The GTPase-activating Protein RGS4 Stabilizes the Transition State for Nucleotide Hydrolysis*

  • David M. Berman
    Footnotes
    Affiliations
    From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • Tohru Kozasa
    Affiliations
    From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • Alfred G. Gilman
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant GM34497, the Lucille P. Markey Charitable Trust, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology. 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.
    Member of the Cell Regulation Graduate Program of University of Texas Southwestern Graduate School of Biomedical Sciences. Supported by the Medical Scientist Training Program and Pharmacological Sciences Training Grant GM07062.
      RGS proteins constitute a newly appreciated group of negative regulators of G protein signaling. Discovered by genetic screens in yeast, worms, and other organisms, two mammalian RGS proteins, RGS4 and GAIP, act as GTPase-activating proteins for members of the Gi family of G protein α subunits. We have purified recombinant RGS4 to homogeneity and demonstrate that it acts catalytically to stimulate GTP hydrolysis by Gi proteins. Furthermore, RGS4 stabilizes the transition state for GTP hydrolysis, as evidenced by its high affinity for the GDP-AlF4-bound forms of G and G and its relatively low affinity for the GTPγS- and GDP-bound forms of these proteins. Consequently, RGS4 is most likely not a downstream effector for activated Gα subunits. All members of the Gi subfamily of proteins tested are substrates for RGS4 (including G and G); the protein has lower affinity for G, and it does not stimulate the GTPase activity of G or G12α.

      INTRODUCTION

      Heterotrimeric G protein
      The abbreviations used are: G proteins
      heterotrimeric guanine nucleotide-binding proteins
      GAP
      GTPase-activating protein
      GTPγS
      guanosine 5′-(3-O-thio)triphosphate
      C12E10
      polyoxyethylene-10-lauryl ether.
      α subunits cycle between inactive, GDP-bound and active, GTP-bound states, and the duration of activation is thus dependent on their intrinsic GTPase activity. Nucleotide hydrolysis by some G proteins is controlled extrinsically by activating proteins known as GAPs, and the G protein GAPs described previously are known effectors of the α subunit with which they interact (
      • Ross E.M.
      ). Thus, phospholipase C-β1 is activated by G and, in turn, increases the kcat for nucleotide hydrolysis by G by roughly 100-fold (
      • Berstein G.
      • Blank J.L.
      • Jhon D.Y.
      • Exton J.H.
      • Rhee S.G.
      • Ross E.M.
      ,
      • Biddlecome G.H.
      • Berstein G.
      • Ross E.M.
      ). G interacts similarly with the γ subunit of a retinal cyclic GMP phosphodiesterase (
      • Arshavsky V.Y.
      • Bownds M.D.
      ,
      • Arshavsky V.Y.
      • Dumke C.L.
      • Zhu Y.
      • Artemyev N.O.
      • Skiba N.P.
      • Hamm H.E.
      • Bownds M.D.
      ,
      • Angleson J.K.
      • Wensel T.G.
      ,
      • Pages F.
      • Deterre P.
      • Pfister C.
      ).
      A family of negative regulators of G protein signaling, so-called RGS proteins, was identified recently as a result of genetic studies in yeast, worms, and other organisms (
      • Roush W.
      ,
      • Chan R.K.
      • Otte C.A.
      ,
      • Chan R.K.
      • Otte C.A.
      ,
      • Weiner J.L.
      • Guttierez-Steil C.
      • Blumer K.J.
      ,
      • Dietzel C.
      • Kurjan J.
      ,
      • Dohlman H.G.
      • Apaniesk D.
      • Chen Y.
      • Song J.P.
      • Nusskern D.
      ,
      • Koelle M.R.
      • Horvitz H.R.
      ,
      • Druey K.M.
      • Blumer K.J.
      • Kang V.H.
      • Kehrl J.H.
      ,
      • Siderovski D.P.
      • Heximer S.P.
      • Forsdyke D.R.
      ,
      • Newton J.S.
      • Deed R.W.
      • Mitchell E.L.D.
      • Murphy J.J.
      • Norton J.D.
      ,
      • Hong J.X.
      • Wilson G.L.
      • Fox C.H.
      • Kehrl J.H.
      ,
      • De Vries L.
      • Mousli M.
      • Wurmser A.
      • Farquhar M.G.
      ). We demonstrated that two of these proteins, RGS4 and GAIP, act as GAPs on several members of the Gi subfamily of α subunits (
      • Berman D.M.
      • Wilkie T.M.
      • Gilman A.G.
      ). However, the mechanism of this effect and the possibility of other functional relationships between these GAPs and their target G proteins remained unclear. We demonstrate herein that purified recombinant RGS4 acts catalytically to accelerate the GTPase activity of Gi protein family members, that RGS4 has high affinity for the transition state complex of the G protein α subunit bound to GDP-AlF4 (but low affinity for GTPγS-α and GDP-α), and that the rank order of affinities of RGS4 for transition state complexes of α subunits is Gi family > Gq≫ Gs. RGS4 has no detectable GAP activity toward G or G12α.

      EXPERIMENTAL PROCEDURES

       Purification and Activation of Proteins

      G, Giα1, and G were expressed and purified as described (
      • Lee E.
      • Linder M.E.
      • Gilman A.G.
      ), as were G and G12α (
      • Kozasa T.
      • Gilman A.G.
      ); Giα1 and G were myristoylated unless stated otherwise. Bovine retinal G was the generous gift of Dr. Heidi Hamm (University of Illinois College of Medicine), while recombinant G was supplied by Dr. John Hepler (this laboratory) (
      • Hepler J.R.
      • Kozasa T.
      • Smrcka A.V.
      • Simon M.I.
      • Rhee S.G.
      • Sternweis P.C.
      • Gilman A.G.
      ). G was activated with 100 µM GTPγS and 10 mM MgSO4 by incubation at 30°C for 60 min. The indicated GDP-bound α subunits were activated with AlF4 by incubation with 20 µM AlCl3, 10 mM NaF, and 10 mM MgSO4 for 10 min on ice.
      RGS4 (hexahistidine-tagged at the amino terminus) was synthesized in Escherichia coli as described previously (
      • Berman D.M.
      • Wilkie T.M.
      • Gilman A.G.
      ), except that cells were incubated for 12 h at 30°C after induction. The bacterial lysate was purified on Ni-NTA resin (Qiagen) as described, except that 500 mM NaCl was included in the first wash. Ammonium sulfate (1.2 M final concentration) was added to the Ni-NTA column eluate, and this solution was applied to a 15-ml Phenyl-Sepharose FPLC column equilibrated with 50 mM Tris-HCl (pH 8), 2 mM dithiothreitol, and 1.2 M (NH4)2SO4. The column was washed with 100 ml of equilibration buffer and eluted with a 100-ml continuous gradient of 50 mM Tris-HCl and 1.2 to 0.84 M (NH4)2SO4. Fractions were analyzed electrophoretically, and protein was pooled and dialyzed into 50 mM NaHepes (pH 8), 1 mM EDTA, and 2 mM dithiothreitol prior to concentration and storage. The yield was 4 mg/liter of bacterial culture. Purified RGS4 was homogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (see Fig. 5), had the anticipated amino-terminal amino acid sequence, and displayed a single mass spectroscopic peak (Mr = 24,183; predicted, 24,188).
      Figure thumbnail gr5
      Fig. 5RGS4 and GDP-AlF4-Giα1 form a high-affinity complex. A, RGS4 (2.2 mg) or Giα1 (2.2 mg; activated with 20 µM AlCl3, 10 mM NaF, and 5 mM MgSO4 for 10 min at 4°C) was applied to a 16/60 Superdex gel filtration column equilibrated and eluted with 50 mM NaHepes (pH 8), 1 mM EDTA, 10 mM NaF, 20 µM AlCl3, 5 mM MgSO4, 2 mM dithiothreitol, and 50 mM NaCl. Fractions (1.5 ml) were collected, and A280 was monitored. Arrows indicate the position of molecular weight standards. B, as in A, except that RGS4 and GDP-AlF4-Giα1 were incubated together prior to gel filtration. Fractions were analyzed electrophoretically, and gels were stained with Coomassie Brilliant Blue. The higher molecular weight band is Giα1; the lower is RGS4. C, as in B, except the Gln204→ Leu mutant of Giα1 was substituted for the wild type protein.

       GAP Assays

      GAP assays were performed using G as the substrate as described previously (
      • Berman D.M.
      • Wilkie T.M.
      • Gilman A.G.
      ). This assay relies on formation of a complex between G and [γ-32P]GTP in the absence of Mg2+ and subsequent initiation of nucleotide hydrolysis by incubation of this complex at 4°C with Mg2+ and RGS4. A single round of hydrolysis of GTP to GDP is then monitored over 5 min by quantification of release of 32Pi.
      Direct measurement of the kcat for GTP hydrolysis by G12α and G required an alternative protocol because of their slow rates of nucleotide exchange. G12α and G were incubated with 5 µM [γ-32P]GTP (20-50 cpm/fmol) and 5 mM EDTA at 30°C for 30 min. Samples were then gel-filtered at 4°C through a Sephadex G-50 spin column (Pharmacia) equilibrated with 50 mM NaHepes (pH 8), 1 mM dithiothreitol, 5 mM EDTA, and 0.1% C12E10 to remove free GTP and Pi. GTPase activity was measured at 15°C in 50 µl of the same solution with addition of 8 mM MgSO4, 1 mM GTP (unlabeled), and the indicated concentration of RGS4.

      RESULTS AND DISCUSSION

       RGS4 Acts Catalytically

      We reported previously that RGS4 and GAIP accelerate the rate of the GTPase reaction catalyzed by selected members of the Gi subfamily of α subunits by at least 40-fold; the concentrations of RGS proteins utilized in that study were in excess of those of the Gα protein substrates. Other GAPs have been shown to act catalytically (
      • Arshavsky V.Y.
      • Bownds M.D.
      ,
      • Lancaster C.A.
      • Taylor-Harris P.M.
      • Self A.J.
      • Brill S.
      • van Erp H.E.
      • Hall A.
      ,
      • Wiesmuller L.
      • Wittinghofer A.
      ,
      • Vogel U.S.
      • Dixon R.A.F.
      • Schaber M.D.
      • Diehl R.E.
      • Marshall M.S.
      • Scolnick E.M.
      • Sigal I.S.
      • Gibbs J.B.
      ), and the same is true of RGS4. Thus, concentrations of RGS4 as low as 2.9 nM were sufficient to enhance the GTPase activity of 70 nM GTP-G (Fig. 1A), and the initial slopes of the curves in Fig. 1A (from 0 to 29 nM RGS4) are directly proportional to the RGS4 concentration. Thus, in this experiment, 1 mol of RGS4 catalyzed the turnover of 6 mol of GTP-G/min at a substrate (GTP-G) concentration that is probably more than 20-fold below the Km of GTP-G for RGS4. It would be necessary to saturate the substrate with RGS4 to estimate the maximal enhancement of the rate of GTP hydrolysis; the rate of hydrolysis of GTP is then too fast to measure with the methods used herein to monitor release of 32Pi. The maximal enhancement of the rate of GTP hydrolysis observed in this experiment was greater than 40-fold.
      Figure thumbnail gr1
      Fig. 1RGS4 acts catalytically to stimulate the GTPase activity of G. A, G (200 nM; 10 pmol/sample) was incubated with 1 µM [γ-32P]GTP for 20 min at 20°C and then transferred to an ice bath for 5 min. The indicated concentrations of RGS4 (plus Mg2+) were added at zero time, and release of 32Pi was determined over the indicated time course at 4°C. (The estimated concentration of GTP-G was 70 nM.) Inset, the initial rates of Pi release are plotted against the amount of RGS4. B, varying concentrations of GTP-G were incubated with 2.1 nM RGS4 and 15 mM MgSO4 at 4°C, and the linear release of 32Pi was monitored over 1 min (7 time points). These initial rates are plotted against the concentration of GTP-G (determined by the maximal amount of Pi released). Inset, Lineweaver-Burk analysis of these data. These data represent one of two similar experiments.
      Variation of the substrate (GTP-G) concentration permitted estimation of the Km for the interaction of GTP-G with RGS4 and the maximal rate of turnover of GTP-G by RGS4 (Fig. 1B). These values are 2.5 µM and 14/s, respectively, but should be considered estimates, since the substrate concentration was varied over a relatively limited range. This turnover rate could be limited by the maximal intrinsic rate of GTP hydrolysis by G.

       RGS4 Stabilizes the Transition State for GTP Hydrolysis

      The crystal structures of the GDP-AlF4 complexes of Giα1 (
      • Coleman D.E.
      • Berghuis A.M.
      • Lee E.
      • Linder M.E.
      • Gilman A.G.
      • Sprang S.R.
      ) and G (
      • Sondek J.
      • Lambright D.G.
      • Noel J.P.
      • Hamm H.E.
      • Sigler P.B.
      ) revealed that AlF4 occupies the position normally taken by the γ-phosphate of GTP but, remarkably, that the fluorine atoms assume a square planar configuration about the central aluminum atom, in contrast to the tetrahedral geometry of a phosphate group. The AlF4 complex is octahedrally coordinated to a β-phosphate oxygen and to the putative hydrolytic water molecule. Furthermore, an Arg residue and a Gln residue that are known to be critical for catalysis are dramatically reoriented in the GDP-AlF4 structure (compared to their positions in the GTPγS-bound protein), contacting the fluorine atoms and the hydrolytic water. These facts (and others) argue that GDP-AlF4 is not a GTP analog but rather mimics the trigonal-bipyramidal species that is presumed to appear at or near the transition state of the SN2 reaction.
      These considerations have prompted examination of the interactions of RGS4 with the GDP-, GTPγS-, and GDP-AlF4-bound forms of G by testing the capacity of these proteins to compete with GTP-G for its GAP. The basic assay is seen in Fig. 2A. Addition of 2 µM GDP-G or GTPγS-G to 200 nM [γ-32P]GTP-G had little or no observable effect on nucleotide hydrolysis stimulated by 29 nM RGS4. However, 2 µM GDP-AlF4-G completely blocked the effect of RGS4, presumably by formation of a high affinity complex with the GAP. Free guanine nucleotide or the combination of AlCl3, NaF, and MgSO4 had no effect on nucleotide hydrolysis in these assays.
      Figure thumbnail gr2
      Fig. 2Inhibition of the effect of RGS4 by GDP-AlF4-G. G was incubated with 100 µM GTPγS at 30°C for 1 h or with 20 µM AlCl3, 10 mM NaF, and 5 mM MgSO4 for 15 min on ice. A, the substrate for RGS4 was 200 nM [γ-32P]GTP-G. RGS4 (29 nM) was incubated on ice with 2 µM GTPγS-G (▪), 2 µM GDP-AlF4-G (•), 2 µM GDP-G (▴), or buffer (∘) prior to incubation with [γ-32P]GTP-G. One reaction mixture did not contain RGS4 protein (□). Release of 32Pi was determined as described under “Experimental Procedures.” The data shown are representative of three such experiments. B, the substrate was 140 nM [γ-32P]GTP-G. RGS4 (29 nM) was incubated with the indicated concentrations of GTPγS-G (▪) or GDP-AlF4-G (•) prior to addition of substrate. The time course of GTP hydrolysis was determined under these conditions, and each point of the graph represents the rate determined by curve fitting to nine such time points. The data shown represent one of two similar experiments.
      Competition assays (at 150 nM GTP-G) with different concentrations of GTPγS-G or GDP-AlF4-G highlight the preferential affinity of RGS4 for the transition state conformation of the G protein (Fig. 2B). Stimulated GTP hydrolysis is nearly completely inhibited at concentrations of GDP-AlF4-G that approximate those of RGS4. The apparent Kd of RGS4 for GDP-AlF4-G is thus below 100 nM. Again, GTPγS-G is a poor competitor (Fig. 2B), consistent with the Km for GTP-G estimated in Fig. 1B, as were the GTPγS-bound forms of G, Giα1, and Giα3 (not shown).

       RGS4 Interacts with G Subfamily Members and with G but Not with G or G12α

      The competition assay just described was utilized to examine the interactions of RGS4 with other G protein α subunit family members. We demonstrated previously that RGS4 stimulates GTP hydrolysis by Giα1, Giα2, Giα3, and G. Giα1 and G were similarly effective in the competition assay as the GDP-AlF4-bound species (Fig. 3). Two additional Gi subfamily members were also tested for interactions with RGS4. GDP-AlF4-G was an effective competitor (Fig. 3), indistinguishable from Giα1 and G. The kcat for GTP hydrolysis by G was measured directly and was increased from its normal low value of 0.02/min to 0.12/min by 12 nM RGS4 (Fig. 4A). It thus seems likely that the GTPase activity of all Gi subfamily members is stimulated by RGS4. We have attempted direct estimation of affinities between GDP-AlF4-bound α subunits and RGS4 by observation of surface plasmon resonance (Pharmacia Biosensor). Appropriate protein-protein interactions were detected, but the rate of dissociation of hexahistidine-tagged RGS4 from the Ni-NTA-derivatized chips of the Biosensor instrument was faster than the rate of dissociation of GDP-AlF4-G from RGS4. Thus, we can only estimate an upper limit for the Kd from these experiments, roughly 100 nM.
      Figure thumbnail gr3
      Fig. 3Specificity of the inhibition of RGS4 by various GDP-4-Gα protein complexes. The substrate was 140 nM [γ-32P]GTP-G. RGS4 (29 nM) was incubated with GDP-AlF4 complexes of G (▾), Giα1 (▴), G (•), G (•), or G (▪) for 5 min on ice prior to incubation with the substrate. The time course of GTP hydrolysis was determined under the indicated conditions, and each point of the graph represents the rate determined by curve fitting to nine such time points. The RGS4-stimulated control rate of GTP hydrolysis ranged from 3.5/min to 6.5/min in various experiments. The data shown represent one of two similar experiments with each competitor protein.
      Figure thumbnail gr4
      Fig. 4RGS4 stimulates the GTPase activity of G but not G12α. [γ-32P]GTP G and G12α substrates were prepared as described under “Experimental Procedures.” A, [γ-32P]GTP-G (2.4 nM) was incubated with (•) or without (▪) 12 nM RGS4, and the rate of GTP hydrolysis was measured at 15°C. B, [γ-32P]GTP-G12α (3 nM) was incubated with (•) or without (▪) 0.6 µM RGS4, and the initial rate of GTP hydrolysis was measured at 15°C. In both panels, data shown are averages of duplicate determinations from a single experiment, which is representative of three such experiments.
      We also noted previously the inability of RGS4 (or GAIP) to stimulate GTP hydrolysis by G; accordingly, the GDP-AlF4-bound form of G did not compete with G (Fig. 3). Of interest, GDP-AlF4-G did interact with RGS4, although its apparent affinity for the protein is 10- to 100-fold or more lower than are those of the Gi subfamily members. However, the assumption that GDP-AlF4-bound complexes of all G protein α subunits are transition-state mimics may be unwarranted. Gq must be reconstituted with an appropriate receptor to examine the effect of RGS4 on nucleotide hydrolysis; the rate of nucleotide exchange is too low to permit preparation of GTP-G substrate by the present methods. A high concentration of RGS4 did not stimulate GTP hydrolysis by G12α, which proceeded with a kcat of 0.06/min at 15°C (Fig. 4B).

       RGS4 Forms a High-affinity Complex with GDP-4-Giα1

      The data presented above strongly imply formation of a high affinity complex between RGS4 and the GDP-AlF4-bound forms of various G proteins. To demonstrate this directly and to prepare material for crystallographic analysis, we have performed gel filtration chromatography on mixtures of RGS4 and nonmyristoylated Giα1; the latter protein has been crystallized previously in various conformations. Gel-filtered separately, Giα1 and RGS4 elute from a Superdex 200 column at positions consistent with their monomeric molecular weights (Fig. 5A). After incubation of GDP-AlF4-Giα1 with a modest molar excess of RGS4 on ice for 10 min, the gel filtration profile reveals formation of a complex of the two proteins, migrating with an apparent molecular weight of 70,000; free, excess RGS4 is also seen in its monomeric position (Fig. 5B). The GTPase activity of the Gln204→ Leu mutant of Giα1 is impaired severely. This protein does not interact with AlF4, and its GTPase activity is not affected by RGS4 (
      • Berman D.M.
      • Wilkie T.M.
      • Gilman A.G.
      ,
      • Coleman D.E.
      • Berghuis A.M.
      • Lee E.
      • Linder M.E.
      • Gilman A.G.
      • Sprang S.R.
      ). Consistent with these observations, this protein does not form a high affinity complex with RGS4 when incubated with AlCl3, NaF, and MgSO4 (Fig. 5C); the elution profiles of the two proteins correspond to those observed in Fig. 5A.
      We conclude that RGS4 has a relatively low affinity (greater than 1 µM) for its substrates, GTP-G family members, but interacts directly and with high affinity with the transition-state conformations of these α subunits. Stabilization of the transition state lowers the activation energy barrier for hydrolysis of GTP, accounting for the large rate enhancements that are seen. We surmise that RGS4 is not an effector for G proteins, based on the relatively poor affinity of RGS4 for their GTPγS-bound forms compared to those of other known effectors of G protein α subunits. We note, however, that some other RGS proteins are considerably larger than are RGS4 or GAIP, and generalization of this point may be unwarranted. Finally, we call attention to the recent observation by Mittal et al. (
      • Mittal R.
      • Zhmadian M.R.
      • Goody R.S.
      • Wittinghofer A.
      ) that wild-type p21ras protein interacts with AlF4 only in the presence of its GAPs. Thus, the ground state of p21ras must be too distant, conformationally, from the transition state to recognize AlF4 (unlike heterotrimeric G proteins), but GAPs for low molecular weight GTPases and RGS proteins both appear to act predominantly by stabilizing the transition states for nucleotide hydrolysis and not by elevating the energy level of the enzyme-substrate complex.

      Acknowledgments

      We thank Drs. Andre Raw, Carmen Dessauer, Thomas Wilkie, and Stephen Sprang for helpful suggestions.

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