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

Michael Natochin, Randall L. McEntaffer, and Nikolai O. ArtemyevDagger

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Members of the RGS family serve as GTPase-activating proteins (GAPs) for heterotrimeric G-proteins and negatively regulate signaling via G-protein-coupled receptors. The recently resolved crystal structure of RGS4 bound to Gialpha 1 suggests two potential mechanisms for the GAP activity of RGS proteins as follows: stabilization of the Gialpha 1 switch regions by RGS4 and the catalytic action of RGS4 residue Asn128. To elucidate a role of the Asn residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Galpha binding domain of human retinal RGS (hRGSr) containing the key Asn at position 131, and we have carried out mutational analysis of Asn131. Synthetic peptide hRGSr-(123-140) retained its ability to bind the AlF4--complexed transducin alpha -subunit, Gtalpha ·AlF4-, but failed to elicit stimulation of Gtalpha GTPase activity. Wild-type hRGSr stimulated Gtalpha GTPase activity by ~10-fold with an EC50 value of 100 nM. Mutant hRGSr proteins with substitutions of Asn131 by Ser and Gln had a significantly reduced affinity for Gtalpha but were capable of substantial stimulation of Gtalpha GTPase activity, 80 and 60% of Vmax, respectively. Mutants hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131 were able to accelerate Gtalpha GTPase activity only at very high concentrations (>10 µM) which appears to correlate with a further decrease of their affinity for transducin. Two mutants, hRGSr-His131 and hRGSr-Delta 131, had no detectable binding to transducin. Mutational analysis of Asn131 suggests that the stabilization of the G-protein switch regions rather than catalytic action of the Asn residue is a key component for the RGS GAP action.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The intensity and duration of signaling via heterotrimeric G-proteins is regulated at multiple levels. The key reaction in termination of G-protein-mediated signaling is the intrinsic GTPase activity of Galpha subunits that convert the active GTP-bound conformation of G-protein alpha  subunits (Galpha ·GTP) to the inactive Galpha ·GDP conformation. The GTPase activity of two G-proteins, Gq and transducin, is stimulated by their effectors, phospholipase Cbeta and cGMP phosphodiesterase (PDE),1 respectively (1-3). A novel class of GTPase-activating proteins (GAPs) for heterotrimeric G-proteins called RGS has been identified (4-6). Strong evidence suggests that members of this family, GAIP, RGS4, RGS1, RGS10 and others, negatively regulate G-protein signaling by stimulating GTPase activity of G-proteins, particularly those from Gi and Gq families (7-9). RGS proteins from yeast to mammals share a highly conserved RGS domain that provides relatively broad specificity of different RGS proteins toward members of the two G-protein classes in vitro. Tissue expression patterns and diverse domains outside the RGS segment may play an important role in determining specificity of RGS proteins in vivo (10-12). Precise mechanisms of RGS GAP activity are not yet clear. The transition state during GTP hydrolysis is thought to be mimicked by the AlF4--bound conformation of Galpha subunits (13, 14). It has been demonstrated that many RGS proteins interact preferentially with the AlF4--bound conformation of Galpha subunits and thus may accelerate GTP hydrolysis through stabilization of the transitional state of G-proteins (8, 15, 16).

Recently, the crystal structure of RGS4 bound to Gialpha 1·AlF4- has been solved at a resolution of 2.8 Å (17). This structure provides the first structural insights into the mechanism of RGS protein action. The conserved RGS core forms three distinct sites of interaction with the three switch regions of Gialpha 1 suggesting that stabilization of the switch regions and Galpha residues directly involved in GTP hydrolysis may be a major component of RGS GAP activity (17). Furthermore, RGS proteins could also contribute catalytic residues to the active site and thus enhance the GTPase rate constant. The conserved residue Asn128 of RGS4 makes a contact with the side chain of Gln204 of Gialpha 1 which stabilizes and orients the hydrolytic water molecule in the transitional state of Gialpha 1 (17). Asn128 also may be localized within hydrogen-bonding distance of the hydrolytic water molecule for nucleophilic attack on the GTP gamma -phosphate (17).

In this study we evaluate a potential catalytic role of the Asn residue for Galpha GTPase acceleration by RGS proteins using the interaction between human retinal RGS (hRGSr) protein and transducin as a model system and mutational analysis of Asn131 of hRGSr which is equivalent to Asn128 of RGS4.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- GTP and GTPgamma S were products of Boehringer Mannheim. Blue-Sepharose and PD-10 Sephadex G-25 columns were obtained from Pharmacia Biotech Inc. [gamma -32P]GTP (>5000 Ci/mmol) was purchased from Amersham Corp. [35S]GTPgamma S (1250 Ci/mmol) was obtained from NEN Life Science Products. All other chemicals were acquired from Sigma.

Preparation of Rod Outer Segment (ROS) Membranes, Gtalpha ·GTPgamma S, Gtalpha ·GDP, and hRGSr-- Bovine ROS membranes were prepared as described previously (18). Hypotonically washed ROS membranes (dROS) depleted of PDE subunits were prepared as described in Ref. 2. Transducin, Gtalpha beta gamma , was extracted from ROS membranes using GTP as described in Ref. 19. The Gtalpha ·GTPgamma S was extracted from ROS membranes using GTPgamma S and purified by chromatography on Blue-Sepharose CL-6B by the procedure described in Ref. 20. Gtalpha ·GDP 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 -20 °C or without glycerol at -80 °C.

Site-directed Mutagenesis of hRGSr-- Mutagenesis of Asn131 residue of hRGSr was performed using PCR amplifications from the pGEX-KG-hRGSr template (22) with 3'-antisense primer ATGCCTCGAGACTCAGGTGTGTGAGG (unique XhoI site is underlined) and the 5' primers: XXXATTGACCATGAGACCCGCGAGC. XXX indicates nucleotides that generate substitutions of Asn131 (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 µl of reaction mixture containing 1 ng of the pGEX-KG-hRGSr plasmid, 3 units of AmpliTaq DNA polymerase (Perkin-Elmer), 25 mM Tris-(hydroxymethyl)-methylaminopropane sulfonic acid, pH 9.3, 2 mM MgCl2, 1 mM 2-mercaptoethanol, 200 µM of dNTPs, and 0.5 µM primers. Conditions for PCR were as follows: 94 °C for 3 min, 30 cycles of 94 °C for 1 min, 64 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 3 min. The PCR products (~220 base pairs) were blunt-ended with Klenow fragment and digested with XhoI. Wild-type hRGSr cDNA was subcloned into XbaI/XhoI sites of pBluescript polylinker. The resulting construct was digested with HincII and XhoI and ligated with the XhoI-digested PCR products carrying mutations. 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 expression. Mutant GST-hRGSr proteins were expressed in DH5alpha Escherichia coli cells, and the GST portion was removed as described earlier (22). Typical yields of purified hRGSr and hRGSr mutants, except for a mutant with deletion of Asn131, were 5-6 mg/liter of culture. Deletion of Asn131 led to an ~4-5-fold reduction in expression of soluble recombinant protein suggesting that the residue at position 131 may be important to the stability and proper folding of RGS proteins.

Binding of Transducin to GST-hRGSr and Mutants-- Gtalpha ·GTPgamma S or Gtalpha ·GDP (10 µg) were incubated with hRGSr or its mutants (50 µg) immobilized on glutathione-agarose in 100 µl of 20 mM Tris-HCl buffer (pH 8.0), containing 100 mM NaCl, 2 mM MgSO4, 6 mM 2-mercaptoethanol, and 5% glycerol (buffer A). Where indicated, the buffer contained 30 µM AlCl3 and 10 mM NaF. After incubation for 20 min at 25 °C, the agarose beads were spun and washed twice 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 dROS membranes containing 5 µM rhodopsin and 0.4 µM transducin essentially as described in Refs. 22 and 23. Transducin concentration of 0.4 µM was determined using the [35S]GTPgamma S binding assay as described previously (22). Bleached dROS membranes were mixed with different concentrations of the tested peptides, hRGSr or hRGSr mutants, and preincubated for 5 min at 25 °C. The GTPase reaction was initiated by addition of 100 nM [gamma -32P]GTP (~5 × 104 dpm/pmol) in a total volume of 20 µl. At 5, 10, 20, 40, and 60 s aliquots of the reaction mixture were withdrawn and quenched with 7% perchloric acid. Nucleotides were then precipitated using activated Norit A charcoal (10% w/v) in 50 mM sodium phosphate buffer (pH 7.5), and 32Pi formation was measured by liquid scintillation counting. The GTPase rate constants were calculated by fitting the experimental data to an exponential function: % GTP hydrolyzed = 100 (1 - e-kt), where k is a rate constant for GTP hydrolysis.

Peptide Synthesis-- A peptide, CSEAPKEVNIDHETRELT, corresponding to residues 123-140 of hRGSr was custom made by Genosys Biotechnologies Inc. The N and C termini of the peptide were acetylated and amidated, respectively. The peptide was purified by reverse-phase high pressure liquid chromatography on a preparative Dynamax-300A column (Rainin). The purity and chemical formula of the peptide were confirmed by fast atom bombardment-mass spectrometry and analytical high pressure liquid chromatography. Preparation of synthetic peptides corresponding to residues 21-31, 461-491, 492-516, and 517-541 of rod PDE alpha -subunit was described previously (24).

Miscellaneous-- Protein concentrations were determined by the method of Bradford (25) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Laemmli (26) 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 ± S.E. of triplicate measurements.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of Synthetic Peptide hRGSr-(123-140)-- The Gialpha binding region of RGS4 containing Asn128 resides in the loop alpha 5-alpha 6 of RGS4 and contains 7 amino acid residues making contacts with all three switch regions of G-protein (17). The number of interactions is sufficient to ensure a relatively high affinity between the corresponding synthetic peptide and Galpha , provided that the peptide is able to adopt a functional conformation. For the preliminary testing of the catalytic role of Asn131 of hRGSr, the hRGSr-(123-140)-peptide was synthesized. The length of the peptide was chosen to allow the Galpha contact residues to be flanked by at least 3 terminal residues. hRGSr-(123-140) was first examined for its ability to stimulate GTPase activity of transducin in suspensions of dROS membranes containing 5 µM rhodopsin and 0.4 µM transducin. dROS membranes lacked intrinsic catalytic PDEalpha beta and inhibitory PDEgamma subunits. Use of such ROS avoided interference of PDEgamma effects with effects of RGS protein or RGS peptide (22, 27, 28). The peptide at concentrations of up to 2 mM had no effect on GTPase activity of transducin (not shown). To determine if hRGSr-(123-140) is capable of binding to transducin, we investigated effects of the hRGSr peptide on the stimulation of GTPase activity of transducin by hRGSr. Fig. 1 shows that hRGSr-(123-140) was able to compete with hRGSr for binding to Gtalpha resulting in a dose-dependent (IC50 = 1.6 ± 0.3 mM) decrease of the stimulated GTPase activity of transducin. hRGSr-(123-140) in the same range of concentrations had no notable effect on the basal transducin GTPase activity (Fig. 1). Because the competition experiments were carried out at a concentration of hRGSr causing half-maximal stimulation of the GTPase activity, the affinity of hRGSr-(123-140) for Gtalpha can be estimated as 0.8 mM. In control experiments, four unrelated peptides (24) corresponding to residues 21-31, 461-553, 492-516, and 517-541 of the rod PDE alpha -subunit (at concentrations of 8 mM) had no effect on hRGSr-stimulated GTPase activity of transducin (not shown).


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Fig. 1.   Effect of hRGSr-(123-140)-peptide on basal and hRGSr-stimulated GTPase activity of transducin. dROS membranes (5 µM rhodopsin and 0.4 µM transducin) were incubated for 5 min with increasing concentrations of peptide hRGSr-(123-140) with (squares) or without (circles) addition of 100 nM hRGSr. The GTPase reaction was initiated by addition of 100 nM [gamma -32P]GTP. Calculated GTPase rate constants are plotted as a function of peptide concentration. The competition curve (squares, IC50 = 1.6 ± 0.3 mM; Hill slope = 0.64) fits the data with r = 0.98.

Binding of hRGSr Mutants with Substitutions of Asn131 to Different Conformations of Gtalpha -- Recently, we have shown that similar to other characterized RGS proteins, hRGSr binds with high affinity to the AlF4- conformations of transducin and very weakly to the GTPgamma S and GDP-bound conformations (22). We evaluated the interaction between hRGSr mutants with substitutions of Asn131 by Ser, Gln, Ala, Leu, His, Asp as well as the mutant with deletion of Asn131 and transducin using precipitation of Gtalpha with the GST-hRGSr mutant proteins immobilized on glutathione-agarose beads. Mutations hRGSr-Ser131 and hRGSr-Gln131 led to a reduction in affinity of the corresponding GST fusion proteins for Gtalpha ·AlF4- (Fig. 2A). Mutants hRGSr-Leu131, hRGSr-Asp131, and hRGSr-Ala131 showed a more significant decrease in their affinity for the Gtalpha conformation (Fig. 2A). hRGSr-His131 and hRGSr-Delta 131 failed to co-precipitate Gtalpha ·AlF4-. Mutations of Asn131 could potentially alter hRGSr interaction with Gtalpha ·GTPgamma S and Gtalpha ·GDP since the RGS4 Asn residue makes contact with the switch I and II regions of Gialpha 1 (17). We have tested this possibility by preincubating mutant GST-hRGSr containing beads with both conformations of Gtalpha . None of the seven hRGSr mutants has demonstrated enhanced affinity for either conformation of Gtalpha compared with the native GST-hRGSr (Fig. 2, B and C).


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Fig. 2.   Binding of GST-hRGSr and its mutants to Gtalpha . SDS-polycrylamide gel (12%) stained with Coomassie Blue. Binding of Gtalpha GDP·AlF4- (A), Gtalpha ·GTPgamma S (B), Gtalpha ·GDP (C), to GST-hRGSr or its mutants immobilized on glutathione-agarose was performed as described under "Experimental Procedures." Lane 1, Gtalpha ; lane 2, GST-hRGSr; mutants of hRGSr with substitution of Asn131: lane 3, Ala; lane 4, Asp; lane 5, His; lane 6, Leu; lane 7, Gln; lane 8, Ser; lane 9, Delta ; lane 10, glutathione-agarose without bound GST-RGS protein (control). w.t., wild type.

Stimulation of GTPase Activity of Transducin by Mutant hRGSr-- Effects of hRGSr mutants with substitutions of Asn131 were tested in dROS membranes containing 5 µM rhodopsin and 0.4 µM transducin. Under these conditions, the calculated rate of GTP hydrolysis by transducin was 0.025 ± 0.004 s-1 (Fig. 3). The rates of transducin GTPase activity were then determined in the presence of increasing concentrations of hRGSr or individual hRGSr mutants and plotted as a function of their concentration. Wild-type hRGSr purified after cleavage of GST-hRGSr with thrombin stimulated GTPase activity of transducin by ~10-fold to a maximal rate k = 0.27 ± 0.01 s-1 with an EC50 value of 101 ± 14 nM (Fig. 3). All hRGSr mutants had substantially reduced ability to stimulate the GTPase activity of transducin. The tested mutants can be arbitrarily separated into three groups. Two of the mutants, hRGSr-Ser131 and hRGSr-Gln131, were relatively potent, and saturation of their GAP effect could be achieved at 10-40 µM concentration of mutant. hRGSr-Ser131 mutant was the most effective and stimulated Gtalpha GTPase activity with an EC50 value of 1.34 ± 0.17 µM and Vmax ~80% (k = 0.22 ± 0.01 s-1). The mutant hRGSr-Gln131 was capable of accelerating the Gtalpha GTPase activity to Vmax of 60% (k = 0.16 ± 0.01 s-1) with an EC50 value of 3.9 ± 1.1 µM (Fig. 3). Three mutants, hRGSr-Leu131, hRGSr-Ala131, and hRGSr-Asp131, began to cause acceleration of Gtalpha GTPase activity only at very high concentrations (>10 µM) (Fig. 3). We were unable to practically achieve saturation of the GAP activity by these mutants due to the very high protein concentrations required. Two mutants, hRGSr-His131 and hRGSr-Delta 131, did not show GAP activity at the concentration tested (40 µM). Interestingly, the potency of hRGSr mutants in stimulating Gtalpha GTPase activity (Fig. 3) appears to correlate well with their ability to bind and precipitate Gtalpha ·AlF4- (Fig. 2A),


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Fig. 3.   Stimulation of GTPase activity of transducin by mutant hRGSr. GTPase activity of transducin was measured in suspensions of dROS (5 µM rhodopsin and 0.4 µM transducin) in the presence of increasing concentrations of hRGSr or its mutants. Calculated GTPase rate constants are plotted as a function of hRGSr or mutant concentration. hRGSr, closed squares; hRGSr-Ser131, open squares; hRGSr-Gln131, closed diamonds; hRGSr-Asp131, open diamonds; hRGSr-Leu131, closed triangles; hRGSr-Ala131, open triangles; hRGSr-His131, closed circles; and hRGSr-Delta 131, open circles.

Competition between hRGSr and hRGSr Mutants in Stimulation of Gtalpha GTPase Activity-- Experiments in Fig. 2 have suggested that hRGSr mutants with substitutions of Asn131 have impaired binding to Gtalpha ·AlF4-. The binding assay may, however, not be sufficiently sensitive to detect relatively weak interactions. To determine if the drastically reduced ability of some RGS mutants to stimulate the GTPase activity of transducin correlates with the lack of mutant binding to transducin, we carried out competition experiments. The hRGSr mutants incapable of accelerating Gtalpha GTPase activity were examined for their ability to block stimulation of GTPase activity of transducin by hRGSR. Fig. 4 demonstrates that none of the tested mutants, hRGSr-Ala131, hRGSr-His131, and hRGSr-Delta 131, at 5 µM concentration, had any effect on stimulation of GTPase activity of transducin by 50 nM hRGSr. These data suggest that the hRGSr mutants that produced no stimulation of Gtalpha GTPase activity lost their binding to Gtalpha .


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Fig. 4.   hRGSr mutants do not block stimulation of GTPase activity of transducin by hRGSr. Rate constants of GTPase activity of transducin were determined in suspensions of dROS membranes (5 µM rhodopsin and 0.4 µM transducin) with and without addition of 50 nM hRGSr and 5 µM each of the following mutants: hRGSr-Ala131, hRGSr-His131, and hRGSr-Delta 131.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Molecular mechanisms of RGS protein action as GAP for heterotrimeric GTP-binding proteins are not well understood. Studies on the Ras-specific p120GAP suggest that the Ras GAP donates conserved Arg789 residue to the RAS catalytic site (29, 30) thus providing the catalytic mechanism for p120GAP activity. The crystal structure of RGS4 bound to Gialpha 1·AlF4- has suggested two mechanisms for RGS GAP activity toward heterotrimeric G-proteins (17). Interaction of RGS protein with the G-protein switch regions indicates that the mechanism of the GTPase activation by RGS may primarily be a reduction in the free energy of the transitional state via stabilization of Galpha switch regions and residues directly involved in GTP hydrolysis (17). An additional putative mechanism for the RGS GAP activity would be a donation of the catalytic residue to the active site of Galpha . The only residue that RGS4 introduces into the active site of Gialpha 1 is Asn128. Although Asn128, in contrast to the Ras GAP Arg789 or an intrinsic Arg in Galpha subunits, does not directly interact with GDP and AlF4- (13, 14, 17, 30), it makes a contact with the side chain of Gln204 of Gialpha 1, which stabilizes and orients the hydrolytic water molecule in the transitional state of Gialpha 1. Conceivably, Asn128 is within hydrogen-bonding distance of the hydrolytic water molecule and may bind and orient it for nucleophilic attack of the gamma -phosphate of GTP (17).

To probe the role of Asn131 of hRGSr for the mechanism of RGS protein GAP activity, we initially synthesized a peptide of hRGSr corresponding to the region of interaction between Gialpha 1·AlF4- and RGS4 containing Asn128. We reasoned that if the catalytic role of the Asn residue is a major component of RGS GAP activity, then perhaps a peptide containing the catalytic residue would alone be capable of eliciting the stimulation of GTPase activity. Our data demonstrated that hRGSr peptide-(123-140) containing catalytic Asn131 retained the ability to bind hRGSr but failed to accelerate the GTPase activity of transducin. This indicates that the interaction of at least two and likely all three Galpha binding regions of RGS protein is required to stimulate Galpha GTPase activity. Consistent with this conclusion is the recent finding that even short deletions within the RGS domain of RGS4 destroyed its GAP activity (31).

Further analysis of the role of Asn131 of hRGSr was carried out using mutational substitutions of this residue. The major result from testing all hRGSr mutants is that replacement of Asn131 with other residues dramatically decreases the affinity of mutant hRGSr binding to Gtalpha . Substitution of Asn131 by Ser was intriguing because the Asn residue is not absolutely conserved in RGS proteins, and some RGS proteins, including GAIP, have a Ser at this position (5, 17, 31). Serine has proven to be the best substitution for Asn with respect of retaining the GAP activity of hRGSr protein. The hRGSr-Ser131 mutant had more than 10-fold lower affinity for Gtalpha but can stimulate its GTPase activity nearly as well as native hRGSr (Vmax ~80%). Therefore, it is not surprising that this residue was evolutionary selected instead of Asn for some RGS proteins. Interestingly, the reported concentrations of the Ser containing RGS domain of retina-specific RET-RGS1 (1 µM) and GAIP (5 µM) required for the half-maximal GTPase stimulation of transducin are comparable to the EC50 value for hRGSr-Ser131 (28, 32).

An indication that the Asn residue may indeed serve to some extent as a catalytic residue was provided by the hRGSr-Gln131 mutant. The effects of this mutant on GTPase activity of transducin nearly reached a plateau at only ~60% Vmax of that observed with hRGSr. However, effects of other hRGSr mutants argue against a catalytic role of the Asn residue as the key component of the RGS GAP activity. The hRGSr-Leu131 and hRGSr-Ala131 mutants at very high concentrations started to have a stimulatory effect on Gtalpha 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 Asn131 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 stabilization of the transition state for GTP hydrolysis because its replacement or deletion leads to a drastic reduction in hRGSr affinity for Gtalpha .

In addition to their role as GAPs, RGS proteins may act as antagonists for some G-protein effectors, particularly for phospholipase Cbeta . RGS4 has been shown to block activation of phospholipase Cbeta by Gqalpha GTPgamma S (33). In another study, RGS4 inhibited inositol phosphate synthesis activated by AlF4- in COS-7 cells overexpressing Gq (34). Tesmer et al. (17) have suggested that the RGS proteins lacking the Asn residue may better serve as inhibitors of effector binding than as GAPs. This would appear to be a likely scenario if replacements of the Asn residue resulted in a loss of GAP activity without a concurrent reduction of the RGS protein affinity for activated Galpha subunits. The results of this work suggest that the main consequence of Asn replacement is an impairment of binding between mutated hRGSr protein and Gtalpha . Furthermore, none of the hRGSr mutants have shown enhanced affinity to the active Gtalpha ·GTPgamma S conformation which could be indicative of the potential of such a mutant to serve as an antagonist for the G-protein effector.

This study only begins to address the questions, introduced by the first crystal structure between G-protein and RGS protein, about the mechanism of RGS protein GAP activity (17). Further biochemical analysis coupled with resolution of other crystal structures between activated Galpha subunits and RGS proteins would ultimately define a role of the critical Asn residue.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant EY-10843. The services provided by the Diabetes and Endocrinology Research Center of the University of Iowa were supported by National Institutes of Health Grant DK-25295.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.

Dagger To whom correspondence and reprint requests should be addressed: Dept. of Physiology and Biophysics, University of Iowa College of Medicine, 5-660 Bowen Science Bldg., Iowa City, IA 52242.. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: Nikolai-Artemyev{at}UIOWA.EDU.

1 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; Gtalpha , rod G-protein (transducin) alpha -subunit; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PCR, polymerase chain reaction.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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