Substitution of transducin ser202 by asp abolishes G-protein/RGS interaction.

Known RGS proteins stimulate GTPase activity of Gi and Gq family members, but do not interact with Gsalpha and G12alpha. To determine the role of specific Galpha residues for RGS protein recognition, six RGS contact residues of chimeric transducin alpha-subunit (Gtalpha) corresponding to the residues that differ between Gialpha and Gsalpha have been replaced by Gsalpha residues. The ability of human retinal RGS (hRGSr) to bind mutant Gtalpha subunits and accelerate their GTPase activity was investigated. Substitutions Thr178 --> Ser, Ile181 --> Phe, and Lys205 --> Arg of Gtalpha did not alter its interaction with hRGSr. The Lys176 --> Leu mutant had the same affinity for hRGSr as Gtalpha, but the maximal GTPase stimulation by hRGSr was reduced by approximately 2.5-fold. The substitution His209 --> Gln led to a 3-fold decrease in the affinity of hRGSr for the Gtalpha mutant without significantly affecting the maximal GTPase enhancement. The Ser202 --> Asp mutation abolished Gtalpha recognition by hRGSr. A counteracting replacement of Glu129 by Ala in hRGSr did not restore the interaction of hRGSr with the Gtalpha Ser202 --> Asp mutant. Our data suggest that the Ser residue at position 202 of Gtalpha is critical for the specificity of RGS proteins toward Gi and Gq families of G-proteins. Consequently, the corresponding residue, Asp229 of Gsalpha, is likely responsible for the inability of RGS proteins to interact with Gsalpha.

Heterotrimeric GTP-binding proteins (G-proteins) are components of many major signaling systems that are used by cells to transduce a variety of signals from specific cell surface receptors to intracellular effector proteins. Regulation of Gprotein GTPase activity represents an important mechanism for establishing proper signal duration. A novel class of proteins called RGS 1 for regulators of G-protein signaling has been identified (1)(2)(3)(4)(5). Evidence has been accumulated that members of this family negatively regulate signaling via G i and G q -like G-proteins by stimulating their GTPase activity (6 -10). Identification of RGS proteins has helped to solve a long standing discrepancy between the fast signal termination in vivo and relatively slow intrinsic GTPase rates typically observed under in vitro conditions (6,11). However, no RGS protein or other GTPase-activating protein (GAP) specific toward G s ␣ has been described to date (9,10). The recently solved crystal structure of RGS4 bound to G i ␣ 1 ⅐AlF 4 Ϫ provides the first structural insights into the mechanism of RGS protein GAP function and offers a starting point for studying the structural basis of the specificity of known RGS proteins (12). RGS4 interacts with the switch regions of G i ␣ 1 that are likely to have a similar general conformation with the corresponding regions of G s ␣ (12). The incompetence of RGS proteins to bind and stimulate the GTPase activity of G s ␣ therefore originates from the differences between amino acid residues of G i ␣ 1 contacting RGS4 and corresponding residues of G s ␣.
In this study we investigate molecular determinants of the specificity of RGS/G-protein interaction using transducin ␣-subunit (G t ␣) as a prototypical member of the G i family and a human homologue (hRGSr) of mouse retinal mRGSr (13,14). We have carried out mutational analysis of specific amino acid residues of chimeric G t ␣ corresponding to the RGS contact residues that are different between G i ␣ and G s ␣ to determine their specific role for RGS protein recognition.

EXPERIMENTAL PROCEDURES
Materials-GTP was a product of Boehringer Mannheim. [␥-32 P]GTP (Ͼ5000 Ci/mmol) was purchased from Amersham Corp. All other chemicals were acquired from Sigma.
Preparation of Rod Outer Segment (ROS) Membranes, G t ␤␥ and hRGSr-Bovine ROS membranes were prepared as described previously (15). Urea-washed ROS membranes (uROS) were prepared according to protocol in Ref. 16. G t ␤␥ was prepared by the procedure described in Ref. 17. GST-hRGSr and hRGSr were prepared and purified as described previously (14). The purified proteins were stored in 40% glycerol at Ϫ20°C or without glycerol at Ϫ80°C.
Site-directed Mutagenesis of Chimeric G t ␣-Mutagenesis of G t ␣ residues was performed using the vector for expression of His 6 -tagged G t ␣/G i ␣ 1 chimera 8 (Chi8) as a template for PCR amplifications (18). The G t ␣ Lys 176 3 Leu and Thr 178 3 Ser substitutions were introduced using 5Ј-primer 1 and 3Ј-primers 2 and 3, respectively, for PCR amplification (see below). The PCR products were digested with BsmBI and subcloned into the BsmBI-digested pHis 6 Chi8. Primer 3 also contained silent mutations creating the unique XbaI site that was used to make the Ile 181 3 Phe mutant. The 5Ј-primer 4 and 3Ј-primer 5 were used to obtain the PCR product carrying the Ile 181 3 Phe mutation. The product was cut with XbaI and HindIII and subcloned into the XbaI/HindIIIdigested pHis 6 Chi8 Thr 178 3 Ser. The Ser 202 3 Asp and Lys 205 3 Arg substitutions were introduced by PCR-directed mutagenesis using 5Јprimer 6 and 3Ј-primers 7 and 8, respectively, followed by insertion of the NcoI/BamHI-digested PCR products into pHis 6 Chi8. Mutation His 209 3 Gln was generated using 5Ј-primer 9 and 3Ј-primer 5 and subcloning of the PCR product into the BamHI and HindIII sites of pHis 6 Chi8. The sequences of all mutants were verified by automated DNA sequencing at the University of Iowa DNA Core Facility. Chi8 and all mutants were expressed and purified as described previously (18). The purified proteins were tested in the trypsin protection assay as described (19). The following primers were used to generate mutant Site-directed Mutagenesis of hRGSr-A substitution Glu 129 3 Ala of hRGSr was performed using PCR amplifications from the pGEX-KG-hRGSr template (14) similarly as described (20). GST-hRGSr and the mutant were expressed in DH5␣ Escherichia coli cells, and the GST portion was removed as described earlier (14).
Binding of Chimeric G t ␣ and Its Mutants to GST-hRGSr-Chi8 or its mutants (1 M final concentration) were mixed with glutathione-agarose retaining ϳ10 g of GST-hRGSr in 200 l of 20 mM HEPES buffer (pH 7.6) containing 100 mM NaCl, 2 mM MgCl 2 , 30 M AlCl 3 , and 10 mM NaF (buffer A). After incubation for 20 min at 25°C, the agarose beads were spun down, washed three times with 1 ml of buffer A, and the bound proteins were eluted with a sample buffer for SDS-polyacrylamide gel electrophoresis.
Single Turnover GTPase Assay-Single turnover GTPase activity measurements were carried out in suspensions of uROS membranes (5 M rhodopsin) reconstituted with chimeric G t ␣ or its mutants (2 M) and G t ␤␥ (1 M) essentially as described in Refs. 14 and 21. Bleached uROS membranes were mixed with different concentrations of hRGSr or hRGSrGlu 129 3 Ala and preincubated for 5 min at 25°C. The GTPase reaction was initiated by addition of 100 nM [␥-32 P]GTP (ϳ4 ϫ 10 5 dpm/pmol). The GTPase rate constants were calculated by fitting the experimental data to an exponential function: % GTP hydrolyzed ϭ 100(1 Ϫ e Ϫkt ), where k is a rate constant for GTP hydrolysis.
Miscellaneous-Protein concentrations were determined by the method of Bradford (22) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (23) in 12% acrylamide gels. Rhodopsin concentrations were measured using the difference in absorbance at 500 nm between "dark" and bleached ROS preparations. Fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prizm (version 2) software. The results are expressed as the mean Ϯ S.E. of triplicate measurements.

Effects of hRGSr on GTPase Activity of G t ␣ Mutants-Six
residues directly interacting with RGS4 are different in G i ␣ 1 and G s ␣ (12). These residues correspond to Lys 176 , Thr 178 , Ile 181 , Ser 202 , Lys 205 , and His 209 of G t ␣. Except for a conservative substitution, G t ␣ Ile 181 /G i ␣ 1 Val 185 , these residues are identical in G t ␣ and G i ␣ 1 . To analyze functional consequences of the replacement of these G t ␣ residues by corresponding G s ␣ residues we took advantage of the efficient expression of functional G t ␣/G i ␣ 1 chimeras in E. coli (18). All the G t ␣ mutants were made based on Chi8 that contains 80% of G t ␣ amino acid sequence, including all three G t ␣ switch regions (18). Analysis of Chi8 GTPase activity showed properties similar to native G t ␣. The GTP hydrolysis by Chi8 alone or in the presence of uROS was negligible (not shown). In the presence of both, uROS and G t ␤␥, the basal GTPase activity of Chi8 was 0.016 Ϯ 0.002 s Ϫ1 (Fig. 1). A similar rate of GTP hydrolysis (0.019 s Ϫ1 ) was observed earlier for holotransducin, G t ␣␤␥, reconstituted with uROS under similar conditions (14). This suggests that despite a lack of myristoylation and the His 6 -tag attached to the N terminus, Chi8 was competent to interact with G t ␤␥ and light-activated rhodopsin. The GTPase activity of Chi8 was substantially enhanced in the presence of hRGSr. Addition of 1 M hRGSr led to acceleration of the GTPase activity by almost 8-fold (k ϭ 0.126 Ϯ 0.018 s Ϫ1 ) (Fig. 1). Stimulation of GTPase activity of transducin by hRGSr under similar conditions was ϳ10-fold (14). Furthermore, the dose dependence of the stimulation of Chi8 GTPase activity by hRGSr yielded an EC 50 value of 109 Ϯ 15 nM ( Fig. 2A), which correlates well with the EC 50 value of 85 nM for the effect of hRGSr on transducin (24). Effects of hRGSr on the GTPase activity of Chi8 suggest that this chimeric G-protein was an appropriate target for the sitedirected mutagenesis.
Binding of G t ␣ Mutants to GST-hRGSr-Binding between the G t ␣ mutants and hRGSr was examined using precipitation of mutants by glutathione-agarose beads containing immobi-

Mutational Analysis of RGS Contact Residues in G t ␣/G i ␣ Chimera 4301
lized GST-hRGSr. hRGSr, as many other RGS proteins, binds with high affinity to the AlF 4 Ϫ conformation of G-protein ␣-subunits (7,9,14). The binding assay demonstrated that GST-hRGSr in the presence of AlF 4 Ϫ was able to precipitate nearly stoichiometric amounts of Chi8, and all of the G t ␣ mutants, except G t ␣ Ser 202 3 Asp (Fig. 3A). The competence of hRGSr to efficiently precipitate the mutant Lys 176 3 Leu is consistent with the EC 50 value of 129 nM for the stimulation of its GTPase activity, even though the maximal GTPase enhancement by hRGSr for this mutant was substantially decreased. G t ␣ Ser 202 3 Asp failed to bind GST-hRGSr using this assay (Fig. 3A). The failure of G t ␣ Ser 202 3 Asp to bind GST-hRGSr is not caused by its inability to bind AlF 4 Ϫ and assume an active conformation. Chi8 and the Ser 202 3 Asp mutant demonstrated equivalent degrees of protection of their switch II region from tryptic cleavage upon binding of AlF 4 Ϫ (Fig. 3B). The binding data indicate correlation between the stimulatory effects of hRGSr on the G t ␣ mutants in the GTPase assay and ability of hRGRr to bind these mutants. The deficiency of hRGSr to stimulate GTPase activity of G t ␣ Ser 202 3 Asp has resulted from the loss of the affinity of this interaction. However, the Lys 176 3 Leu substitution appeared to produce a different result. The reduction in the maximal GTPase acceleration of G t ␣ Lys 176 3 Leu occurred without a concurrent decrease in affinity of the G-protein/RGS interaction.
Effects of the hRGSr Mutant Glu 129 3 Ala on GTPase Activity of Chimeric G t ␣ and Its Ser 202 3 Asp Mutant-Based on the crystal structure of RGS4 bound to G i ␣ 1 ⅐AlF 4 Ϫ (12), a residue Ser 202 makes a contact with hRGSr residue Glu 129 . We have tested the possibility that a complementary replacement of hRGSr residue Glu 129 by Ala would restore the ability of hRGSr to interact and stimulate GTPase activity of G t ␣ Ser 202 3 Asp. hRGSr Glu 129 3 Ala was fully active toward Chi8 and five of its mutants, but deficient of any GAP activity toward G t ␣ Ser 202 3 Asp (not shown). Similarly, to the wild type hRGSr, the Glu 129 3 Ala mutant failed to bind G t ␣ Ser 202 3 Asp, whereas its binding to Chi8 was intact (Fig. 3C). DISCUSSION Since its recent discovery, the family of RGS proteins has been rapidly growing. Those RGS proteins that have already been extensively characterized share a common specificity pattern. These RGS proteins interact with G-protein ␣-subunits from G i and G q families but have no activity toward G s (6 -8, 10) and G 12 (9). Both possibilities remain open: a member(s) of the RGS family capable of interaction with G s ␣ (G 12 ␣) has not been yet identified or characterized, or none of the RGS proteins would be a GAP for G s ␣ (G 12 ␣). The answer to this question lies in understanding the structural details and requirements for RGS/G-protein interaction.
The crystal structure of the complex of RGS4 with G i ␣ 1 ⅐AlF 4 Ϫ has revealed a structural basis for the inability of RGS4 to interact with G s ␣. Six amino acid residues from the RGS/Gprotein interface are different between G i ␣ and G s ␣ (12). Three of these residues, corresponding to Thr 178 , Ser 202 , and His 209 in G t ␣ are conserved among the G i ␣, G t ␣, G q ␣, and G z ␣ subunits that are known to interact with RGS. Another two G t ␣ residues, Ile 181 and Lys 205 , have homologous substitutions. Ile 181 is substituted by Val in G i ␣ and G z ␣, and Lys 205 is replaced by Arg in G q ␣. To identify the residue(s) critical for the failure of G s ␣ to interact with RGS proteins, we replaced the RGS contact residues in G t ␣ by corresponding residues in G s ␣ and examined the ability (EC 50 and V max ) of hRGSr to stimulate GTPase activity of these mutants. hRGSr is a human homologue (hRGSr) of mouse retinal mRGS, which was originally thought to be a retina-specific RGS protein, but later it was found in other tissues as well (13,25). Like other characterized RGS proteins, hRGSr interacts with G i -and G q -like ␣-subunits, but does not bind G s ␣ (24). Substitutions Thr 178 3 Ser, Ile 181 3 Phe, and Lys 205 3 Arg did not significantly alter the activity of hRGSr toward these mutants. While this was not unexpected for the conservative replacement Lys 205 3 Arg, it was rather surprising for the Thr 178 3 Ser mutant. The corresponding G i ␣ 1 Thr 182 residue interacts with seven invariant or highly conserved residues of RGS4 and, thus, even homologous substitution by Ser could have had a major impact on the G␣/RGS interaction (12). It appears that Ser may substitute Thr 178 Mutational Analysis of RGS Contact Residues in G t ␣/G i ␣ Chimera 4302 suitably in most of the RGS contacts. Another substitution that did not interfere with the affinity of G t ␣ binding to hRGSr is Lys 176 3 Leu. This is consistent with the lack of conservation at this position between G t ␣, G q ␣, and G z ␣. Interestingly, however, this mutation led to a substantial reduction in the GTPase V max value elicited by hRGSr. Perhaps the lower stimulated GTPase activity of the Lys 176 3 Leu mutant reflects an intrinsic partial impairment of the catalytic site not evident from the basal GTPase activity. The adjacent G t ␣ Thr 177 residue is intimately involved in the GTP hydrolysis (26) and may not be fully stabilized in the RGS/G t ␣ Lys 176 3 Leu complex. The Lys 176 3 Leu mutation highlights the possibility that G s ␣ may have a limited ability for stimulation by RGS proteins assuming there is one that binds G s ␣. A modest decrease in the affinity for hRGSr without significantly affecting the maximal degree of the GTPase rate acceleration was observed for G t ␣ His 209 3 Gln. The most severe outcome for the G t ␣/hRGSr interaction was caused by the Ser 202 3 Asp mutation. This mutation resulted in the loss of hRGSr binding. The crystal structure of G i ␣ 1 with RGS4 provides a rationale for such an outcome (12). A negative charge introduced by the Asp residue might be repelled by the negative charge of the counteracting Glu 129 residue of hRGSr, which corresponds to the Glu 126 residue of RGS4. However, the Glu residue is not absolutely conserved in RGS proteins. A number of RGS proteins, RGS1, RGS6, and RGS7, have residues other than Glu at this position. Small uncharged residues such as the Ala residue in RGS7 might be the most accommodating residue for Asp. We found that the Glu 129 3 Ala substitution in hRGSr cannot rescue the ability of hRGSr to interact with G t ␣ Ser 202 3 Asp. Perhaps, additional residue(s) such as Asn 131 of hRGSr (Asn 128 of RGS4) also interferes with the Asp side chain. RGS4 Asn 128 makes a contact with G i ␣ 1 Ser 206 (Ser 202 of G t ␣). The RGS Asn residue is critical for the RGS/G␣ interaction (12), and may only be substituted by Ser, though with a notable loss of the RGS affinity for G t ␣ (20). Quite possibly, an interference of the G␣ Asp residue with the network of interactions involving the hRGSr Asn 131 residue is also responsible for the lack of interaction between hRGSr and G t ␣ Ser 202 3 Asp.
The degree of impairment of the RGS/G␣ interaction in the Ser 202 3 Asp mutant allows us to speculate that the corresponding Asp 229 of G s ␣ is mainly responsible for the inability of G s ␣ to interact with characterized RGS proteins. Other differ-ences in RGS contact residues between G s ␣ and the G i -like ␣-subunits could be more easily accommodated by limited variability of different RGS domains. Our results do not support a likelihood that one of the currently identified RGS proteins may serve as a GAP for G s ␣. Nevertheless, they provide a direction toward identification of potential candidates for interaction with G s ␣ among yet undiscovered RGS proteins.