A Point Mutation in Galpha o and Galpha i1 Blocks Interaction with Regulator of G Protein Signaling Proteins*

Keng-Li LanDagger , Noune A. SarvazyanDagger , Ronald Taussig§, Robert G. Mackenzie, Paul R. DiBelloparallel , Henrik G. Dohlmanparallel , and Richard R. NeubigDagger **Dagger Dagger

From the Dagger  Departments of Pharmacology, ** Internal Medicine/Hypertension, and § Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109,  Parke-Davis Research Division of Warner Lambert Company, Ann Arbor, Michigan 48105, and parallel  Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha  subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1sst that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alpha o and alpha i1 was done. G184S alpha o and G183S alpha i1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G right-arrow S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alpha o and alpha i1 to compete with fluorescein isothiocyanate-alpha o binding to glutathione S-transferase-RGS4. The G right-arrow S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alpha o and alpha i1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins initiates signals elicited by numerous hormone, neurotransmitter, and sensory stimuli (1). Receptors activate G proteins by stimulating the release of GDP from the alpha  subunit, allowing GTP to bind and to induce dissociation of the G protein alpha  and beta gamma subunits, which interact with effector proteins to modulate cellular responses (2-5).

The duration and strength of receptor-generated physiological responses are regulated by the rate at which GTP is hydrolyzed by alpha  subunit (6, 7). It has been known for some time that the physiological turn-off of some G protein-mediated signals is faster than would be predicted from the in vitro GTPase activity of isolated G protein subunits (8, 9). The solution to this paradox appears to reside in the newly recognized family of regulator of G protein signaling (RGS)1 proteins, first identified genetically in the yeast Saccharomyces cerevisiae and in the nematode, Caenorhabditis elegans (10-13). At least 19 RGS protein cDNAs have been identified in mammalian tissues, all sharing a homologous carboxyl-terminal region of ~120 amino acid residues termed the RGS domain (13-15). Biochemical studies with alpha i and alpha q family of G proteins demonstrated that RGS4 and Galpha -interacting protein (GAIP) act as GTPase accelerating proteins (GAPs) (16, 17), which could account for inhibition of G protein-mediated responses (15). GAP activity of Sst2 for Gpa1 has also been recently demonstrated (18). The mechanism by which GTPase activity is enhanced by RGS appears to be the stabilization of the transition state conformation of Galpha for nucleotide hydrolysis (19, 20). RGS4 also directly inhibits the interaction of the GTPgamma S-bound alpha q subunit with phospholipase Cbeta , presumably by binding to the effector region of activated alpha q (16).

A mutant yeast Galpha subunit, Gpa1sst, was recently identified in a screen for novel strains showing the "supersensitive to pheromone" (sst) phenotype. It has a single glycine to serine mutation and escapes from negative regulation by the RGS protein, Sst2 (21). Since many RGS proteins affect Gi-family G proteins, and the crystal structure of the RGS4·alpha i1 complex was recently reported, we wanted to see if the corresponding G right-arrow S mutation in alpha i1 and alpha o would produce insensitivity to RGS. A major objective was to obtain a detailed biochemical and mechanistic analysis of this newly identified class of mutations.

We report that G alpha i1 and alpha o subunit G right-arrow S mutants are insensitive to GTPase activation by two different RGS proteins. Quantitative flow cytometry studies demonstrated that a >30-100-fold reduction in affinity of RGS for the alpha  subunit transition state is the mechanism of the insensitivity. In future studies, these mutant alpha  subunits should be useful for evaluating the role of endogenous RGS proteins in the kinetics and function of Gi family members. Given the existence of nearly 20 RGS proteins of which at least 5 act on Gi-family proteins, it would be difficult to inactivate all of them to determine the physiological role of endogenous RGS proteins in Gi signaling. Thus, a Gi alpha  subunit insensitive to RGS proteins can be used to assess the combined role of all RGS proteins in Gi function in vivo.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

DNA Construction and Mutagenesis-- The G184S mutation in the alpha o sequence was introduced by the megaprimer polymerase chain reaction mutagenesis technique using mutagenic antisense primer 5'-GGTTTCTACGATCGAAGTTGTTTTGAC-3' as described (22, 23). The G183S mutation in alpha i1 was constructed by overlap-extension polymerase chain reaction using sense and antisense mutagenic primers 5'-AGTGAAAACGACGTCAATTGTGGAAACC-3' and 5'-GGTTTCCACAATTGACGTCGTTTTCACT-3', respectively. The coding region of rat RGS4 was amplified by polymerase chain reaction from an Expressed Sequence Tag obtained from The Institute of Genomic Research and cloned into the pGEX-2T expression vector. A restriction fragment comprising nucleotides 913-1358 of the complete human RGS7 cDNA2 was cloned into the GST expression vector pgGSTag2. The expressed protein contained the RGS domain (nucleotides 985-1341) but only a portion of the long amino terminus of RGS7.

Purification of His6alpha Subunits and GST-RGS Proteins-- All Galpha subunits in this paper were expressed in Escherichia coli and purified as amino-terminal His6 constructs by a modification of the method of Lee et al. (24). The GST-RGS4 and -RGS7 fusion proteins were purified as described (25). The bacterial supernatant was incubated with glutathione-agarose beads (Amersham Pharmacia Biotech) at 4 °C overnight. After washing with phosphate-buffered saline, the GST-RGS4 was eluted with 10 mM glutathione in phosphate-buffered saline and dialyzed against 50 mM Tris and 1 mM EDTA, pH 7.4. The fusion proteins were cleaved by incubation overnight at 4 °C with 10 units of thrombin/mg of fusion protein followed by incubation with glutathione-agarose to remove GST and any uncleaved GST-RGS4.

GAP Assays-- [gamma -32P]GTP (1 µM) was allowed to bind to 50 nM alpha o for 20 min at room temperature (23-24 °C) or 50 nM alpha i1 for 15 min at 30 °C. After lowering the temperature to 4 °C, the hydrolysis reaction was started by the addition of MgSO4 and GTPgamma S to final concentrations of 15 mM and 200 µM in the presence or absence of 100 nM RGS4 or RGS7. Aliquots (50 µl) were diluted in 1 ml of 15% (w/v) charcoal solution (50 mM NaH2PO4, pH 2.3, 0 °C) at the indicated time points. The amount of [gamma -32P]Pi released at each time point was fit to an exponential function, cpm(t) = cpmo + Delta cpm × (1 - e-kt).

Binding of alpha  to GST-RGS4-agarose-- Wild type or mutant GDP-bound alpha  (1 µM) was mixed with GST-RGS4 fusion protein (1 µM) bound to glutathione-agarose beads (5 × 105 beads/ml) in a final volume of 100 µl of HEDML buffer (50 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 10 mM MgSO4, 20 ppm deionized Lubrol) with 0.05% bovine serum albumin in the presence or absence of 20 µM AlCl3 and 10 mM NaF (HEDML/AF). After 2 h, the beads were washed three times with 1 ml of ice-cold HEDML/AF buffer with bovine serum albumin, and bound products were separated by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue stain.

Competition Binding of the alpha  Subunit to GST-RGS4-- Labeling of purified His6 alpha o with fluorescein isothiocyanate (FITC) was conducted as described (26). Glutathione-agarose beads (Amersham) with sizes between 35 and 78 µm were prepared for flow cytometry by filtering through stainless steel Tyler sieves in HEDML buffer. Two nM GST or GST-RGS4 fusion protein was bound to the beads (5 × 104/ml) for 20 min at room temperature in HEDML buffer. Beads were then washed and incubated with 4 nM FITC-alpha o with the indicated amounts of unlabeled alpha  subunit in 100 µl of HEDML/AF buffer at room temperature for 2.5 h. The amount of FITC-alpha o bound to the GST-RGS4 was quantitated on a Becton Dickinson FACScan as described (26). Data are presented as a fraction of the control fluorescence after subtracting nonspecific binding (~20% of the total). Results were fit to a one-site competition binding function, and the IC50 for the competitors were calculated using Prism version 2.01 for Windows 95 (Graphpad Software, San Diego, CA).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Nucleotide Binding to and RGS-stimulated Hydrolysis by alpha  Subunit-- The time course of nucleotide binding to alpha  subunits was determined by use of the fluorescent nucleotide derivative, methylanthraniloyl GTPgamma S as described (27). Similar rates of binding were seen for both the G right-arrow S mutants and the wild type proteins. Rate constants were 0.20 ± 0.06 and 0.21 ± 0.08 min-1 for alpha o and 0.049 ± 0.002 and 0.12 ± 0.03 min-1 for alpha i1, wild type and mutant, respectively. The kcat values for wild type and G right-arrow S mutant proteins in the absence of RGS were very similar (Fig. 1 and Table I). In the presence of 100 nM RGS4, the reaction was completed by the first time point for wild type alpha o and alpha i1 (Fig. 1). Even at 4 °C, the reaction was too fast to measure with a rate constant greater than 5 min-1. There was no effect of RGS4 on the kcat of either G right-arrow S mutant (Fig. 1, B and D, and Table I). The RGS domain fragment of RGS7 (100 nM) increased the kcat of wild type alpha i1 by ~9-fold, whereas there was no effect on the alpha i1 G right-arrow S (Fig. 1C and Table I).


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Fig. 1.   Effect of RGS proteins on single-turnover GTP hydrolysis by wild type (WT) and mutant alpha  subunits. Single-turnover GTP hydrolysis by 50 nM alpha o (A and B) or alpha i1 (C and D) was initiated by the addition of MgSO4 and GTPgamma S in the presence or absence of 100 nM RGS4 or truncated RGS7. Base-line [gamma -32P]GTP hydrolysis was measured before the reaction (~30% of total) and was subtracted from the data. GTP hydrolysis at the indicated time points was calculated as a fraction of the total GTP hydrolyzed measured at 30 min. Experiments have been replicated at least three times. The data were fitted to a single exponential association function (Graphpad Prism), and the results are listed in Table I.

                              
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Table I
Single turnover rates for alpha  subunit with and without RGS
Rate constants were determined from exponential fits of the data in Fig. 1. Values are the mean ± S.E. of three determinations except where noted.

GAP Activity at Increasing Concentrations of RGS4-- To determine if GAP activity could be restored at higher concentrations of RGS4, GTP hydrolysis at 1 min was measured with 50 nM Galpha subunit and 0-3 µM RGS4. The EC50 values for RGS4 were 5 ± 1 and 10 ± 4 nM for wild type alpha o and alpha i1, respectively (Fig. 2). There was only a slight increase in GTP hydrolyzed by either G right-arrow S mutant alpha  subunit, even at the maximum concentration of RGS4 (Fig. 2).


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Fig. 2.   GAP activity at increasing concentrations of RGS4. Single-turnover GTP hydrolysis by wild type and mutant alpha  subunits was measured at 1 and 30 min after the addition of MgSO4 and GTPgamma S with increasing concentrations of RGS4. Conditions were the same as in Fig. 1. The amount of GTP hydrolyzed at 1 min was normalized to maximal GTP hydrolysis determined at 30 min. Data represent the mean ± S.E. of three experiments, each done in duplicate.

Reduced GAP Activity Is Due to Reduced Affinity-- To determine whether the marked reduction in sensitivity of mutant alpha  to RGS4 was due to decreased binding, we tested co-precipitation of alpha  and RGS4. In the absence of AlF4-, GDP-bound alpha  subunit did not bind to GST-RGS4 (Fig. 3). In the presence of AlF4-, wild type alpha o and alpha i1 showed substantial binding to GST-RGS4 immobilized on glutathione-agarose beads.3 In contrast, there was no detectable binding of either of the G right-arrow S mutant alpha  subunits. To more quantitatively characterize the interaction of alpha  subunits with RGS4, we used a recently developed flow cytometry approach (26). Binding of FITC-labeled alpha o to GST-RGS4 on glutathione-agarose beads was detected by measuring the amount of fluorescence associated with the beads in a FACScan flow cytometer (see "Experimental Procedures"). Unlabeled wild type and mutant alpha  subunits were added to compete for the binding of FITC-alpha o to RGS4. All binding was done in AlF4--containing buffer with 4 nM FITC-His6alpha o and 2 nM GST-RGS4. Nonspecific binding in the presence of a 50-fold excess of unlabeled alpha o represented less than 20% of the total binding. Similarly, GTPgamma S-bound FITC-alpha o did not show any specific binding (data not shown). Wild type alpha o and alpha i1 reduced the binding of FITC-alpha o, with IC50s of 30 and 96 nM, respectively (and 95% confidence intervals of 18-35 and 60-150 nM; see Fig. 4). As observed for the effects of RGS4 on GTPase activity, we were unable to demonstrate significant interactions of the alpha i1 G right-arrow S mutant protein with RGS4 up to 3 µM alpha  subunit. Because of the limited purity and amounts of alpha o G right-arrow S, the highest concentration used was 300 nM. At this concentration, there was an ~20% decrease in bound FITC-alpha o. Thus the affinity for both G right-arrow S mutant alpha  subunits is at least 30-100-fold lower than that of wild type.


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Fig. 3.   Mutant alpha  does not bind to RGS4. GST-RGS4 fusion protein (1 µM) was bound to glutathione-agarose beads (5×105 beads/ml) and incubated with 1 µM GDP-bound alpha . The reaction was done in 100 µl of HEDML buffer with 0.05% bovine serum albumin in the presence or absence of AlF4-. Top, the total amount of alpha  used in each reaction is shown. Bottom, pellets were prepared as described under "Experimental Procedures." The bound proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining. A representative result from three separate experiments is shown. WT, wild type.


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Fig. 4.   Affinity of alpha  subunits for GST-RGS4. Binding of FITC-alpha o was measured by flow cytometry as described under "Experimental Procedures." Samples contained 4 nM FITC-alpha o, 2 nM GST-RGS4 on glutathione-agarose beads, and the indicated concentrations of unlabeled alpha  subunits. All incubations were done in the in the presence of AlF4- at room temperature for 2.5 h. Data are presented as a fraction of control with nonspecific binding (~20% of total) subtracted. Experiments have been replicated twice in duplicate. The data for wild type (WT) proteins were fit a to one-site competition function.

Generality of G right-arrow S Mutation in Galpha -RGS Interactions-- In this report, we identify a G right-arrow S point mutation in the conformationally flexible "switch I" region of alpha o and alpha i1 that completely eliminates the interaction of these alpha  subunit with RGS proteins. This mutation should be useful in determining the role of endogenous RGS proteins in the physiological functioning of G proteins. This is especially important for Gi, since the kinetics of Gi-mediated regulation of effectors (e.g. adenylyl cyclase (28) and potassium channels (9)) are faster than expected for the in vitro GTP hydrolysis rates in the absence of RGS proteins. Deactivation of potassium currents was recently shown to be accelerated by overexpressed RGS1, 3, or 4 in oocytes and Chinese hamster ovary cells (29, 30). These data show that exogenous RGS proteins can alter G protein function, but the question of whether normal cellular concentrations of RGS proteins alter the kinetics of ion channel function has not been answered experimentally. With the alpha  subunit mutation described here, it is now possible to directly address that question.

This class of G right-arrow S mutations was first described in yeast by DiBello et al. (21). They identified, in a genetic screen for the supersensitive phenotype, a G right-arrow S mutation in the yeast alpha  subunit (Gpa1sst) that resulted in insensitivity to 1) the functional effects of the yeast RGS protein, Sst2, and 2) the biochemical effects of the Galpha -interacting protein. The loss of function due to these G right-arrow S mutations is selective, since our mutant alpha  subunits retain nearly normal intrinsic GTPase activity and kinetics of GDP release. The corresponding mutation in alpha q prevented the RGS7-mediated reduction of phospholipase C activation in co-transfection studies (21). These latter data also demonstrate that the G right-arrow S mutation in alpha q does not disrupt effector coupling. In preliminary data,4 a myristolylated mutant G right-arrow S alpha i1 inhibited forskolin stimulated type IV adenylyl cyclase activity. Thus, mutating this glycine residue has profound and consistent effects on four different alpha  subunits and their interactions with four different RGS proteins.

Structural Basis of Glycine to Serine Effects-- In the crystal structure of the alpha i1·RGS4 complex (19), the switch I region of alpha  interacts with three of the four different segments of the RGS consensus domain, but there are also contacts with switch II and switch III. Glycine 183 is located in the switch I region of alpha i1, forming a turn just before the beta 1 strand (31, 33). Interestingly, Natochin and Artemyev (32) recently found that the mutation of Ser-202 in switch II of transducin prevents interaction with retinal specific RGS. Glycine 183 provides a substantial contribution to the buried surface area between alpha i1 and RGS4 (see Ref. 21 for details). There is direct contact of glycine 183 with the Calpha and Cbeta of serine 85 and Calpha of tyrosine 84 of the RGS protein. Introduction of the hydroxymethyl side chain of serine would sterically hinder the formation of a tight complex of alpha  and RGS. Also, threonine 182, which is directly adjacent to glycine 183 mutated in the alpha i1 G right-arrow S mutant, exhibits the greatest change in accessibility of any alpha  residue upon forming the alpha i1·RGS4 complex (19). Thus the G right-arrow S mutation may also disrupt local protein conformation and prevent threonine 182 from interacting with the highly conserved residues in RGS.

The glycine at position 183 in alpha i1 is highly conserved among all alpha  subunits. There are only two exceptions; in alpha 7 of Dictyostelium discoidum and in open reading frame B0207.3 of C. elegans it is replaced by a serine (34, 35). Interestingly, this is the same residue found in the Gpa1sst, suggesting that these proteins may be naturally occurring sst variants that are insensitive to modulation by RGS proteins. The contact site in RGS is similarly conserved. Either serine or cysteine is present at the position equivalent to 85 in RGS4 where the Galpha glycine interacts. Druey and Kehrl (36) recently showed that modification of asparagine 88 and leucine 159 in RGS4 disrupted alpha  subunit binding and GAP activity. In the three-dimensional structure, both residues are very close to serine 85 where glycine 183 in Galpha makes contact (19).

Binding of alpha  Subunits to GST-RGS4-- Both in co-precipitation and fluorescence competition studies, the affinity of the AlF4--bound mutant alpha  subunits for RGS4 are dramatically reduced. With the flow cytometry method, we obtained quantitative measures of subunit affinities. The IC50 values of wild type alpha o and alpha i1 (30 and 96 nM, respectively) are similar to the KD of 45 nM determined by surface plasmon resonance for transducin binding to retinal-specific RGS (37). Our IC50 is significantly higher than the KD estimated from on and off rates by plasmon resonance for RGS4 and alpha i1 (38). The differences between the latter data and ours may be due to methodological differences (kinetic versus equilibrium determination or direct binding versus competition methods). In any case, the 30-100-fold lower affinity of the G right-arrow S mutant Galpha proteins is very clear.

Function of RGS7-- Our data also include the first biochemical demonstration of GAP activity by RGS7. At 100 nM, the effect of the RGS7 RGS domain on GTP hydrolysis by alpha i1 is significantly less than the effect of RGS4. Interestingly, the FITC-alpha o did not show detectable binding to the GST-RGS7 fusion protein (data not shown). These results are probably due to a lower affinity of RGS7 for alpha o compared with RGS4. This may be due in part to the lack of full-length RGS7 in the expression construct.

In summary, our data show a dramatic disruption in both RGS binding and RGS-mediated GAP activity when the glycine in the switch 1 region of alpha i1 or alpha o is mutated to serine. The effect of the G right-arrow S mutation is both specific in that it only disrupts RGS binding and quite general in the range of alpha  subunit and RGS proteins that it effects. The introduction of this mutation into G protein alpha  subunits can be used in conjunction with expression or transgenic animal studies to evaluate the physiological role of endogenous RGS proteins in the function of a given G protein.

    ACKNOWLEDGEMENTS

The authors thank Dr. Albrecht Moritz for providing the GST-RGS7 expression construct and Masakatsu Nanamori for expression and purification of the alpha o protein. The expression vectors pQE/alpha o and the pQE6/His6alpha i1 were generously provided by Dr. M. E. Linder (Washington University).

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM 39561 (to R. R. N.), GM 53645 (to R. T.), and GM 55316 (to H. G. D.).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 Dagger To whom correspondence and reprint requests should be addressed: Dept. of Pharmacology, 1301 MSRB III, 1150 W. Med. Ctr. Dr., Ann Arbor, MI 48109-0632. Tel.: 313-763-3650; Fax: 313-763-4450; E-mail: RNeubig{at}umich.edu.

1 The abbreviations used are: RGS, regulator of G protein signaling; FITC, fluorescein 5-isothiocyanate; GAP, GTPase activating protein; GST, glutathione S-transferase; GTPgamma S, guanosine 5'-O-(3- thiotriphosphate).

2 A. Moritz and R. Taussig, unpublished information.

3 The apparent excess of bound Galpha over GST-RGS4 protein was due to incomplete elution of the GST-RGS4 from the beads in this experiment.

4 M. Nanamori, K-L.. Lan, and R. R. Neubig, unpublished information.

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Abstract
Introduction
Procedures
Results & Discussion
References

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