformation of Galpha or destabilize the GTP-bound ground state (25).

The crystal structures of the N-terminally truncated rgRGS domain of p115RhoGEF (26) and that of its homolog, PDZRhoGEF (27), were recently determined. These domains are comprised of 11 alpha  helices; the N-terminal 7 helices form a bilobal fold similar to that in classic RGS domains. The remaining 4 helices pack against the latter to generate a single globular domain. The core of the rgRGS domain from p115RhoGEF, which encompasses two of the three segments involved in Galpha binding, shows high structural similarity to the corresponding segments in RGS proteins of known structure. Accordingly, mutation of two p115RhoGEF residues that correspond, in RGS4, to side chains that interact with switch I and switch II, were found to diminish GAP activity (26). These observations led to the inference that the general features of the protein-protein interface observed in the published structures of RGS·Galpha complexes are likely be preserved in the interaction of rgRGS with Galpha 13. Although the N terminus of p115RhoGEF is not present in the crystal structure, it is required for GAP activity (15), and structural modeling of the rgRGS·Galpha 13 complex suggests that N-terminal residues of the rgRGS domain might interact with the helical domain and switch regions of Galpha 13 (26).

Here, we describe an extensive mutagenic analysis performed to define the residues in the rgRGS domain which interact with Galpha 13 and convey GAP activity. These studies focused on both the globular helical domain of the rgRGS and the N-terminal residues that precede the RGS box. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Galpha 13 and the interactions of RGS4 and RGS9 for their Galpha substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Placement of the same mutations in the context of the p115RhoGEF molecule produces the same deficiencies in GAP activity as observed with the rgRGS domain alone but shows no attenuation of the regulation of Rho exchange activity by Galpha 13.

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Expression Plasmids-- Complementary DNA oligomers encoding single or double amino acid changes from the wild type p115RhoGEF gene were used to generate point mutants by PCR. The mutagenesis template consisted of amino acids 1-252 of human p115RhoGEF (rgRGS domain) subcloned into the pGEX-KG vector. The sequences of primers used are available upon request. The QuikChange KitTM from Stratagene was used in a four-step procedure to generate mutant cDNA. Plasmids encoding the desired mutations were selected and verified by sequencing. Selected mutated rgRGS DNAs were also subcloned into the full-length p115RhoGEF gene in the pFastbac1 vector (Invitrogen), which has been modified to provide an N-terminal EE tag (EYMPME) (8) for production of baculovirus and subsequent expression in Spodoptera frugiperda (Sf9) cells.

Expression and Purification of Proteins-- All p115RhoGEF proteins were expressed in transformed BL21 (DE3) or DH5alpha strains of E. coli or in cultured Sf9 cells infected with recombinant baculoviruses. The rgRGS domains were produced in E. coli as fusion proteins with glutathione S-transferase (GST). Cells were grown in LB medium at 37 °C to A600 ~0.6 and induced for 3 h at 30 °C with 0.2 mM isopropyl-1-thiogalactopyranoside for expression of mutant proteins. Bacteria were then pelleted by centrifugation and lysed for 30 min in solution A (25 mM NaHepes, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, and protease inhibitors (2.5 µg/ml leupeptin, 1 µg/ml pepstatin A, 21 µg/ml phenylmethylsulfonyl fluoride, 21 µg/ml Na-p-tosyl-L-lysine chloromethyl ketone, 21 µg/ml tosylphenylalanyl ketone, and 21 µg/ml Na-p-tosyl-L-arginine methyl ester)) containing 2 mg/ml lysozyme, 1 mg/ml DNase I, and 5 mM MgCl2. GST-tagged rgRGS proteins were purified by chromatography through glutathione-Sepharose 4B (Amersham Biosciences) in solution A and elution with solution A containing 15 mM reduced glutathione.

Full-length p115RhoGEFs containing mutated rgRGS domains were expressed in Sf9 cells during 48 h of infection with recombinant baculoviruses. The expressed proteins were purifed from lysates by chromatography with immobilized antibodies to the EE tag (BabCO) as described elsewhere (8).

Galpha 13 was expressed in Sf9 cells and purified as described previously (28). RhoA was expressed in Sf9 cells with an N-terminal hexahistidine (His6) tag and purified from lysates by isolation with Ni2+-nitriloacetic acid resin (Qiagen) in 25 mM NaHepes, pH 7.5, 2.5 mM beta -mercaptoethanol, 50 mM NaCl, and protease inhibitors. Elution was facilitated by a step gradient of increasing imidazole concentration, with His6-RhoA being released at ~75 mM.

GAP Assays for Galpha 13-- 2 µM Galpha 13 was loaded with 5 µM [gamma -32P]GTP (50-100 cpm/fmol) for 15 min at 30 °C in solution B (20 mM NaHepes, pH 8.0, 5 mM EDTA, 1 mM dithiothreitol, and 0.05% polyoxyethylene 10 lauryl ether (Lubrol)). Free [gamma -32P]GTP and 32Pi were removed rapidly by centrifugation of the samples at 4 °C through Sephadex G-50 that had been equilibrated with solution B. Hydrolysis of GTP was initiated by adding Galpha 13 loaded with [gamma -32P]GTP (final concentration of 1-5 nM) to solution B containing 10 mM MgSO4, 1 mM GTP, and 1-100 nM p115RhoGEF rgRGS domain (wild type or mutant) in a 50-µl reaction volume. After incubation for the indicated times at 4 °C, reactions were quenched with 750 µl of 5% (w/v) NoritA in 50 mM NaH2PO4. The mixtures were then centrifuged at 3,000 rpm for 5 min, and 400 µl of supernatant containing 32Pi was counted by liquid scintillation spectrometry.

G Protein Binding Assays-- The purified GST-tagged rgRGS domains were bound to 20 µl of glutathione-Sepharose 4B (packed beads) by gentle mixing in 200 µl of solution C (20 mM NaHepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 100 mM NaCl) for 30 min at 4 °C. During that time, Galpha 13 was diluted to 2 µM in solution D (20 mM NaHepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM GDP, and 0.05% Lubrol) and then gel filtered rapidly by centrifugation through Sephadex G-50 that had been equilibrated with the same solution. This removes residual activating ligands in the preparation. Samples of glutathione-Sepharose 4B containing bound GST-tagged rgRGS proteins were then pelleted in a microfuge for 30 s and washed with 200 µl of solution D to remove unbound protein. 200-µl binding reactions were prepared by mixing the immobilized rgRGS proteins and gel-filtered Galpha 13 in solution D either with or without AMF (30 µM AlCl3, 5 mM MgCl2, and 5 mM NaF). Samples were mixed gently for 30 min at 4 °C, and unbound Galpha 13 was then removed by two 200-µl washes of solution D either with or without AMF. The washed beads were finally resuspended with 40 µl of solution D and then boiled in SDS sample buffer.

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Mutations of the rgRGS Helical Domain That Affect GAP Activity toward Galpha 13-- Structural modeling of the rgRGS·Galpha 13 complex predicted that residues of loops L3, L5, helix alpha 8, and loop L11 would interact with the switch regions of Galpha 13 (Fig. 1A) (26). To test this hypothesis, we mutated residues located within this predicted interaction surface of the p115RhoGEF rgRGS domain and measured the GAP activity of the mutant domains toward Galpha 13. Unless stated otherwise, "p115rgRGS" refers to the protein fragment encompassing residues 1-252 of p115RhoGEF. Mutations are identified by the one-letter code for the residue in wild type p115rgRGS, followed by the position of the residue in the amino acid sequence and the one-letter code for the residue to which it was mutated (e.g. E71A has glutamic acid in position 71 mutated to an alanine). Residues chosen for mutagenesis (Fig. 1B) fall into three categories. The first group includes residues predicted to form direct and specific contacts with the switch regions of Galpha 13, according to structural modeling studies of the rgRGS·Galpha 13 complex (Glu-71, Arg-111, Pro-113, Pro-115, Pro-116, Glu-155, and Lys-214). The second group of residues in the predicted interface region includes those that are charged, have solvent accessible surface areas greater than 25 Å2, and are conserved in the rgRGS domains but not in the classical RGS proteins (Gln-69, Asp-156, Lys-160, Arg-161, Glu-212, and Glu-213). A third group of residues includes surface residues that are predicted to project toward, but not necessarily contact, switch regions of Galpha 13 (Phe-70, Arg-152, and Ser-159). In most instances, residues were mutated to alanine or residues with opposite charge. Three of the mutations, F70A, P113K, and R152E, were also designed to reflect the differences in sequence between p115RhoGEF and two rgRGS-containing proteins with little or no GAP activity, GTRAP48 and PDZRhoGEF. None of the residues chosen for mutagenesis is involved in extensive packing interactions. Hence, alterations of these residues are therefore not expected to disrupt the tertiary structure of p115rgRGS.


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Fig. 1.   Tertiary and primary structure of p115rgRGS. A, the tertiary structure of p115rgRGS (26) is depicted using the coloring scheme published for RGS4 (5); the N-terminal 42 residues and C-terminal 19 residues of the rgRGS domain are not present in the structure. Helices that comprise structural elements of the RGS box are colored orange, green, and blue. L3, L5, and helix alpha 8 in RGS4 and RGS9 form contacts with Galpha i1 and Galpha t, respectively (5, 7) (see "Results"). The C-terminal helical bundle (colored red) is unique to rgRGS domains. B, the amino acid sequences of rgRGS domains were aligned with two members of the RGS family for which crystal structures have been determined. Sequence alignments performed using ClustalW (31) were modified on the basis of structural superposition (26). Colored bars set above the sequences represent helices with color codes that match the ribbon diagram shown in A. Gray bars represent helices in the structure of RGS4 (5). Residues in RGS4 or RGS9 (7) which are involved in contacts (closer than 4.0 Å) with Galpha i1 or Galpha t, respectively, are colored in red. Residues in rgRGS proteins which are conserved and have solvent-accessible surface areas greater than 25 Å2 in the structure of p115rgRGS are colored in purple, and those conserved with solvent-accessible surface areas less than 25 Å2 are colored in blue. Residues marked with asterisks were targeted for site-directed mutagenesis.

Initial experiments were conducted with constructs encompassing only the p115rgRGS domain (residues 1-252). 21 mutated p115rgRGS domains were expressed in E. coli. All of the mutants were expressed at a high level (>1 mg/liter) and as soluble proteins (data not shown) as would be expected for properly folded protein domains. The GAP activities of the recombinant p115rgRGS mutants were measured by the single turnover GTPase assay for Galpha 13 (see "Materials and Methods"). Examples of these assays, which assessed the effect of 10 nM mutant p115rgRGS protein on the time dependence at 4 °C of hydrolysis of GTP bound to Galpha 13, are shown in Fig. 2, A and B. At this concentration and conditions, the wild type rgRGS domain stimulated the GTPase activity of Galpha 13 by ~5-10-fold and caused almost complete hydrolysis of the bound GTP by 2 min. The total amount of GTP hydrolyzed differed from assay to assay, largely because of variations in GTP loading caused by weak nucleotide affinity and the high intrinsic GTPase rate of Galpha 13 (29); thus, the final concentration of [gamma -32P]GTP-loaded Galpha 13 used in the assays ranged from 1 to 10 nM.


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Fig. 2.   Effect of mutations within the RGS box of p115rgRGS on GAP activity. A, Galpha 13 was loaded with [gamma -32P]GTP (final concentration, 1-5 nM), and hydrolysis of the bound GTP was initiated by mixing with buffer containing 10 mM MgSO4 and 1 mM GTP (basal, closed circles). Samples were incubated for the indicated times at 4 °C and then quenched as described under "Materials and Methods." 32Pi evolved from the reactions was counted by liquid scintillation spectrometry. As indicated, 10 nM p115rgRGS domains were included in the reaction: wild type p115rgRGS (closed diamonds), D156A mutant (open circles), K160A mutant (closed inverted triangles), R161A mutant (open inverted triangles), E212A mutant (closed squares), or E213A mutant (open squares). B, the time course of GTP hydrolysis, as described in A, is shown for basal (closed circles), wild type p115rgRGS (open diamonds), Q69A mutant (open circles), F70A mutant (closed inverted triangles), F70Y mutant (open inverted triangles), R152A mutant (closed squares), R152E mutant (open squares), or S159A mutant (closed diamonds). C, the apparent initial rate of GTP hydrolysis catalyzed by Galpha 13 in the presence of each p115rgRGS mutant (10 nM) is represented as a percentage of the initial rate of GTP hydrolysis by Galpha 13 in the presence of 10 nM wild type p115rgRGS. D, the quantity of 32Pi released in the assay as described in A after a 2-min incubation was determined in the presence of the indicated concentration of mutant or wild type p115rgRGS·wild type p115rgRGS (closed circles), Q69A mutant (open circles), F70A mutant (closed inverted triangles), E71K mutant (open inverted triangles), D156A mutant (closed squares), or E71K/P113K mutant (open squares).

A summary of the results with the 21 mutant domains tested under the conditions described above is shown in Fig. 2C. Several mutations, including R111A, E155K, E212A, E213A, K214A, and K214E, had no effect on GAP activity. In contrast, three mutations, Q69A, F70A, and D156A, appear largely to abolish GAP activity, whereas other mutations had more modest effects (apparent decreases in GAP activity of 20-60% of the wild type domain). GAP activities of mutants designed to target specific Galpha 13 interaction sites are consistent with the involvement of the structurally conserved L3 and L5 loops and also the less well conserved helix, alpha 8. However, the lack of effects by mutations in L11 from the C-terminal helical region does not support a role for this structural element in the GAP activity of the rgRGS domain.

The L3 loop of the rgRGS domain is a likely site for interaction with Galpha 13, yet its conformation differs from that of RGS4. The group 1 residue in this loop, Glu-71, is proposed to engage in electrostatic and van der Waals interactions with Lys-204 of Galpha 13 and is conserved among rgRGS proteins (Fig. 1B). Accordingly, mutants in which Glu-71 was replaced by either alanine or lysine have 50% of the GAP activity toward Galpha 13 compared with the wild type rgRGS domain (Fig. 2C). Nonconservative mutations of the group 2 and group 3 residues in L3, Q69A, and F70A, respectively, caused large reductions in GAP activity. Both residues are exposed to solvent and are in close proximity to switch I of Galpha 13 in the model of the rgRGS·Galpha 13 complex, although their interaction partners cannot be predicted. Gln-69 is absolutely conserved among known rgRGS proteins (Fig. 1B), whereas Phe-70 is conserved only in p115RhoGEF, Lsc, and LARG, but replaced by alanine in PDZRhoGEF and GTRAP48. The potential interactive role of this residue is supported by the retention of about 50% of GAP activity by the conservative mutation of Phe-70 to tyrosine.

Structural and mutagenic studies of RGS proteins indicate that the residue corresponding to Asn-128 in RGS4 is important for GAP activity. A proline residue occupies the analogous position (residue 113 in p115RhoGEF) in the rgRGS domains with strong GAP activity but is replaced by lysine in GTRAP48 (Fig. 1B). Mutation of Pro-113 in L5 to either an alanine or a lysine reduces GAP activity (Fig. 2C), but the effect is not as severe as that caused by mutations in the L3 or alpha 8 regions. Other mutations of group 1 residues in L5, including R111A and P115G/P116G, had little effect on GAP activity (Fig. 2C).

Residues outside the alpha 3-L5 core region of the rgRGS domain align poorly in tertiary structure with RGS4. Therefore, although alpha 8 is a probable interaction partner for switch I of Galpha 13, only one residue in this helix, Glu-155, could be included in group 1. Mutation of this residue had little effect on GAP activity. In contrast, mutation of one of the two neighboring group 2 residues, Arg-152, reduced GAP activity by 50%. Arg-152 is replaced by glutamate in PDZRhoGEF and GTRAP48, two rgRGS proteins with very weak GAP activity toward Galpha 13. Replacing Arg-152 with glutamate in p115rgRGS resulted in only a modest reduction in activity similar to replacement with alanine. Three charged group 3 residues were selected for mutagenesis in alpha 8. Of these, substitution of Asp-156, a residue in alpha 8 which is conserved among rgRGS proteins (Fig. 1B), severely reduced GAP activity. However, structure-based modeling reveals no residues in Galpha 13 poised to interact directly with Asp-156 in the rgRGS domain. Mutation of other charged residues in alpha 8, Glu-155, Ser-159, and Arg-161, had little effect.

To elucidate the extent to which certain mutations affect GAP activity of the rgRGS domain, we measured the quantity of GTP hydrolyzed by Galpha 13 within a 2-min period as a function of rgRGS concentration (Fig. 2D). At a sufficiently high concentration, all of the mutated rgRGS domains were capable of stimulating Galpha 13 to the extent observed for the wild type domain. Thus, the D156A, Q69A, and F70A mutations do not substantially alter the efficacy of rgRGS as a GAP for Galpha 13, but strongly reduce the potency of the domain for this function. Even the most debilitated mutant, p115rgRGS (D156A), exhibited GAP activity that was comparable with that of the wild type protein at concentrations that were 100-1,000-fold greater than Galpha 13 in the assay.

A Map of the Contact Surface of the rgRGS Core Domain for Interaction with Galpha 13-- In Fig. 3A, the solvent-accessible surface of rgRGS is color-coded according to the relative loss of GAP activity that results from mutation of the underlying residues. The relative GAP activity of a mutant p115rgRGS domain is quantified as the apparent initial velocity of the GTPase reaction catalyzed by Galpha 13 in the presence of the mutant normalized by the rate in the presence of wild type p115rgRGS (% V0WT) (for examples, see Fig. 2, A and B). The mutagenesis data identify a functionally critical surface on the rgRGS domain which is centered on three key residues from the RGS box: Gln-69 and Phe-70 from L3, and Asp-156 from alpha 8. The functional map of rgRGS is grossly similar to that of RGS4 as charted by Srinivasa and colleagues (16) (Fig. 3B) but differs in the extent to which mutations at corresponding positions affect GAP activity. For example, mutation of Asn-128 in the L5 loop of RGS4 severely impairs GAP activity, whereas the corresponding P113A mutation is only modestly debilitating in rgRGS. Conversely, the Q69A and F70A substitutions generate severe effects in rgRGS, whereas mutation of the structurally equivalent residues in RGS4 only reduce GAP activity to ~30% of wild type. On the other hand, Asp-156 in rgRGS and Arg-167 in RGS4 occupy equivalent positions in alpha 8, and both are critical for GAP activity of the respective domains.


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Fig. 3.   Relative GAP activity of p115rgRGS and RGS4 mutants mapped on the Galpha binding surface. A, the solvent accessible surface of p115rgRGS (excluding residues 1-42, which are not present in the crystal structure) was computed using the program GRASP (32). The surface of the protein presumed to interact with Galpha 13 faces the viewer. Residues that were mutated are labeled. The surface corresponding to each solvent accessible residue is color-coded from light pink to deep red, in proportion to the loss of GAP activity as measured by % V0WT (Fig. 2C) upon mutation (see "Results"). The surfaces of residues that were not mutated, or where mutation had no significant affect on GAP activity (% V0WT > 80%), are rendered in light gray. B, the corresponding surface representation of RGS4, color-coded according to GAP activity as in A, using relative activity data obtained for mutants of RGS4 reported by Srinivasa et al. in Table I of Ref. 16. The activity data obtained in the presence of 200 nM RGS4 were used to color code this surface diagram. Only those solvent-accessible residues mutated to alanine are color-coded in this diagram. In both panels, circles labeled and color-coded according to the scheme used in Fig. 1A enclose structural elements that form the RGS4·Galpha i1 binding site or the proposed p115rgRGS·Galpha 13 binding site.

Mutations of Residues N-terminal to the RGS Box in the rgRGS Domain-- Residues 15-41 of p115rgRGS are required for GAP activity toward Galpha 13 (15). This region contains a hydrophobic sequence followed by a 16-residue segment containing 9 acidic residues (Figs. 1B and 8A). Within this region, sequence conservation between p115RhoGEF and GTRAP48, which possesses only weak GAP activity, is low, and fewer negatively charged side chains are present in GTRAP48. To define the contribution of residues within the acidic sequences to the GAP activity of rgRGS, a series of single site mutations was generated within the segment extending from residue 27 to residue 45. Also included for mutagenesis were 2 amino acids at the N-terminal border of the functional rgRGS domain, Ser-14 and Arg-15. Within this set, negatively charged glutamic acid residues were mutated to lysine; the remaining amino acids were mutated to alanine. All of the mutant p115rgRGS domains could be expressed as soluble proteins in E. coli and were assayed for GAP activity toward Galpha 13 as described above. Residues 27-31 were identified as crucial to the GAP activity of the rgRGS domain. Mutation of Glu-27 or Glu-29 to lysine severely impaired GAP activity (Fig. 4, A and B) as did the mutation of Asp-28 or Phe-31 to alanine. Mutations of the charged residues immediately flanking this segment reduced GAP activity to ~50% that of wild type, whereas mutations of residues 40-45 had no effect. Substitution of Ser-14 and Arg-15 did not reduce GAP activity appreciably. The concentration dependence of GAP activity was also measured for p115rgRGS bearing single mutations in residues 27-31 (Fig. 4D). In contrast to mutations within the RGS box region, mutation of these residues appeared to reduce efficacy as well as potency severely.


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Fig. 4.   Effect of mutations within the N terminus of p115rgRGS on GAP activity. A, the time course of GTP hydrolysis by Galpha 13, as described in Fig. 2A, is shown for the basal condition (closed circles) or inclusion of the 10 nM wild type (open squares), E32K mutant (open circles), E34K mutant (closed inverted triangles), E27K mutant (open inverted triangles), or E29K mutant (closed squares) form of the p115rgRGS domain. B, the time course of GTP hydrolysis by Galpha 13, as described in Fig. 2A, is shown for basal conditions (closed circles) or inclusion of the 10 nM wild type (open squares), S44A mutant (open circles), Q45A mutant (closed inverted triangles), D28A mutant (open inverted triangles), or F31A mutant (closed squares) form of the p115rgRGS domain. C, the apparent initial rate of GTP hydrolysis catalyzed by Galpha 13 in the presence of each p115rgRGS mutant (10 nM) is represented as a percentage of the initial rate of GTP hydrolysis by Galpha 13 in the presence of wild type p115rgRGS domain. D, the quantity of 32Pi released in the assay described in A after a 2-min incubation was determined in the presence of the indicated concentration of mutant or wild type p115rgRGS: wild type p115rgRGS (closed squares), E27K (closed circles), D28A mutant (open circles), E29K mutant (closed inverted triangles), or F31A mutant (open inverted triangles).

Mutations of Residues Required for rgRGS GAP Activity Also Reduce Affinity for Galpha 13-- Recombinant p115rgRGS proteins, which possess single mutations from the N terminus (E27K, D28A, E29K, F31A, E32K, or E34K) of the rgRGS domain or from the RGS box region (Q69A, F70A, F70Y, E71K, or D156A), or the double mutation (E71K/P113K), were tested for their ability to bind Galpha 13 in a pull-down assay. Immobilized p115rgRGS proteins were incubated with Galpha 13 in the presence of GDP and Mg2+ or GDP with Mg2+ and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP> (Fig. 5). Both wild type and mutant p115rgRGS domains bound only poorly or not at all to Galpha 13·GDP in the absence of Mg2+ and AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. Although the wild type rgRGS domain bound efficiently to the activated alpha  subunit (Galpha 13·GDP·Mg2+·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>), the E27K, D28A, E29K, F31A, and D156A mutant proteins failed to bind appreciably. In the GAP assays described in the previous sections, these mutations severely reduced GAP activity of the p115rgRGS domain. Two mutations in the RGS domain, Q69A and F70A, which reduced the potency but not the efficacy of p115rgRGS in the GAP assay, bound to Galpha 13·GDP·Mg2+·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>, albeit to a lesser extent than wild type p115rgRGS. Other mutants tested in the pull down assay, which showed only modest reduction in the GAP activity, retain their ability to bind Galpha 13·GDP·Mg2+·AlF<UP><SUB>4</SUB><SUP>−</SUP></UP>. Mutations that severely impair the GAP activity of rgRGS also strongly diminish its ability to bind to activated Galpha 13; in no case do we observe an inactive rgRGS mutant that retains its full ability to bind Galpha 13.


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Fig. 5.   Binding of Galpha 13 to rgRGS domain mutants. Purified GST-tagged rgRGS domains (20 pmol) were bound to glutathione-Sepharose 4B (see "Materials and Methods"). Washed beads were incubated with gel-filtered GDP-bound Galpha 13 (20 pmol) prepared in the presence or absence of AMF and mixed gently for 30 min at 4 °C. The beads were then washed, and the relative amount of Galpha 13 that remained bound to the immobilized rgRGS domains was determined by immunoblot with B860 anti-Galpha 13 antiserum after separation by SDS-PAGE.

Activities of p115RhoGEF Containing Point Mutations in the rgRGS Domain-- Several of the single residue mutations characterized for the rgRGS domain were inserted into the holo-p115RhoGEF protein to determine their effect in this context. As shown in Fig. 6A, these mutations are as debilitating to the GAP activity of the holoprotein as they are to that of the rgRGS domain alone. Thus, mutations F31A and E27K caused almost total loss of GAP activity, whereas three other mutations (D156A, D28A, and E29K) showed impaired potency but substantial efficacy at higher concentrations. As with the rgRGS domain, these mutations also reduced the affinity of the holoprotein for activated Galpha 13 (Fig. 6B). This indicates that the rgRGS domain confers the dominant elements required for binding of p115RhoGEF to Galpha 13.


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Fig. 6.   GAP and Galpha 13 binding activities of p115RhoGEF containing mutations in the rgRGS domain. A, Galpha 13 was loaded with [gamma -32P]GTP (final concentration, 1-5 nM), and hydrolysis of the bound GTP was initiated as described in Fig. 2 either in the absence or presence of the indicated concentrations of wild type (WT) p115RhoGEF or the exchange factor containing the indicated mutations in its rgRGS domain: wild type (closed circles), D156A mutant (open circles), E27K mutant (closed inverted triangles), D28A mutant (open inverted triangles), E29K mutant (closed squares), or F31A mutant (open squares). Hydrolysis of GTP was measured for 2 min at 4 °C. B, binding of Galpha 13 to the mutant proteins was done as described in Fig. 5 except the Glu-tagged p115RhoGEFs were bound to Sepharose containing immobilized anti-Glutag IgG.

In contrast to the impairment of binding and GAP activities, the point mutations described above did not affect the ability of activated Galpha 13 to stimulate the Rho exchange activity of p115RhoGEF (for examples, see Fig. 7). Among the mutants tested, stimulation of exchange activity by either the wild type or mutant proteins ranged from 3- to 5-fold with an EC50 for Galpha 13 between 5 and 15 nM. There is no apparent decline in potency which correlates with the observed effects on activities of the rgRGS domain. Thus, although the rgRGS domain is required for regulation of Rho exchange by Galpha 13 (15), binding of the domain to the alpha  subunit does not appear to be coupled to this effect.


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Fig. 7.   Stimulation of p115RhoGEF mutants by Galpha 13. Exchange of guanine nucleotide on Rho was assessed by binding of [35S]GTPgamma S (30) either in the absence or presence of p115RhoGEF and the addition of increasing concentrations of activated Galpha 13 as indicated. Reactions contained 2 µM His6-RhoA and 20 nM p115RhoGEF when indicated: wild type (open circles), F31A mutant (closed inverted triangles), D28A (open inverted triangles), D156A (closed squares), no GEF (closed circles). Reactions were incubated at 30 °C for 3 min. Activation of Galpha 13 was accomplished by preincubation with AMF at 4 °C for 30 min and inclusion of AMF in the exchange assay.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rgRGS domains form a unique and distinct subgroup of the RGS family. Although the rgRGS domains are, in three-dimensional structure, apparent homologs of the RGS domains, their sequences bear little similarity to the RGS consensus sequence (3). The mutagenesis experiments that we have conducted are consistent with the hypothesis that the Galpha 13 interaction surface of the RGS box region in p115RhoGEF is roughly similar to that observed in the structures of RGS4 and RGS9 bound to their Galpha substrates (5, 7). However, the set of rgRGS residues that emerges from the analysis as "hot spots" for GAP activity are not all cognates of the functionally critical residues in RGS4 (16).

The segment extending from L3 to L5 is the most highly conserved structural feature common to the rgRGS and RGS folds. Less well conserved is the segment corresponding to alpha 7-alpha 8 in RGS4, which folds as a single helix, alpha 8, in rgRGS. Residues from both segments are important for GAP activity in both the RGS and rgRGS domains. A triad of residues, Gln-69, Phe-70, and Asp-156, appears to form a functional core in rgRGS. Their counterparts in RGS4 are Glu-83, Tyr-84, and Arg-167, respectively. In the complex with Galpha i1, Arg-167 is involved in a weak or indirect electrostatic interaction with the main chain of Thr-182 in switch I, near the center of the RGS4·Galpha i1 contact region. Glu-83 plays a supporting role through formation of an ion pair with Arg-167 but does not directly contact the G protein. Tyr-84 forms a stacking interaction with His-213 at the C terminus of the switch II helix in Galpha i1. As is true for its counterpart, Asp-156 in p115rgRGS, mutation of Arg-167 causes a severe loss of GAP activity (17), whereas the effects of mutations at positions 83 and 84 are less damaging (16). However, if we create a model of the rgRGS·Galpha 13 complex by superposing both components onto the corresponding structures in RGS4·Galpha i1 (26), none of the core triad residues directly contacts Galpha 13. We therefore suppose that the rgRGS·Galpha 13 complex differs in significant molecular detail from that of RGS4·Galpha i1.

Mutation of Asn-128 in the L5 loop of RGS4 (16) can result in a modest reduction in GAP activity if replaced by serine (the cognate residue in GAIP) but severe losses (>99.9% reductions) if replaced by bulkier residues (18). Yet, mutation of the structurally equivalent residue in p115RhoGEF, Pro-113, to either alanine or lysine, decreases GAP activity only 50% at a 10:1 molar ratio of rgRGS to Galpha 13. Pro-113, and possibly the L5 loop, therefore appear to be less important to the function of rgRGS than equivalent regions of RGS proteins.

The N-terminal 41 residues of p115RhoGEF include a hydrophobic and proline-rich sequence (residues 11-26) followed by a 15-residue segment (amino acids 27-41) containing 9 acidic residues (Fig. 8A). Although the first 12 residues of p115RhoGEF were shown to be dispensable for GAP activity, deletion of the first 41 residues abolished this function (15). Here, we show that the electronegative cluster encompassed by residues 27-30 is crucial to GAP function. Indeed, substitution of any of the first 3 acidic residues in the cluster caused a greater than 99% loss of GAP potency and efficacy. Mutation of the aromatic residue, Phe-31, which is adjacent to the acidic sequence, is equally deleterious. The only mutation within the RGS domain of rgRGS which causes an equivalent degree of impairment is that of Asp-156 in alpha 8, which, of the residues mutated, has the least solvent-accessible (~25 Å2) surface area in the structure of p115rgRGS. Thus, residues within the RGS box and a small cluster in the preceding N-terminal region are both necessary for the GAP activity of rgRGS.


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Fig. 8.   Putative Galpha 13 binding site for the N terminus of p115RhoGEF. A, sequence of the N-terminal 41 residues of p115RhoGEF. Acidic residues are colored red, and basic residues colored blue. B, the complex of p115rgRGS (colored as in Fig. 1A) with Galpha 13 (gray except for switch segments in purple) was modeled as described previously (26). The position of the most N-terminal residue that is defined in the structure of the p115rgRGS domain (residue 44) is labeled. C, the putative binding site for the N terminus of p115RhoGEF (a detailed view of the area shown enclosed by a dotted box in B) is formed by the helical domain and the switch regions of Ga13, and helix alpha 8 of the rgRGS domain.

In the absence of structural information, we speculate that residues N-terminal to the RGS box might function in GAP activity through an electrostatic binding mechanism. A model of the rgRGS·Galpha 13 complex based on that of previously determined RGS·Galpha complexes shows a cluster of positively charged residues contributed by helix alpha 8 of the rgRGS domain and the switch regions and central helix (residues 79-105) of the helical domain of Galpha 13. The latter form an electropositive trough that could provide a binding site for the negatively charged residues that precede the N terminus of the rgRGS domain (Fig. 8, B and C). The electropositive character of helical domain of Galpha 13 appears to be a conserved feature of the G12 class of Galpha subunits. In Galpha i, Galpha o, Galpha q, and Galpha z, which are not substrates for p115RhoGEF, many of the positively charged residues in the helical domain are replaced by neutral or acidic residues. The most critical residues within the RGS domain of rgRGS: Gln-69, Phe-70, and Asp-156, might be supposed to form, with the critical EDEDF segment, a functional core of the rgRGS·Galpha 13 interface. Phe-31, which flanks this highly charged segment, might be accommodated in a hydrophobic pocket, possibly within the helical domain of Galpha 13.

The rgRGS subfamily represented by GTRAP48 and PDZRhoGEF have little or no GAP activity toward Galpha 13. The absence of GAP activity for these two rgRGS proteins may stem from specific sequence differences in either the RGS box or the preceding N-terminal sequence. The overall sequence similarity between p115RhoGEF and GTRAP48 is high (57%) in the RGS box region (Fig. 1B), and many of the substitutions are conservative. Three mutations, F70A, P113K, and R152E, were designed to reflect nonconserved differences in sequence between p115RhoGEF and GTRAP48 in regions hypothesized to form contact sites with Galpha 13. Of these, F70A is sufficient to reduce catalytic efficiency and binding affinity of the rgRGS domain toward Galpha 13 severely. In contrast, differences between the N-terminal sequences of p115RhoGEF and GTRAP48 are so great that it is difficult to align them. Although both active and inactive RhoGEFs possess a sequence consisting of 3 or 4 acidic residues preceding a phenylalanine (residues 27-31 in p115RhoGEF) or tyrosine, the positions of this sequence relative to the N terminus differ. Therefore, spatial mismatch of the N-terminal acidic cluster with respect to its potential binding site on Galpha 13 might explain the reduced GAP activity of GTRAP48.

All of the mutations that diminish the GAP activity of p115rgRGS also reduce its affinity for Galpha 13. A quantitative relationship between affinity and activity cannot be deduced from the data presented here, as is the case for RGS4 (18); however, no mutant capable of binding Galpha 13 is devoid of GAP activity. It therefore appears that the GAP activity of p115rgRGS arises from its ability to bind and stabilize a conformational state of Galpha 13 which is conducive to transition state formation. The mechanism by which rgRGS acts in this role may be in part elucidated by structural studies now in progress. The requirement for specific N-terminal residues outside of the conserved RGS box indicates that the rgRGS domains have developed alternate mechanisms to stabilize such a state in the G12 subfamily of heterotrimeric G proteins.

The rgRGS domain contributes to the intrinsic nucleotide exchange activity of p115RhoGEF as demonstrated by the 60% reduction of this activity when the domain is deleted (30). However the most notable effect that results from this truncation is a total loss of the ability of p115RhoGEF to be stimulated by Galpha 13 (15). One hypothesis is that it is the binding of the rgRGS to Galpha 13 which facilitates this regulation. This is consistent with the observation that the regulation of exchange activity by Galpha 13 was retained when the rgRGS region of GTRAP48 replaced the endogenous rgRGS domain of p115RhoGEF (15). Yet, as we show here, mutations that compromise the ability of the rgRGS domain to bind to Galpha 13 do not affect the susceptibility of p115RhoGEF to activation by Galpha 13. This finding is consistent with a model in which the rgRGS domain plays a structural or allosteric role, for example, by conferring stability upon conformational states of other segments of p115RhoGEF which are required for Galpha 13-mediated stimulation of GEF activity. The reduction of inherent GEF activity upon deletion of the rgRGS domain (30) is evidence for direct intramolecular coupling between the rgRGS and Dbl homology/pleckstrin homology domains. This raises the intriguing possibility that the ability of the rgRGS domain to affect the efficacy of Galpha 13 might itself be subject to regulatory mechanisms such as covalent modification, which have yet to be discovered.

The data presented here also suggest that stimulation of p115RhoGEF by Galpha 13 results from interaction of the alpha  subunit at a surface on p115RhoGEF different from the rgRGS domain. Previous binding studies of Galpha 13 to p115RhoGEF detected a second site for interaction between residues 288 and 760, which includes the Dbl homology and pleckstrin homology domains (15). The surface of Galpha 13 which binds the rgRGS domain and that which engages the second site are most likely distinct. This could resemble the interaction demonstrated by Slep et al. (7), that Galpha t binds the gamma  subunit of cyclic GMP phosphodiesterase and RGS9 simultaneously at separate yet interacting interfaces (7). The mechanisms by which Galpha 13 interacts with regions beyond the rgRGS domain of p115RhoGEF and exploitation of the coupling between this and other domains of the molecule remain to be explored.

    ACKNOWLEDGEMENTS

We thank Stephen Gutowski and Abiola Badejo for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM31954 (to P. C. S.), DK46371 (to S. R. S.), and GM07062 (to C. D. W.), by the Robert A. Welch foundation (to P. C. S. and S. R. S.), the Alfred and Mabel Gilman chair in molecular pharmacology (to P. C. S.), and the John W. and Rhonda K. Pate professorship in biochemistry (to S. R. S.).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.

§ These authors contributed equally to this work.

|| Current address: Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada.

Dagger Dagger To whom correspondence may be addressed: Dept. of Biochemistry, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: stephen.sprang@utsouthwestern.edu.

§§ To whom correspondence may be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-9041. Tel.: 214-648-2835; Fax: 214-648-2971; E-mail: paul.sternweis@utsouthwestern.edu.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212695200

2 T. Kozasa, personal communication.

    ABBREVIATIONS

The abbreviations used are: RGS, regulator of G protein signaling; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GST, glutathione S-transferase; rg, RhoGEF; Rho, Ras homology; % V0WT, initial velocity of a reaction in the presence of the mutant normalized by the rate in the presence of wild type p115rgRGS.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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