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Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins

Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 12, Issue of March 20, pp. 6731–6735, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins* (Received for publication, September 8, 1997, and in revised form, November 20, 1997) Michael Natochin, Randall L. McEntaffer, and Nikolai O. Artemyev‡ From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242 Members of the RGS family serve as GTPase-activat- spectively (1–3). A novel class of GTPase-activating proteins (GAPs) for heterotrimeric G-proteins called RGS has been iden- ing proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled re- tified (4 – 6). Strong evidence suggests that members of this ceptors. The recently resolved crystal structure of RGS4 family, GAIP, RGS4, RGS1, RGS10 and others, negatively reg- a suggests two potential mechanisms for bound to G i 1 ulate G-protein signaling by stimulating GTPase activity of the GAP activity of RGS proteins as follows: stabiliza- G-proteins, particularly those from G and G families (7–9). i q a switch regions by RGS4 and the catalytic tion of the G i 1 RGS proteins from yeast to mammals share a highly conserved . To elucidate a role of the action of RGS4 residue Asn RGS domain that provides relatively broad specificity of differ- Asn residue for RGS GAP function, we have investigated ent RGS proteins toward members of the two G-protein classes effects of the synthetic peptide corresponding to the G in vitro. Tissue expression patterns and diverse domains out- binding domain of human retinal RGS (hRGSr) contain- side the RGS segment may play an important role in determin- ing the key Asn at position 131, and we have carried out ing specificity of RGS proteins in vivo (10 –12). Precise mecha- . Synthetic peptide hRGSr- mutational analysis of Asn nisms of RGS GAP activity are not yet clear. The transition -complexed (123–140) retained its ability to bind the AlF state during GTP hydrolysis is thought to be mimicked by the a-subunit, G azAlF , but failed to elicit stim- transducin t 4 AlF -bound conformation of Ga subunits (13, 14). It has been a GTPase activity. Wild-type hRGSr stimu- ulation of Gt demonstrated that many RGS proteins interact preferentially a GTPase activity by ;10-fold with an EC value lated G t 50 with the AlF -bound conformation of Ga subunits and thus M. Mutant hRGSr proteins with substitutions of 4 of 100 n may accelerate GTP hydrolysis through stabilization of the by Ser and Gln had a significantly reduced affin- Asn transitional state of G-proteins (8, 15, 16). a but were capable of substantial stimulation of ity for G Recently, the crystal structure of RGS4 bound to G a zAlF a GTPase activity, 80 and 60% of V , respectively. G i 1 4 t max 131 131 131 , hRGSr-Ala , and hRGSr-Asp has been solved at a resolution of 2.8 Å (17). This structure Mutants hRGSr-Leu were able to accelerate G a GTPase activity only at very provides the first structural insights into the mechanism of mM) which appears to corre- high concentrations (>10 RGS protein action. The conserved RGS core forms three dis- late with a further decrease of their affinity for trans- tinct sites of interaction with the three switch regions of G a i 1 131 131 and hRGSr-D , had ducin. Two mutants, hRGSr-His suggesting that stabilization of the switch regions and G res- no detectable binding to transducin. Mutational analy- idues directly involved in GTP hydrolysis may be a major suggests that the stabilization of the G- sis of Asn component of RGS GAP activity (17). Furthermore, RGS pro- protein switch regions rather than catalytic action of teins could also contribute catalytic residues to the active site the Asn residue is a key component for the RGS GAP and thus enhance the GTPase rate constant. The conserved action. 128 residue Asn of RGS4 makes a contact with the side chain of Gln of G a which stabilizes and orients the hydrolytic water i 1 molecule in the transitional state of G a (17). Asn also may i 1 The intensity and duration of signaling via heterotrimeric be localized within hydrogen-bonding distance of the hydrolytic G-proteins is regulated at multiple levels. The key reaction in water molecule for nucleophilic attack on the GTP g-phosphate termination of G-protein-mediated signaling is the intrinsic (17). GTPase activity of Ga subunits that convert the active GTP- In this study we evaluate a potential catalytic role of the Asn bound conformation of G-protein a subunits (GazGTP) to the residue for G GTPase acceleration by RGS proteins using the inactive GazGDP conformation. The GTPase activity of two interaction between human retinal RGS (hRGSr) protein and G-proteins, G and transducin, is stimulated by their effectors, transducin as a model system and mutational analysis of phospholipase Cb and cGMP phosphodiesterase (PDE), re- 131 128 Asn of hRGSr which is equivalent to Asn of RGS4. EXPERIMENTAL PROCEDURES * This work was supported by National Institutes of Health Grant Materials—GTP and GTPgS were products of Boehringer Mann- EY-10843. The services provided by the Diabetes and Endocrinology heim. Blue-Sepharose and PD-10 Sephadex G-25 columns were ob- Research Center of the University of Iowa were supported by National tained from Pharmacia Biotech Inc. [g- P]GTP (.5000 Ci/mmol) was Institutes of Health Grant DK-25295. The costs of publication of this purchased from Amersham Corp. [ S]GTPgS (1250 Ci/mmol) was ob- article were defrayed in part by the payment of page charges. This tained from NEN Life Science Products. All other chemicals were ac- article must therefore be hereby marked “advertisement” in accordance quired from Sigma. with 18 U.S.C. Section 1734 solely to indicate this fact. Preparation of Rod Outer Segment (ROS) Membranes, G azGTPgS, ‡ To whom correspondence and reprint requests should be addressed: G azGDP, and hRGSr—Bovine ROS membranes were prepared as de- Dept. of Physiology and Biophysics, University of Iowa College of Med- t scribed previously (18). Hypotonically washed ROS membranes (dROS) icine, 5– 660 Bowen Science Bldg., Iowa City, IA 52242.. Tel.: 319-335- depleted of PDE subunits were prepared as described in Ref. 2. Trans- 7864; Fax: 319-335-7330; E-mail: [email protected]. ducin, G abg, was extracted from ROS membranes using GTP as de- The abbreviations used are: PDE, cGMP phosphodiesterase; RGS proteins, regulators of G-protein signaling; hRGSr, human retinal RGS protein; ROS, rod outer segment(s); dROS, hypotonically washed ROS membranes; G a, rod G-protein (transducin) a-subunit; GTPgS, guanosine 59-O-(3-thiotriphosphate); PCR, polymerase chain reaction. This paper is available on line at http://www.jbc.org 6731 This is an Open Access article under the CC BY license. 131 6732 Functional Role of Asn of Human Retinal RGS scribed in Ref. 19. The G azGTPgS was extracted from ROS membranes using GTPgS and purified by chromatography on Blue-Sepharose CL-6B by the procedure described in Ref. 20. G azGDP was prepared and purified according to protocols in Ref. 21. hRGSr was prepared and purified as described previously (22). The purified proteins were stored in 40% glycerol at 220 °C or without glycerol at 280 °C. Site-directed Mutagenesis of hRGSr—Mutagenesis of Asn residue of hRGSr was performed using PCR amplifications from the pGEX-KG- hRGSr template (22) with 39-antisense primer ATGCCTCGAGACT- CAGGTGTGTGAGG (unique XhoI site is underlined) and the 59 prim- ers: XXXATTGACCATGAGACCCGCGAGC. XXX indicates nucleotides that generate substitutions of Asn (AAC) in hRGSr cDNA by the following amino acid residues: Ala (GCG), Asp (GAT), His (CAT), Leu (CTG), Gln (CAG), Ser (AGC), and deletion mutant (—). PCR reactions were performed in 100 ml of reaction mixture containing 1 ng of the pGEX-KG-hRGSr plasmid, 3 units of AmpliTaq DNA polymerase (Per- M Tris-(hydroxymethyl)-methylaminopropane sulfonic kin-Elmer), 25 m FIG.1. Effect of hRGSr-(123–140)-peptide on basal and hRGSr- acid, pH 9.3, 2 mM MgCl ,1mM 2-mercaptoethanol, 200 mM of dNTPs, stimulated GTPase activity of transducin. dROS membranes (5 mM and 0.5 mM primers. Conditions for PCR were as follows: 94 °C for 3 min, rhodopsin and 0.4 mM transducin) were incubated for 5 min with in- 30 cycles of 94 °C for 1 min, 64 °C for 30 s and 72 °C for 30 s, and a final creasing concentrations of peptide hRGSr-(123–140) with (squares)or extension at 72 °C for 3 min. The PCR products (;220 base pairs) were without (circles) addition of 100 nM hRGSr. The GTPase reaction was blunt-ended with Klenow fragment and digested with XhoI. Wild-type 32 initiated by addition of 100 nM [g- P]GTP. Calculated GTPase rate hRGSr cDNA was subcloned into XbaI/XhoI sites of pBluescript constants are plotted as a function of peptide concentration. The com- polylinker. The resulting construct was digested with HincII and XhoI petition curve (squares,IC 5 1.6 6 0.3 mM; Hill slope 5 0.64) fits the and ligated with the XhoI-digested PCR products carrying mutations. data with r 5 0.98. The mutant sequences were verified by automated DNA sequencing at the University of Iowa DNA Core Facility using the T7 primer and subcloned into the XbaI/XhoI sites of pGEX-KG vector for protein ex- squares criteria using GraphPad Prizm (version 2) software. The re- pression. Mutant GST-hRGSr proteins were expressed in DH5a Esch- sults are expressed as the mean 6 S.E. of triplicate measurements. erichia coli cells, and the GST portion was removed as described earlier RESULTS (22). Typical yields of purified hRGSr and hRGSr mutants, except for a mutant with deletion of Asn , were 5– 6 mg/liter of culture. Deletion of Effects of Synthetic Peptide hRGSr-(123–140)—The G a bind- led to an ;4 –5-fold reduction in expression of soluble recombi- Asn ing region of RGS4 containing Asn resides in the loop a5–a6 nant protein suggesting that the residue at position 131 may be impor- of RGS4 and contains 7 amino acid residues making contacts tant to the stability and proper folding of RGS proteins. with all three switch regions of G-protein (17). The number of Binding of Transducin to GST-hRGSr and Mutants—G azGTPgSor interactions is sufficient to ensure a relatively high affinity G azGDP (10 mg) were incubated with hRGSr or its mutants (50 mg) between the corresponding synthetic peptide and Ga, provided immobilized on glutathione-agarose in 100 mlof20mM Tris-HCl buffer (pH 8.0), containing 100 mM NaCl, 2 mM MgSO ,6mM 2-mercaptoeth- that the peptide is able to adopt a functional conformation. For anol, and 5% glycerol (buffer A). Where indicated, the buffer contained the preliminary testing of the catalytic role of Asn of hRGSr, mM AlCl and 10 mM NaF. After incubation for 20 min at 25 °C, the the hRGSr-(123–140)-peptide was synthesized. The length of agarose beads were spun and washed twice with 1 ml of buffer A, and the peptide was chosen to allow the Ga contact residues to be the bound proteins were eluted with a sample buffer for SDS-polyacryl- flanked by at least 3 terminal residues. hRGSr-(123–140) was amide gel electrophoresis. first examined for its ability to stimulate GTPase activity of Single Turnover GTPase Assay—Single turnover GTPase activity transducin in suspensions of dROS membranes containing 5 measurements were carried out in suspensions of dROS membranes containing 5 mM rhodopsin and 0.4 mM transducin essentially as de- mM rhodopsin and 0.4 mM transducin. dROS membranes lacked scribed in Refs. 22 and 23. Transducin concentration of 0.4 mM was intrinsic catalytic PDEab and inhibitory PDEg subunits. Use S]GTPgS binding assay as described previously determined using the [ of such ROS avoided interference of PDEg effects with effects of (22). Bleached dROS membranes were mixed with different concentra- RGS protein or RGS peptide (22, 27, 28). The peptide at con- tions of the tested peptides, hRGSr or hRGSr mutants, and preincu- centrations of up to 2 mM had no effect on GTPase activity of bated for 5 min at 25 °C. The GTPase reaction was initiated by addition 32 4 transducin (not shown). To determine if hRGSr-(123–140) is of 100 nM [g- P]GTP (;5 3 10 dpm/pmol) in a total volume of 20 ml. At 5, 10, 20, 40, and 60 s aliquots of the reaction mixture were withdrawn capable of binding to transducin, we investigated effects of the and quenched with 7% perchloric acid. Nucleotides were then precipi- hRGSr peptide on the stimulation of GTPase activity of trans- tated using activated Norit A charcoal (10% w/v) in 50 mM sodium ducin by hRGSr. Fig. 1 shows that hRGSr-(123–140) was able P formation was measured by liquid phosphate buffer (pH 7.5), and to compete with hRGSr for binding to G a resulting in a dose- scintillation counting. The GTPase rate constants were calculated by dependent (IC 5 1.6 6 0.3 mM) decrease of the stimulated fitting the experimental data to an exponential function: % GTP hydro- 2kt GTPase activity of transducin. hRGSr-(123–140) in the same lyzed 5 100 (1 2 e ), where k is a rate constant for GTP hydrolysis. range of concentrations had no notable effect on the basal Peptide Synthesis—A peptide, CSEAPKEVNIDHETRELT, corre- sponding to residues 123–140 of hRGSr was custom made by Genosys transducin GTPase activity (Fig. 1). Because the competition Biotechnologies Inc. The N and C termini of the peptide were acetylated experiments were carried out at a concentration of hRGSr and amidated, respectively. The peptide was purified by reverse-phase causing half-maximal stimulation of the GTPase activity, the high pressure liquid chromatography on a preparative Dynamax-300A affinity of hRGSr-(123–140) for G a can be estimated as 0.8 mM. column (Rainin). The purity and chemical formula of the peptide were In control experiments, four unrelated peptides (24) corre- confirmed by fast atom bombardment-mass spectrometry and analyti- sponding to residues 21–31, 461–553, 492–516, and 517–541 of cal high pressure liquid chromatography. Preparation of synthetic pep- the rod PDE a-subunit (at concentrations of 8 mM) had no effect tides corresponding to residues 21–31, 461– 491, 492–516, and 517–541 of rod PDE a-subunit was described previously (24). on hRGSr-stimulated GTPase activity of transducin (not Miscellaneous—Protein concentrations were determined by the shown). method of Bradford (25) using IgG as a standard or using calculated 131 Binding of hRGSr Mutants with Substitutions of Asn to extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophore- Different Conformations of G a—Recently, we have shown that sis was performed by the method of Laemmli (26) in 12% acrylamide similar to other characterized RGS proteins, hRGSr binds with gels. Rhodopsin concentrations were measured using the difference in high affinity to the AlF conformations of transducin and very absorbance at 500 nm between “dark” and bleached ROS preparations. 4 Fitting of the experimental data was performed with nonlinear least weakly to the GTPgS and GDP-bound conformations (22). We 131 Functional Role of Asn of Human Retinal RGS 6733 FIG.2. Binding of GST-hRGSr and its mutants to G a. SDS- FIG.3. Stimulation of GTPase activity of transducin by mutant polycrylamide gel (12%) stained with Coomassie Blue. Binding of G a hRGSr. GTPase activity of transducin was measured in suspensions of GDPzAlF (A), G azGTPgS(B), G azGDP (C), to GST-hRGSr or its mu- 4 t t dROS (5 mM rhodopsin and 0.4 mM transducin) in the presence of tants immobilized on glutathione-agarose was performed as described increasing concentrations of hRGSr or its mutants. Calculated GTPase under “Experimental Procedures.” Lane 1,G a; lane 2, GST-hRGSr; rate constants are plotted as a function of hRGSr or mutant concentra- mutants of hRGSr with substitution of Asn : lane 3, Ala; lane 4, Asp; tion. hRGSr, closed squares; hRGSr-Ser , open squares; hRGSr- lane 5, His; lane 6, Leu; lane 7, Gln; lane 8, Ser; lane 9, D; lane 10, 131 131 131 Gln , closed diamonds; hRGSr-Asp , open diamonds; hRGSr-Leu , glutathione-agarose without bound GST-RGS protein (control). w.t., 131 131 closed triangles; hRGSr-Ala , open triangles; hRGSr-His , closed wild type. circles; and hRGSr-D , open circles. evaluated the interaction between hRGSr mutants with sub- activity by these mutants due to the very high protein concen- stitutions of Asn by Ser, Gln, Ala, Leu, His, Asp as well as trations required. Two mutants, hRGSr-His and hRGSr- the mutant with deletion of Asn and transducin using pre- D , did not show GAP activity at the concentration tested (40 cipitation of G a with the GST-hRGSr mutant proteins immo- mM). Interestingly, the potency of hRGSr mutants in stimulat- bilized on glutathione-agarose beads. Mutations hRGSr-Ser ing G a GTPase activity (Fig. 3) appears to correlate well with and hRGSr-Gln led to a reduction in affinity of the corre- their ability to bind and precipitate G azAlF (Fig. 2A), t 4 sponding GST fusion proteins for G azAlF (Fig. 2A). Mutants t 4 Competition between hRGSr and hRGSr Mutants in Stimu- 131 131 131 hRGSr-Leu , hRGSr-Asp , and hRGSr-Ala showed a lation of G a GTPase Activity—Experiments in Fig. 2 have more significant decrease in their affinity for the G a confor- t suggested that hRGSr mutants with substitutions of Asn 131 131 mation (Fig. 2A). hRGSr-His and hRGSr-D failed to co- have impaired binding to G azAlF . The binding assay may, t 4 2 131 precipitate G azAlF . Mutations of Asn could potentially t 4 however, not be sufficiently sensitive to detect relatively weak alter hRGSr interaction with G azGTPgS and G azGDP since interactions. To determine if the drastically reduced ability of t t the RGS4 Asn residue makes contact with the switch I and II some RGS mutants to stimulate the GTPase activity of trans- regions of G a (17). We have tested this possibility by prein- ducin correlates with the lack of mutant binding to transducin, i 1 cubating mutant GST-hRGSr containing beads with both con- we carried out competition experiments. The hRGSr mutants formations of G a. None of the seven hRGSr mutants has incapable of accelerating G a GTPase activity were examined demonstrated enhanced affinity for either conformation of G a t for their ability to block stimulation of GTPase activity of compared with the native GST-hRGSr (Fig. 2, B and C). transducin by hRGSR. Fig. 4 demonstrates that none of the 131 131 131 Stimulation of GTPase Activity of Transducin by Mutant tested mutants, hRGSr-Ala , hRGSr-His , and hRGSr-D , hRGSr—Effects of hRGSr mutants with substitutions of at 5 mM concentration, had any effect on stimulation of GTPase Asn were tested in dROS membranes containing 5 mM rho- activity of transducin by 50 nM hRGSr. These data suggest that dopsin and 0.4 mM transducin. Under these conditions, the the hRGSr mutants that produced no stimulation of G a calculated rate of GTP hydrolysis by transducin was 0.025 6 GTPase activity lost their binding to G a. 0.004 s (Fig. 3). The rates of transducin GTPase activity were DISCUSSION then determined in the presence of increasing concentrations of hRGSr or individual hRGSr mutants and plotted as a function Molecular mechanisms of RGS protein action as GAP for of their concentration. Wild-type hRGSr purified after cleavage heterotrimeric GTP-binding proteins are not well understood. GAP of GST-hRGSr with thrombin stimulated GTPase activity of Studies on the Ras-specific p120 suggest that the Ras GAP 21 789 transducin by ;10-fold to a maximal rate k 5 0.27 6 0.01 s donates conserved Arg residue to the RAS catalytic site (29, GAP with an EC value of 101 6 14 nM (Fig. 3). All hRGSr mutants 30) thus providing the catalytic mechanism for p120 activ- had substantially reduced ability to stimulate the GTPase ac- ity. The crystal structure of RGS4 bound to G a zAlF has i 1 4 tivity of transducin. The tested mutants can be arbitrarily suggested two mechanisms for RGS GAP activity toward het- separated into three groups. Two of the mutants, hRGSr-Ser erotrimeric G-proteins (17). Interaction of RGS protein with and hRGSr-Gln , were relatively potent, and saturation of the G-protein switch regions indicates that the mechanism of their GAP effect could be achieved at 10 – 40 mM concentration the GTPase activation by RGS may primarily be a reduction in of mutant. hRGSr-Ser mutant was the most effective and the free energy of the transitional state via stabilization of Ga stimulated G a GTPase activity with an EC value of 1.34 6 switch regions and residues directly involved in GTP hydrolysis t 50 0.17 mM and V ;80% (k 5 0.22 6 0.01 s ). The mutant (17). An additional putative mechanism for the RGS GAP ac- max hRGSr-Gln was capable of accelerating the G a GTPase tivity would be a donation of the catalytic residue to the active activity to V of 60% (k 5 0.16 6 0.01 s ) with an EC value site of Ga. The only residue that RGS4 introduces into the max 50 131 128 128 of 3.9 6 1.1 mM (Fig. 3). Three mutants, hRGSr-Leu , hRGSr- active site of G a is Asn . Although Asn , in contrast to the i 1 131 131 789 Ala , and hRGSr-Asp , began to cause acceleration of G a Ras GAP Arg or an intrinsic Arg in Ga subunits, does not GTPase activity only at very high concentrations (.10 mM) (Fig. directly interact with GDP and AlF (13, 14, 17, 30), it makes 3). We were unable to practically achieve saturation of the GAP a contact with the side chain of Gln of G a , which stabilizes i 1 131 6734 Functional Role of Asn of Human Retinal RGS catalytic role of the Asn residue as the key component of the RGS 131 131 GAP activity. The hRGSr-Leu and hRGSr-Ala mutants at very high concentrations started to have a stimulatory effect on G a GTPase activity even though these residues are not expected to form hydrogen bonds which are made by the Asn residue. Our mutational analysis suggests that although Asn of hRGSr may play a catalytic role in the RGS GAP activity, stabilization of the switch regions of G-protein and reduction of the energy of the transition state appear to be the major components of the RGS GAP function. The Asn residue is absolutely essential for the sta- bilization of the transition state for GTP hydrolysis because its replacement or deletion leads to a drastic reduction in hRGSr affinity for G a. In addition to their role as GAPs, RGS proteins may act as antagonists for some G-protein effectors, particularly for phos- pholipase Cb. RGS4 has been shown to block activation of phospholipase Cb by G aGTPgS (33). In another study, RGS4 inhibited inositol phosphate synthesis activated by AlF in FIG.4. hRGSr mutants do not block stimulation of GTPase COS-7 cells overexpressing G (34). Tesmer et al. (17) have activity of transducin by hRGSr. Rate constants of GTPase activity suggested that the RGS proteins lacking the Asn residue may of transducin were determined in suspensions of dROS membranes (5 better serve as inhibitors of effector binding than as GAPs. This mM rhodopsin and 0.4 mM transducin) with and without addition of 50 would appear to be a likely scenario if replacements of the Asn nM hRGSr and 5 mM each of the following mutants: hRGSr-Ala , 131 131 hRGSr-His , and hRGSr-D . residue resulted in a loss of GAP activity without a concurrent reduction of the RGS protein affinity for activated G subunits. The results of this work suggest that the main consequence of and orients the hydrolytic water molecule in the transitional Asn replacement is an impairment of binding between mutated state of G a . Conceivably, Asn is within hydrogen-bonding hRGSr protein and G a. Furthermore, none of the hRGSr mu- i 1 distance of the hydrolytic water molecule and may bind and tants have shown enhanced affinity to the active G azGTPgS orient it for nucleophilic attack of the g-phosphate of GTP (17). conformation which could be indicative of the potential of such To probe the role of Asn of hRGSr for the mechanism of a mutant to serve as an antagonist for the G-protein effector. RGS protein GAP activity, we initially synthesized a peptide of This study only begins to address the questions, introduced hRGSr corresponding to the region of interaction between by the first crystal structure between G-protein and RGS pro- 2 128 G a zAlF and RGS4 containing Asn . We reasoned that if the tein, about the mechanism of RGS protein GAP activity (17). i 1 4 catalytic role of the Asn residue is a major component of RGS GAP Further biochemical analysis coupled with resolution of other activity, then perhaps a peptide containing the catalytic residue crystal structures between activated Ga subunits and RGS would alone be capable of eliciting the stimulation of GTPase ac- proteins would ultimately define a role of the critical Asn tivity. Our data demonstrated that hRGSr peptide-(123–140) con- residue. taining catalytic Asn retained the ability to bind hRGSr but REFERENCES failed to accelerate the GTPase activity of transducin. This indi- 1. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416 – 417 cates that the interaction of at least two and likely all three Ga 2. Angleson, J. K., and Wensel, T. G. (1994) J. Biol. Chem. 269, 16290 –16296 binding regions of RGS protein is required to stimulate Ga GTPase 3. Berstein, G., Blank, J. L., Jhon, D. 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Wieland, T., Chen, C.-K., and Simon, M. I. (1997) J. Biol. Chem. 272, U. S. A. 94, 7216 –7220 8853– 8856 32. Faurobert, E., and Hurley, J. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 93, 28. Nekrasova, E. R., Berman, D. M., Rustandi, R. R., Hamm, H. E., Gilman, A. G., 2945–2950 and Arshavsky, V. Y. (1997) Biochemistry 36, 7638 –7643 33. Hepler, J. R., Berman, D. M., Gilman, A. G., and Kozasa, T. (1997) Proc. Natl. 29. Scheffzek, K., Lautwein, A., Kabsch, W., Ahmadian, M. R., and Wittinghofer, Acad. Sci. U. S. A. 94, 428 – 432 A. (1996) Nature 384, 591–596 34. Yan, Y., Chi, P. P., and Bourne, H. R. (1997) J. Biol. Chem. 272, 11924 –11927 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins

Natochin, Michael; McEntaffer, Randall L.; Artemyev, Nikolai O.
Journal of Biological Chemistry – Mar 1, 1998
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 12, Issue of March 20, pp. 6731–6735, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Mutational Analysis of the Asn Residue Essential for RGS Protein Binding to G-proteins* (Received for publication, September 8, 1997, and in revised form, November 20, 1997) Michael Natochin, Randall L. McEntaffer, and Nikolai O. Artemyev‡ From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242 Members of the RGS family serve as GTPase-activat- spectively (1–3). A novel class of GTPase-activating proteins (GAPs) for heterotrimeric G-proteins called RGS has been iden- ing proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled re- tified (4 – 6). Strong evidence suggests that members of this ceptors. The recently resolved crystal structure of RGS4 family, GAIP, RGS4, RGS1, RGS10 and others, negatively reg- a suggests two potential mechanisms for bound to G i 1 ulate G-protein signaling by stimulating GTPase activity of the GAP activity of RGS proteins as follows: stabiliza- G-proteins, particularly those from G and G families (7–9). i q a switch regions by RGS4 and the catalytic tion of the G i 1 RGS proteins from yeast to mammals share a highly conserved . To elucidate a role of the action of RGS4 residue Asn RGS domain that provides relatively broad specificity of differ- Asn residue for RGS GAP function, we have investigated ent RGS proteins toward members of the two G-protein classes effects of the synthetic peptide corresponding to the G in vitro. Tissue expression patterns and diverse domains out- binding domain of human retinal RGS (hRGSr) contain- side the RGS segment may play an important role in determin- ing the key Asn at position 131, and we have carried out ing specificity of RGS proteins in vivo (10 –12). Precise mecha- . Synthetic peptide hRGSr- mutational analysis of Asn nisms of RGS GAP activity are not yet clear. The transition -complexed (123–140) retained its ability to bind the AlF state during GTP hydrolysis is thought to be mimicked by the a-subunit, G azAlF , but failed to elicit stim- transducin t 4 AlF -bound conformation of Ga subunits (13, 14). It has been a GTPase activity. Wild-type hRGSr stimu- ulation of Gt demonstrated that many RGS proteins interact preferentially a GTPase activity by ;10-fold with an EC value lated G t 50 with the AlF -bound conformation of Ga subunits and thus M. Mutant hRGSr proteins with substitutions of 4 of 100 n may accelerate GTP hydrolysis through stabilization of the by Ser and Gln had a significantly reduced affin- Asn transitional state of G-proteins (8, 15, 16). a but were capable of substantial stimulation of ity for G Recently, the crystal structure of RGS4 bound to G a zAlF a GTPase activity, 80 and 60% of V , respectively. G i 1 4 t max 131 131 131 , hRGSr-Ala , and hRGSr-Asp has been solved at a resolution of 2.8 Å (17). This structure Mutants hRGSr-Leu were able to accelerate G a GTPase activity only at very provides the first structural insights into the mechanism of mM) which appears to corre- high concentrations (>10 RGS protein action. The conserved RGS core forms three dis- late with a further decrease of their affinity for trans- tinct sites of interaction with the three switch regions of G a i 1 131 131 and hRGSr-D , had ducin. Two mutants, hRGSr-His suggesting that stabilization of the switch regions and G res- no detectable binding to transducin. Mutational analy- idues directly involved in GTP hydrolysis may be a major suggests that the stabilization of the G- sis of Asn component of RGS GAP activity (17). Furthermore, RGS pro- protein switch regions rather than catalytic action of teins could also contribute catalytic residues to the active site the Asn residue is a key component for the RGS GAP and thus enhance the GTPase rate constant. The conserved action. 128 residue Asn of RGS4 makes a contact with the side chain of Gln of G a which stabilizes and orients the hydrolytic water i 1 molecule in the transitional state of G a (17). Asn also may i 1 The intensity and duration of signaling via heterotrimeric be localized within hydrogen-bonding distance of the hydrolytic G-proteins is regulated at multiple levels. The key reaction in water molecule for nucleophilic attack on the GTP g-phosphate termination of G-protein-mediated signaling is the intrinsic (17). GTPase activity of Ga subunits that convert the active GTP- In this study we evaluate a potential catalytic role of the Asn bound conformation of G-protein a subunits (GazGTP) to the residue for G GTPase acceleration by RGS proteins using the inactive GazGDP conformation. The GTPase activity of two interaction between human retinal RGS (hRGSr) protein and G-proteins, G and transducin, is stimulated by their effectors, transducin as a model system and mutational analysis of phospholipase Cb and cGMP phosphodiesterase (PDE), re- 131 128 Asn of hRGSr which is equivalent to Asn of RGS4. EXPERIMENTAL PROCEDURES * This work was supported by National Institutes of Health Grant Materials—GTP and GTPgS were products of Boehringer Mann- EY-10843. The services provided by the Diabetes and Endocrinology heim. Blue-Sepharose and PD-10 Sephadex G-25 columns were ob- Research Center of the University of Iowa were supported by National tained from Pharmacia Biotech Inc. [g- P]GTP (.5000 Ci/mmol) was Institutes of Health Grant DK-25295. The costs of publication of this purchased from Amersham Corp. [ S]GTPgS (1250 Ci/mmol) was ob- article were defrayed in part by the payment of page charges. This tained from NEN Life Science Products. All other chemicals were ac- article must therefore be hereby marked “advertisement” in accordance quired from Sigma. with 18 U.S.C. Section 1734 solely to indicate this fact. Preparation of Rod Outer Segment (ROS) Membranes, G azGTPgS, ‡ To whom correspondence and reprint requests should be addressed: G azGDP, and hRGSr—Bovine ROS membranes were prepared as de- Dept. of Physiology and Biophysics, University of Iowa College of Med- t scribed previously (18). Hypotonically washed ROS membranes (dROS) icine, 5– 660 Bowen Science Bldg., Iowa City, IA 52242.. Tel.: 319-335- depleted of PDE subunits were prepared as described in Ref. 2. Trans- 7864; Fax: 319-335-7330; E-mail: [email protected]. ducin, G abg, was extracted from ROS membranes using GTP as de- The abbreviations used are: PDE, cGMP phosphodiesterase; RGS proteins, regulators of G-protein signaling; hRGSr, human retinal RGS protein; ROS, rod outer segment(s); dROS, hypotonically washed ROS membranes; G a, rod G-protein (transducin) a-subunit; GTPgS, guanosine 59-O-(3-thiotriphosphate); PCR, polymerase chain reaction. This paper is available on line at http://www.jbc.org 6731 This is an Open Access article under the CC BY license. 131 6732 Functional Role of Asn of Human Retinal RGS scribed in Ref. 19. The G azGTPgS was extracted from ROS membranes using GTPgS and purified by chromatography on Blue-Sepharose CL-6B by the procedure described in Ref. 20. G azGDP was prepared and purified according to protocols in Ref. 21. hRGSr was prepared and purified as described previously (22). The purified proteins were stored in 40% glycerol at 220 °C or without glycerol at 280 °C. Site-directed Mutagenesis of hRGSr—Mutagenesis of Asn residue of hRGSr was performed using PCR amplifications from the pGEX-KG- hRGSr template (22) with 39-antisense primer ATGCCTCGAGACT- CAGGTGTGTGAGG (unique XhoI site is underlined) and the 59 prim- ers: XXXATTGACCATGAGACCCGCGAGC. XXX indicates nucleotides that generate substitutions of Asn (AAC) in hRGSr cDNA by the following amino acid residues: Ala (GCG), Asp (GAT), His (CAT), Leu (CTG), Gln (CAG), Ser (AGC), and deletion mutant (—). PCR reactions were performed in 100 ml of reaction mixture containing 1 ng of the pGEX-KG-hRGSr plasmid, 3 units of AmpliTaq DNA polymerase (Per- M Tris-(hydroxymethyl)-methylaminopropane sulfonic kin-Elmer), 25 m FIG.1. Effect of hRGSr-(123–140)-peptide on basal and hRGSr- acid, pH 9.3, 2 mM MgCl ,1mM 2-mercaptoethanol, 200 mM of dNTPs, stimulated GTPase activity of transducin. dROS membranes (5 mM and 0.5 mM primers. Conditions for PCR were as follows: 94 °C for 3 min, rhodopsin and 0.4 mM transducin) were incubated for 5 min with in- 30 cycles of 94 °C for 1 min, 64 °C for 30 s and 72 °C for 30 s, and a final creasing concentrations of peptide hRGSr-(123–140) with (squares)or extension at 72 °C for 3 min. The PCR products (;220 base pairs) were without (circles) addition of 100 nM hRGSr. The GTPase reaction was blunt-ended with Klenow fragment and digested with XhoI. Wild-type 32 initiated by addition of 100 nM [g- P]GTP. Calculated GTPase rate hRGSr cDNA was subcloned into XbaI/XhoI sites of pBluescript constants are plotted as a function of peptide concentration. The com- polylinker. The resulting construct was digested with HincII and XhoI petition curve (squares,IC 5 1.6 6 0.3 mM; Hill slope 5 0.64) fits the and ligated with the XhoI-digested PCR products carrying mutations. data with r 5 0.98. The mutant sequences were verified by automated DNA sequencing at the University of Iowa DNA Core Facility using the T7 primer and subcloned into the XbaI/XhoI sites of pGEX-KG vector for protein ex- squares criteria using GraphPad Prizm (version 2) software. The re- pression. Mutant GST-hRGSr proteins were expressed in DH5a Esch- sults are expressed as the mean 6 S.E. of triplicate measurements. erichia coli cells, and the GST portion was removed as described earlier RESULTS (22). Typical yields of purified hRGSr and hRGSr mutants, except for a mutant with deletion of Asn , were 5– 6 mg/liter of culture. Deletion of Effects of Synthetic Peptide hRGSr-(123–140)—The G a bind- led to an ;4 –5-fold reduction in expression of soluble recombi- Asn ing region of RGS4 containing Asn resides in the loop a5–a6 nant protein suggesting that the residue at position 131 may be impor- of RGS4 and contains 7 amino acid residues making contacts tant to the stability and proper folding of RGS proteins. with all three switch regions of G-protein (17). The number of Binding of Transducin to GST-hRGSr and Mutants—G azGTPgSor interactions is sufficient to ensure a relatively high affinity G azGDP (10 mg) were incubated with hRGSr or its mutants (50 mg) between the corresponding synthetic peptide and Ga, provided immobilized on glutathione-agarose in 100 mlof20mM Tris-HCl buffer (pH 8.0), containing 100 mM NaCl, 2 mM MgSO ,6mM 2-mercaptoeth- that the peptide is able to adopt a functional conformation. For anol, and 5% glycerol (buffer A). Where indicated, the buffer contained the preliminary testing of the catalytic role of Asn of hRGSr, mM AlCl and 10 mM NaF. After incubation for 20 min at 25 °C, the the hRGSr-(123–140)-peptide was synthesized. The length of agarose beads were spun and washed twice with 1 ml of buffer A, and the peptide was chosen to allow the Ga contact residues to be the bound proteins were eluted with a sample buffer for SDS-polyacryl- flanked by at least 3 terminal residues. hRGSr-(123–140) was amide gel electrophoresis. first examined for its ability to stimulate GTPase activity of Single Turnover GTPase Assay—Single turnover GTPase activity transducin in suspensions of dROS membranes containing 5 measurements were carried out in suspensions of dROS membranes containing 5 mM rhodopsin and 0.4 mM transducin essentially as de- mM rhodopsin and 0.4 mM transducin. dROS membranes lacked scribed in Refs. 22 and 23. Transducin concentration of 0.4 mM was intrinsic catalytic PDEab and inhibitory PDEg subunits. Use S]GTPgS binding assay as described previously determined using the [ of such ROS avoided interference of PDEg effects with effects of (22). Bleached dROS membranes were mixed with different concentra- RGS protein or RGS peptide (22, 27, 28). The peptide at con- tions of the tested peptides, hRGSr or hRGSr mutants, and preincu- centrations of up to 2 mM had no effect on GTPase activity of bated for 5 min at 25 °C. The GTPase reaction was initiated by addition 32 4 transducin (not shown). To determine if hRGSr-(123–140) is of 100 nM [g- P]GTP (;5 3 10 dpm/pmol) in a total volume of 20 ml. At 5, 10, 20, 40, and 60 s aliquots of the reaction mixture were withdrawn capable of binding to transducin, we investigated effects of the and quenched with 7% perchloric acid. Nucleotides were then precipi- hRGSr peptide on the stimulation of GTPase activity of trans- tated using activated Norit A charcoal (10% w/v) in 50 mM sodium ducin by hRGSr. Fig. 1 shows that hRGSr-(123–140) was able P formation was measured by liquid phosphate buffer (pH 7.5), and to compete with hRGSr for binding to G a resulting in a dose- scintillation counting. The GTPase rate constants were calculated by dependent (IC 5 1.6 6 0.3 mM) decrease of the stimulated fitting the experimental data to an exponential function: % GTP hydro- 2kt GTPase activity of transducin. hRGSr-(123–140) in the same lyzed 5 100 (1 2 e ), where k is a rate constant for GTP hydrolysis. range of concentrations had no notable effect on the basal Peptide Synthesis—A peptide, CSEAPKEVNIDHETRELT, corre- sponding to residues 123–140 of hRGSr was custom made by Genosys transducin GTPase activity (Fig. 1). Because the competition Biotechnologies Inc. The N and C termini of the peptide were acetylated experiments were carried out at a concentration of hRGSr and amidated, respectively. The peptide was purified by reverse-phase causing half-maximal stimulation of the GTPase activity, the high pressure liquid chromatography on a preparative Dynamax-300A affinity of hRGSr-(123–140) for G a can be estimated as 0.8 mM. column (Rainin). The purity and chemical formula of the peptide were In control experiments, four unrelated peptides (24) corre- confirmed by fast atom bombardment-mass spectrometry and analyti- sponding to residues 21–31, 461–553, 492–516, and 517–541 of cal high pressure liquid chromatography. Preparation of synthetic pep- the rod PDE a-subunit (at concentrations of 8 mM) had no effect tides corresponding to residues 21–31, 461– 491, 492–516, and 517–541 of rod PDE a-subunit was described previously (24). on hRGSr-stimulated GTPase activity of transducin (not Miscellaneous—Protein concentrations were determined by the shown). method of Bradford (25) using IgG as a standard or using calculated 131 Binding of hRGSr Mutants with Substitutions of Asn to extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophore- Different Conformations of G a—Recently, we have shown that sis was performed by the method of Laemmli (26) in 12% acrylamide similar to other characterized RGS proteins, hRGSr binds with gels. Rhodopsin concentrations were measured using the difference in high affinity to the AlF conformations of transducin and very absorbance at 500 nm between “dark” and bleached ROS preparations. 4 Fitting of the experimental data was performed with nonlinear least weakly to the GTPgS and GDP-bound conformations (22). We 131 Functional Role of Asn of Human Retinal RGS 6733 FIG.2. Binding of GST-hRGSr and its mutants to G a. SDS- FIG.3. Stimulation of GTPase activity of transducin by mutant polycrylamide gel (12%) stained with Coomassie Blue. Binding of G a hRGSr. GTPase activity of transducin was measured in suspensions of GDPzAlF (A), G azGTPgS(B), G azGDP (C), to GST-hRGSr or its mu- 4 t t dROS (5 mM rhodopsin and 0.4 mM transducin) in the presence of tants immobilized on glutathione-agarose was performed as described increasing concentrations of hRGSr or its mutants. Calculated GTPase under “Experimental Procedures.” Lane 1,G a; lane 2, GST-hRGSr; rate constants are plotted as a function of hRGSr or mutant concentra- mutants of hRGSr with substitution of Asn : lane 3, Ala; lane 4, Asp; tion. hRGSr, closed squares; hRGSr-Ser , open squares; hRGSr- lane 5, His; lane 6, Leu; lane 7, Gln; lane 8, Ser; lane 9, D; lane 10, 131 131 131 Gln , closed diamonds; hRGSr-Asp , open diamonds; hRGSr-Leu , glutathione-agarose without bound GST-RGS protein (control). w.t., 131 131 closed triangles; hRGSr-Ala , open triangles; hRGSr-His , closed wild type. circles; and hRGSr-D , open circles. evaluated the interaction between hRGSr mutants with sub- activity by these mutants due to the very high protein concen- stitutions of Asn by Ser, Gln, Ala, Leu, His, Asp as well as trations required. Two mutants, hRGSr-His and hRGSr- the mutant with deletion of Asn and transducin using pre- D , did not show GAP activity at the concentration tested (40 cipitation of G a with the GST-hRGSr mutant proteins immo- mM). Interestingly, the potency of hRGSr mutants in stimulat- bilized on glutathione-agarose beads. Mutations hRGSr-Ser ing G a GTPase activity (Fig. 3) appears to correlate well with and hRGSr-Gln led to a reduction in affinity of the corre- their ability to bind and precipitate G azAlF (Fig. 2A), t 4 sponding GST fusion proteins for G azAlF (Fig. 2A). Mutants t 4 Competition between hRGSr and hRGSr Mutants in Stimu- 131 131 131 hRGSr-Leu , hRGSr-Asp , and hRGSr-Ala showed a lation of G a GTPase Activity—Experiments in Fig. 2 have more significant decrease in their affinity for the G a confor- t suggested that hRGSr mutants with substitutions of Asn 131 131 mation (Fig. 2A). hRGSr-His and hRGSr-D failed to co- have impaired binding to G azAlF . The binding assay may, t 4 2 131 precipitate G azAlF . Mutations of Asn could potentially t 4 however, not be sufficiently sensitive to detect relatively weak alter hRGSr interaction with G azGTPgS and G azGDP since interactions. To determine if the drastically reduced ability of t t the RGS4 Asn residue makes contact with the switch I and II some RGS mutants to stimulate the GTPase activity of trans- regions of G a (17). We have tested this possibility by prein- ducin correlates with the lack of mutant binding to transducin, i 1 cubating mutant GST-hRGSr containing beads with both con- we carried out competition experiments. The hRGSr mutants formations of G a. None of the seven hRGSr mutants has incapable of accelerating G a GTPase activity were examined demonstrated enhanced affinity for either conformation of G a t for their ability to block stimulation of GTPase activity of compared with the native GST-hRGSr (Fig. 2, B and C). transducin by hRGSR. Fig. 4 demonstrates that none of the 131 131 131 Stimulation of GTPase Activity of Transducin by Mutant tested mutants, hRGSr-Ala , hRGSr-His , and hRGSr-D , hRGSr—Effects of hRGSr mutants with substitutions of at 5 mM concentration, had any effect on stimulation of GTPase Asn were tested in dROS membranes containing 5 mM rho- activity of transducin by 50 nM hRGSr. These data suggest that dopsin and 0.4 mM transducin. Under these conditions, the the hRGSr mutants that produced no stimulation of G a calculated rate of GTP hydrolysis by transducin was 0.025 6 GTPase activity lost their binding to G a. 0.004 s (Fig. 3). The rates of transducin GTPase activity were DISCUSSION then determined in the presence of increasing concentrations of hRGSr or individual hRGSr mutants and plotted as a function Molecular mechanisms of RGS protein action as GAP for of their concentration. Wild-type hRGSr purified after cleavage heterotrimeric GTP-binding proteins are not well understood. GAP of GST-hRGSr with thrombin stimulated GTPase activity of Studies on the Ras-specific p120 suggest that the Ras GAP 21 789 transducin by ;10-fold to a maximal rate k 5 0.27 6 0.01 s donates conserved Arg residue to the RAS catalytic site (29, GAP with an EC value of 101 6 14 nM (Fig. 3). All hRGSr mutants 30) thus providing the catalytic mechanism for p120 activ- had substantially reduced ability to stimulate the GTPase ac- ity. The crystal structure of RGS4 bound to G a zAlF has i 1 4 tivity of transducin. The tested mutants can be arbitrarily suggested two mechanisms for RGS GAP activity toward het- separated into three groups. Two of the mutants, hRGSr-Ser erotrimeric G-proteins (17). Interaction of RGS protein with and hRGSr-Gln , were relatively potent, and saturation of the G-protein switch regions indicates that the mechanism of their GAP effect could be achieved at 10 – 40 mM concentration the GTPase activation by RGS may primarily be a reduction in of mutant. hRGSr-Ser mutant was the most effective and the free energy of the transitional state via stabilization of Ga stimulated G a GTPase activity with an EC value of 1.34 6 switch regions and residues directly involved in GTP hydrolysis t 50 0.17 mM and V ;80% (k 5 0.22 6 0.01 s ). The mutant (17). An additional putative mechanism for the RGS GAP ac- max hRGSr-Gln was capable of accelerating the G a GTPase tivity would be a donation of the catalytic residue to the active activity to V of 60% (k 5 0.16 6 0.01 s ) with an EC value site of Ga. The only residue that RGS4 introduces into the max 50 131 128 128 of 3.9 6 1.1 mM (Fig. 3). Three mutants, hRGSr-Leu , hRGSr- active site of G a is Asn . Although Asn , in contrast to the i 1 131 131 789 Ala , and hRGSr-Asp , began to cause acceleration of G a Ras GAP Arg or an intrinsic Arg in Ga subunits, does not GTPase activity only at very high concentrations (.10 mM) (Fig. directly interact with GDP and AlF (13, 14, 17, 30), it makes 3). We were unable to practically achieve saturation of the GAP a contact with the side chain of Gln of G a , which stabilizes i 1 131 6734 Functional Role of Asn of Human Retinal RGS catalytic role of the Asn residue as the key component of the RGS 131 131 GAP activity. The hRGSr-Leu and hRGSr-Ala mutants at very high concentrations started to have a stimulatory effect on G a GTPase activity even though these residues are not expected to form hydrogen bonds which are made by the Asn residue. Our mutational analysis suggests that although Asn of hRGSr may play a catalytic role in the RGS GAP activity, stabilization of the switch regions of G-protein and reduction of the energy of the transition state appear to be the major components of the RGS GAP function. The Asn residue is absolutely essential for the sta- bilization of the transition state for GTP hydrolysis because its replacement or deletion leads to a drastic reduction in hRGSr affinity for G a. In addition to their role as GAPs, RGS proteins may act as antagonists for some G-protein effectors, particularly for phos- pholipase Cb. RGS4 has been shown to block activation of phospholipase Cb by G aGTPgS (33). In another study, RGS4 inhibited inositol phosphate synthesis activated by AlF in FIG.4. hRGSr mutants do not block stimulation of GTPase COS-7 cells overexpressing G (34). Tesmer et al. (17) have activity of transducin by hRGSr. Rate constants of GTPase activity suggested that the RGS proteins lacking the Asn residue may of transducin were determined in suspensions of dROS membranes (5 better serve as inhibitors of effector binding than as GAPs. This mM rhodopsin and 0.4 mM transducin) with and without addition of 50 would appear to be a likely scenario if replacements of the Asn nM hRGSr and 5 mM each of the following mutants: hRGSr-Ala , 131 131 hRGSr-His , and hRGSr-D . residue resulted in a loss of GAP activity without a concurrent reduction of the RGS protein affinity for activated G subunits. The results of this work suggest that the main consequence of and orients the hydrolytic water molecule in the transitional Asn replacement is an impairment of binding between mutated state of G a . Conceivably, Asn is within hydrogen-bonding hRGSr protein and G a. Furthermore, none of the hRGSr mu- i 1 distance of the hydrolytic water molecule and may bind and tants have shown enhanced affinity to the active G azGTPgS orient it for nucleophilic attack of the g-phosphate of GTP (17). conformation which could be indicative of the potential of such To probe the role of Asn of hRGSr for the mechanism of a mutant to serve as an antagonist for the G-protein effector. RGS protein GAP activity, we initially synthesized a peptide of This study only begins to address the questions, introduced hRGSr corresponding to the region of interaction between by the first crystal structure between G-protein and RGS pro- 2 128 G a zAlF and RGS4 containing Asn . We reasoned that if the tein, about the mechanism of RGS protein GAP activity (17). i 1 4 catalytic role of the Asn residue is a major component of RGS GAP Further biochemical analysis coupled with resolution of other activity, then perhaps a peptide containing the catalytic residue crystal structures between activated Ga subunits and RGS would alone be capable of eliciting the stimulation of GTPase ac- proteins would ultimately define a role of the critical Asn tivity. Our data demonstrated that hRGSr peptide-(123–140) con- residue. taining catalytic Asn retained the ability to bind hRGSr but REFERENCES failed to accelerate the GTPase activity of transducin. This indi- 1. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416 – 417 cates that the interaction of at least two and likely all three Ga 2. Angleson, J. K., and Wensel, T. G. (1994) J. Biol. Chem. 269, 16290 –16296 binding regions of RGS protein is required to stimulate Ga GTPase 3. Berstein, G., Blank, J. L., Jhon, D. 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Published: Mar 1, 1998

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