Mutagenesis of the Conserved Residue Glu259 of Gsalpha Demonstrates the Importance of Interactions between Switches 2 and 3 for Activation*

Dennis R. WarnerDagger §, Rianna RomanowskiDagger , Shuhua Yu, and Lee S. Weinstein

From the Dagger  Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, NINDS and the  Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Top
Abstract
Introduction
References

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha -subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha -E259A), Gln (Gsalpha -E259Q), Asp (Gsalpha -E259D), or Val (Gsalpha -E259V), and the properties of in vitro translation products were examined. The Gsalpha -E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha -E259D stimulated adenylyl cyclase normally in the presence of GTPgamma S but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgamma S was a more effective activator than AlF4- for all mutants, with Gsalpha -E259D being the least severely impaired. For Gsalpha -E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha -E259D and Gsalpha -E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgamma S binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgamma S binding was impaired, suggesting that they bound AlF4- more poorly. GTPgamma S bound to purified Gsalpha -E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha -E259V bind to beta gamma at 0 °C and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.

    INTRODUCTION
Top
Abstract
Introduction
References

Heterotrimeric guanine nucleotide-binding proteins (G proteins)1 couple heptahelical receptors to intracellular effectors and are composed of three subunits (alpha , beta , and gamma ) (reviewed in Refs. 1-3). The alpha -subunits, which are distinct for each G protein, bind guanine nucleotide and modulate the activity of specific downstream effectors. For Gs, these include the stimulation of adenylyl cyclase and modulation of ion channels (4, 5). In the inactive state, GDP-bound alpha -subunit is associated with a beta gamma -dimer. Upon receptor activation, the alpha -subunit undergoes a conformational change resulting in the exchange of GTP for GDP and dissociation from beta gamma . While GTP is bound, the alpha -subunit interacts with and regulates specific effectors. An intrinsic GTPase activity within the alpha -subunit hydrolyzes bound GTP to GDP, returning the G protein to the inactive state. Analogs of GTP, such as GTPgamma S and GDP-AlF4-, lock the G protein in the active state.

X-ray crystal structures reveal that G protein alpha -subunits have two domains, a ras-like GTPase domain, which includes the regions for guanine nucleotide binding and effector interaction, and a helical domain, which may prevent release of GDP in the inactive state (6-12). Comparison of the crystal structures of inactive (GDP-bound) and activated (GTPgamma S- or AlF4--bound) alpha -subunits demonstrates three regions (named switches 1, 2, and 3), the conformation of which changes upon switching from the inactive to active state. The movement of switches 1 and 2 is directly related to the presence of the gamma -phosphate group, whereas switch 3 has no direct contact with bound guanine nucleotide. Upon activation, switches 2 and 3 move toward each other, and the two regions form multiple interactions that presumably stabilize the active state (7, 10). Switch 3 residues also make contacts with the helical domain that are important for high affinity guanine nucleotide binding (10, 15). At least for transducin, this region may also be important in effector activation (13).

We have previously shown that substitutions of the switch 3 residue Arg258 impairs activation by receptor or AlF4- (15).2 The latter effect was the direct result of decreased GDP binding due to loss of contacts between the Arg258 side chain and residues within the helical domain. The adjacent residue (Glu259) is invariant in all known G protein alpha -subunits and is predicted to be important in activation, because it makes interactions with switch 2 residues in the active state (7, 12). Moreover, this residue is mutated to a valine in a patient with Albright hereditary osteodystrophy (16). In the present report, we provide evidence that substitution of Glu259 also leads to impaired activation, particularly by receptor or AlF4-. However, impaired activation of these mutants by AlF4- is not the result of decreased GDP binding (as is the case for the Arg258 mutants) but rather is the result of a decreased ability to bind the AlF4- moiety. The crystal structure of GTPgamma S-bound Gsalpha reveals interactions between the acidic side chain of Glu259 and basic residues within switch 2 that are important in maintaining the active state and in binding of AlF4- (12). Although adjacent switch 3 residues in Gsalpha (Arg258 and Glu259) are both critical for activation, the mechanisms by which mutations of these residues result in defective activation are distinct.

    EXPERIMENTAL PROCEDURES

Construction of Gsalpha Plasmids and in Vitro Transcription/Translation-- To generate Gsalpha Glu259 mutants, polymerase chain reaction was performed as described previously (15) using linearized vector containing wild type Gsalpha cDNA as template. The upstream primer was 5'-GACAAAGTCAACTTCCACATGTTTGACGTGGGTGGCCAGCGCGATGAACG-3', and the downstream mutagenic primers were as follows: 5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCACCCGGATGACCATGTTG-3' for E259V, 5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCGCCCGGATGACCATGTTG-3' for E259A, 5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCTGCCGGATGACCATGTTG-3' for E259Q, and 5'-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCGTCCCGGATGACCATGTTG-3' for E259D. Each polymerase chain reaction product was digested with HincII andSse8387I and ligated into the transcription vector pBluescript II SK (Stratagene, La Jolla, CA) that contained wild type human Gsalpha cDNA (splice variant Gsalpha -1, Ref. 17) in which the same HincII-Sse8387I restriction fragment had been removed. Mutations were verified by DNA sequencing, and synthesis of full-length Gsalpha from each construct was confirmed by immune precipitation of in vitro translated products with RM antibody, directed against the carboxyl-terminal decapeptide of Gsalpha (18). In vitro transcription/translation was performed on Gsalpha plasmids as described previously (15, 19) using the TNT-coupled transcription/translation system from Promega, with the exception that in most experiments, no RNase inhibitor was added.

Adenylyl Cyclase Assays-- Wild type and mutant Gsalpha in vitro transcription/translation products (10 µl of translation medium) were reconstituted into 25 µg of purified S49 cyc plasma membranes and tested for stimulation of adenylyl cyclase in the presence of various agents as indicated in Table I (15, 19, 20). Reactions were incubated for 15 min at 30 °C, and the amount of [32P]cAMP produced was measured as described previously (21).

Trypsin Protection Assays-- Limited trypsin digestion of in vitro translated Gsalpha was performed as described previously (15, 19). Briefly, 1 µl of in vitro translated [35S]methionine-labeled Gsalpha was incubated in incubation buffer (20 mM HEPES, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol) with or without 100 µM GTPgamma S or 10 mM NaF/10 µM AlCl3 at various temperatures for 1 h and then digested with 200 µg/ml tosyl-L-phenylyalanine chloromethyl ketone-trypsin for 5 min at 20 °C. In some experiments, GDP was also included in the preincubation, and in other experiments the MgCl2 concentration was varied. Reactions were terminated by boiling in Laemmli buffer. Digestion products were separated on 10% SDS-polyacrylamide gels, and the amount of 38-kDa protected fragment was measured by phosphorimaging. The percentage of protection is the signal in 38-kDa protected band divided by the signal in the undigested full-length Gsalpha band × 100.

Sucrose Density Gradient Centrifugation-- [35S]Methionine-labeled Gsalpha was synthesized, and rate zonal centrifugation was performed on linear 5-20% sucrose gradients (200 µl) as described previously (19, 22). Gradients were prepared in 20 mM HEPES, pH 8.0, 1 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 0.1% Lubrol-PX. Six-µl fractions were obtained and analyzed by SDS-polyacrylamide gel electrophoresis, and the relative amount of Gsalpha in each fraction was quantified as described previously (19). To assess the ability of Gsalpha to bind to Gbeta gamma , in vitro translation products were preincubated for 1 h at 0 °C in the presence or absence of Gbeta gamma (20 µg/ml) prior to centrifugation. In order to optimize separation between free alpha -subunit and heterotrimer, 0.15% (w/v) CHAPS was substituted for Lubrol-PX in the preincubations and gradients, and the samples were centrifuged at 120,000 rpm (627,000 × g at the maximum radial distance from the center of rotation (Rmax) in a TLA-120.2 rotor (Beckman). Gbeta gamma was isolated from bovine brain (23).

Expression and Purification of Gsalpha from Escherichia coli-- Plasmid pQE60, containing the long form of bovine Gsalpha cDNA with a hexa-histidine extension at the carboxyl terminus, was a generous gift of A. G. Gilman and R. K. Sunahara. The Glu259 residue was mutated by site-directed mutagenesis using the Quickchange kit (Statagene). Each mutated cDNA was sequenced to confirm the presence of the desired mutation and to rule out polymerase chain reaction artifacts. After transformation into E. coli strain JM109, cultures were grown, Gsalpha expression was induced, and Gsalpha proteins were purified as described previously (15, 24), except that [GDP] was only 10 µM in the storage buffer.

Guanine Nucleotide Binding Assays-- Assays measuring the rate of binding of GTPgamma S were performed as described previously (15, 25). Briefly, 1-2 pmol of purified Gsalpha was incubated at 37 °C in a final volume of 2 ml containing 1 µM [35S]GTPgamma S (5,000-10,000 cpm/pmol) in 25 mM HEPES, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 0.01% Lubrol-PX with or without 10 mM NaF/10 µM AlCl3. At various times, 50-µl aliquots were removed and diluted with 2 ml of ice-cold stop solution (25 mM Tris-HCl, 100 mM NaCl, 25 mM MgCl2, and 100 µM GTP) and maintained on ice until all samples were collected. Samples were then filtered under vacuum through nitrocellulose filters (Millipore) and washed twice with 10 ml of stop solution without GTP, and filters were dissolved in 10 ml of scintillation mixture. To determine the effect of Mg2+ on the rate of GTPgamma S dissociation, ~2.5 pmol of purified Gsalpha was loaded with [35S]GTPgamma S at 30 °C for 45 min in the presence of various free Mg2+ concentrations. After addition of 100 µM cold GTPgamma S, bound [35S]GTPgamma S was determined at various time points as described above. koff for GTPgamma S dissociation was determined by fitting the data to the function y = ae-kt + b using the software GraphPad Prism, version 2.01. Free Mg2+ concentrations were calculated as described (26).

    RESULTS

Substitution of Gsalpha Glu259 Leads to Decreased Activation-- Gsalpha Glu259 substitution mutants were cloned into the transcription vector pBluescript, and the in vitro transcription/translation products were compared with those of Gsalpha -WT in various biochemical assays. We substituted Glu259 with valine (Gsalpha -E259V) because a mutation encoding this substitution has been identified in a patient with Albright hereditary osteodystrophy (16), a human disorder associated with heterozygous loss-of-function mutations of Gsalpha (27, 28). Because the presence of an amino acid with a bulky and branched side chain (valine) may introduce nonspecific steric effects, we also generated and analyzed additional mutants in which Glu259 was replaced by alanine (Gsalpha -E259A), glutamine (Gsalpha -E259Q), or aspartate (Gsalpha -E259D). In Gsalpha -E259A, the acidic side chain was removed, whereas in Gsalpha -E259Q it is converted to a residue in which the carboxyl group is replaced by a neutral amide group. In Gsalpha -E259D, the charge of the residue at position Glu259 is maintained, but the length of the side chain is shortened by one methylene group.

After reconstitution of translation products into purified S49 cyc membranes (which lack endogenous Gsalpha ), Gsalpha -E259V had markedly decreased ability to stimulate adenylyl cyclase in the presence of GTPgamma S, AlF4-, or activated receptor (isoproterenol + GTP) (Table I). For Gsalpha -E259A and -E259Q, the ability to stimulate adenylyl cyclase was moderately reduced in the presence of GTPgamma S (~40% of Gsalpha -WT) and more markedly reduced in the presence of AlF4- or activated receptor. Stimulation of adenylyl cyclase by Gsalpha -E259D was normal in the presence of GTPgamma S but moderately reduced in the presence of AlF4- or activated receptor. Although the severity of the defect varied depending on which specific residue replaced Glu259, for each Gsalpha -Glu259 mutant, GTPgamma S was the most effective activator and AlF4- the least effective activator.

                              
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Table I
Adenylyl cyclase stimulation by Gsalpha mutants
In vitro transcription/translation products were mixed with purified cyc- membranes and assayed for adenylyl cyclase stimulation as described under "Experimental Procedures." Results are expressed as the mean ± S.D. (sigma n - 1) of triplicate determinations and are corrected for the relative level of synthesis of each mutant to Gsalpha -WT. Gsalpha -E259V, -E259A, -E259Q, and -E259D were synthesized to 73, 78, 74, and 91% of Gsalpha -WT levels, respectively, as determined by in vitro translation with [35S]methionine, SDS-PAGE, and phosphorimaging. Background values determined from mock transcription/translation reactions (in pmol of cAMP/ml of translation medium/15 min: GTP, 29 ± 1; isoproterenol, 39 ± 5; GTPgamma S, 39 ± 2; and AlF4-, 64 ± 6) were subtracted from each determination.

We next examined the ability of AlF4- or GTPgamma S to protect each mutant from trypsin digestion, which measures the ability of each agent to bind to Gsalpha and induce the active conformation (29). In the inactive, GDP-bound state, two arginine residues within switch 2 (most likely Arg228 and Arg231, based upon sequence homology with transducin) are sensitive to trypsin digestion, leading to the generation of low molecular weight fragments. When Gsalpha attains the active conformation, these residues are inaccessible to trypsin digestion (7) and therefore trypsinization of activated Gsalpha generates a partially protected 38-kDa product. Gsalpha -WT was well protected by AlF4- or GTPgamma S at temperatures up to 37 °C (Fig. 1, Table II). At 30 °C, Gsalpha -E259V, -E259A, and -E259Q showed little protection by GTPgamma S and no protection by AlF4- (Fig. 1). In contrast, both GTPgamma S and AlF4- were able to protect Gsalpha -E259D, with GTPgamma S being a more efficient activator than AlF4- (Fig. 1, Table II). Consistent with the results of the cyc reconstitution assays, AlF4- was less effective than GTPgamma S in protecting all Gsalpha -E259 mutants from trypsin digestion.


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Fig. 1.   Trypsin protection of in vitro translated Gsalpha -E259 mutants in the presence of GTPgamma S or AlF4-. In vitro translates were digested with tosyl-L-phenylyalanine chloromethyl ketone-trypsin (200 µg/ml) for 5 min at 20 °C after 1-h preincubations at 30 °C in the presence of GTPgamma S or AlF4-. For each Gsalpha , the full-length undigested Gsalpha (52 kDa) is shown in the far left lane (no trypsin), and complete digestion in the absence of activators is demonstrated in the second lane (no adds.). The two right lanes show the amount of the 38-kDa protected band generated after trypsin digestion in the presense of either GTPgamma S (100 µM) or AlF4-. The smaller products in the left lane are due to initiation of protein synthesis at downstream methionine codons. Quantitation of trypsin protection assays for Gsalpha -E259D is presented in Table II.

                              
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Table II
Effect of temperature and GDP on AlF4--induced trypsin protection
These data were obtained from experiments of the type presented in Fig. 1. The amount of the 38-kDa trypsin-stable Gsalpha fragment was determined by phosphorimaging, and for Gsalpha -WT, it is expressed as a percent of undigested Gsalpha (mean ± S.E.). No protection was observed when AlF4- and GTPgamma S were excluded. Maximum trypsin protection has a theoretical limit of 71%, based on the removal of 2 of 7 total methionine residues by trypsin. For Gsalpha -E259D, the data are expressed as percentage of wild type at each condition (mean ± S.E.). The number of experiments performed for each condition is shown in the right column.

Because the Gsalpha -E259D encoded the most subtle structural change and had the smallest activation defect, we studied the ability of this mutant to be protected by GTPgamma S and AlF4- at various temperatures and in the presence or absence of excess GDP (Table II). For Gsalpha -R258 mutants, the activation defect in the presence of AlF4- was more severe at higher temperatures and was reversible in the presence of excess GDP (15). Although raising the temperature had little effect on the ability of GTPgamma S to protect Gsalpha -E259D from trypsin protection, temperature had a profound effect on protection by AlF4-, being 85, 49, and 7% of Gsalpha -WT at 25, 30, and 37 °C, respectively. At 37 °C, addition of 2 mM GDP was able to somewhat reverse the defect in activation by AlF4-, although not to the extent that it was able to reverse the defect in the Gsalpha -R258 mutants (15). Interestingly, addition of GDP lowered the ability of AlF4- to protect Gsalpha -E259D at 25 and 30 °C (Table II). Although this effect was consistently observed, we have no good explanation for this observation.

Substitution of Gsalpha Glu259 Has Little Effect on the Rate of GDP Release in the Basal State-- The impaired activation of Gsalpha -Glu259 mutants by AlF4- could result from decreased affinity for AlF4-, decreased ability for the GDP-AlF4- complex to activate the mutant Gsalpha s, or decreased ability of the mutant Gsalpha s to maintain the GDP-bound state because GDP binding is a prerequisite for AlF4- binding and activation. For the Gsalpha -Arg258 mutants, impaired activation by AlF4- is primarily the result of impaired GDP binding (15). The inability of GDP to significantly reverse the AlF4--induced activation defect in Gsalpha -E259D suggests that this defect is not due to defective GDP binding.

To directly evaluate the rate of GDP release in the basal state, we expressed and purified bovine Gsalpha -WT, -E259A, and -E259D, each with a carboxyl-terminal hexahistidine tag, from E. coli and examined the time course of GTPgamma S binding. The rate of GTPgamma S binding has been shown to be limited by the rate of GDP dissociation, and the experimentally determined values of these two rates are essentially identical (30, 31). This assay has also been previously used as a measure of the GDP dissociation rate in other Gsalpha mutants(15, 32). Substitution of Glu259 had little effect on the rate of GDP release in the basal state, as the time course of GTPgamma S binding at 37 °C (in the absence of AlF4-) is essentially identical for Gsalpha -WT and -E259D, whereas the rate of GTPgamma S binding for Gsalpha -E259A is only increased minimally (Fig. 2). Consistent with these results, the rate of increase of trypsin protection of Gsalpha -WT and -E259D in vitro translation products in the presence of GTPgamma S was also identical (data not shown). These results demonstrate that unlike substitutions of Arg258, the rate of GDP release is not significantly altered by substitution of Glu259, and therefore the impaired activation by AlF4- is not primarily due to decreased GDP binding.


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Fig. 2.   Time course of GTPgamma S binding to purified Gsalpha s in the presence or absence of AlF4-. Bovine Gsalpha -WT, -E259A, and -E259D, each with a carboxyl-terminal hexahistidine extension, were expressed and purified from E. coli, and the time course of GTPgamma S binding for each was determined either in the presence (filled symbols) or absence (open symbols) of AlF4-. Gsalpha -WT (black-square and ), Gsalpha -E259A (bullet  and open circle ), and Gsalpha -E259D (black-triangle and triangle ) were incubated with 1 µM [35S]GTPgamma S (~10,000 cpm/pmol) at 37 °C for varying times, and the amount of bound GTPgamma S was determined as described under "Experimental Procedures." For each Gsalpha , each data point (with or without AlF4-) was normalized to maximal binding at 10 min in the absence of AlF4-. Each data point is the mean ± S.D. of triplicate determinations. This experiment was representative of three experiments. The Bmax values in the absence of AlF4- were as follows: Gsalpha -WT, 3 pmol; Gsalpha -E259A, 2 pmol; and Gsalpha -E259D, 1 pmol.

Substitution of Gsalpha Glu259 Decreases AlF4- Binding-- We next examined the ability of AlF4- to interact with mutant Gsalpha s in the GDP-bound state to determine whether the decreased activation of Gsalpha -E259 mutants by AlF4- is due to impaired AlF4- binding. It has been shown previously that the rate and extent of GTPgamma S binding to G alpha -subunits is markedly reduced in the presence of AlF4-, presumably because the GDP-AlF4- complex bound to Galpha is more stable than GDP alone (8). Because Gsalpha -WT, -E259D, and -E259A have similar rates of GTPgamma S binding in the absence of AlF4-, the time course of GTPgamma S binding in the presence of AlF4- should reflect the ability of each form of Gsalpha to interact with AlF4-. Similar to previously reported observations (8), the rate and extent of GTPgamma S binding to Gsalpha -WT was markedly reduced in the presence of AlF4- (Fig. 2). In contrast, AlF4- only partially reduced the rate and extent of GTPgamma S binding to Gsalpha -E259D and had a minimal effect on the GTPgamma S binding curve for Gsalpha -E259A (Fig. 2). These results are consistent with the results of adenylyl cyclase and trypsin protection assays, which demonstrate that AlF4--induced activation is severely impaired in Gsalpha -E259A but only partially impaired in Gsalpha -E259D and suggest that the decreased ability of AlF4- to activate Gsalpha -E259 mutants is primarily due to decreased ability of the mutants to maintain AlF4- in the guanine nucleotide binding pocket.

Effect of Mg2+ Concentration on Activation by AlF4- and GTPgamma S Binding-- Substitution of Gsalpha Arg231, a residue in switch 2 that interacts with switch 3 residues in the active state, leads to a defect in activation by AlF4- that is more pronounced at low Mg2+ concentrations (33). We therefore examined the effect of varying Mg2+ concentration on the ability of AlF4- to protect Gsalpha -E259D from trypsin digestion. In the trypsin protection experiments shown in Fig. 1 and Table II, the MgCl2 concentration was 10 mM (~9 mM free Mg2+). Lowering the MgCl2 concentration to 2 mM (~1 mM free Mg2+) had no effect on the ability of AlF4- to protect Gsalpha -WT at 30 °C (Fig. 3). In contrast, lowering the MgCl2 concentration below 8 mM (~7 mM free Mg2+) further impaired the ability of AlF4- to protect Gsalpha -E259D in a concentration-dependent manner. Increasing the MgCl2 concentration up to 100 mM did not reverse the defect at 37 °C (data not shown). These results are similar to those observed for the Gsalpha -R231 mutant (33) and demonstrate that, like this mutant, the GDP-AlF4--bound form of Gsalpha -E259D has a lower apparent affinity for Mg2+ than Gsalpha -WT.


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Fig. 3.   Effect of MgCl2 concentration on trypsin protection of Gsalpha -WT and -E259D in the presence of AlF4-. Trypsin protection experiments were performed on Gsalpha -WT and -E259D after incubation for 1 h at 30 °C in the presence of AlF4- and various concentrations of MgCl2 ranging from 0 to 50 mM. The top two panels show the gels from a representative experiment. The bottom panel shows the percentage of protection for Gsalpha -WT and -E259D as a function of MgCl2 concentration. Each data point represents the mean ± S.D. of three experiments.

We next examined the effect of lowering the Mg2+ concentration on the dissociation of GTPgamma S from Gsalpha -E259D to determine whether or not the Mg2+ dependence was specific for the GDP-AlF4--bound form. The apparent Kd of GTPgamma S-Gsalpha -WT for Mg2+ is very low (5-10 nM), and binding of GTPgamma S is essentially irreversible in the presence of micromolar concentrations of Mg2+ (34). Consistent with previously published results (34), no dissociation of GTPgamma S from Gsalpha -WT was observed at free Mg2+ concentrations of 30 µM or higher (Fig. 4 and data not shown), although GTPgamma S dissociated rapidly (koff = 2.5 min-1) in the absence of Mg2+ (5 mM EDTA). For Gsalpha -E259D, GTPgamma S binding was essentially irreversible in the presence of 1 mM free Mg2+, but in contrast to Gsalpha -WT, GTPgamma S clearly dissociated from Gsalpha -E259D in the presence of 30 µM free Mg2+ (Fig. 4, koff = 0.05 min-1). Dissociation of GTPgamma S from Gsalpha -E259D (koff = 3.7 min-1) was similar to that of Gsalpha -WT in the absence of Mg2+ (5 mM EDTA). Therefore, like GDP-AlF4--Gsalpha -E259D, GTPgamma S-Gsalpha -E259D appears to have decreased affinity for Mg2+, although the defects are apparent in the millimolar range for the former and micromolar range for the latter.


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Fig. 4.   Effect of free Mg2+ concentration on dissociation of GTPgamma S from purified Gsalpha s. Bovine Gsalpha -WT and -E259D, each with a carboxyl-terminal hexahistidine extension, were expressed and purified from E. coli, and the time course of GTPgamma S dissociation was determined for each at various free Mg2+ concentrations. Gsalpha -WT (closed symbols) and Gsalpha -E259D (open symbols) were preloaded with [35S]GTPgamma S (5,000-10,000 cpm/pmol) at 30 °C for 45 min in the presence of 1 mM (open circle ), 30 µM (black-square and ), or no (black-triangle and triangle ) free Mg2+ (5 mM EDTA was added for the no Mg2+ condition). After addition of 100 µM cold GTPgamma S, the amount of bound [35S]GTPgamma S was determined at various time points. Each data point is the mean ± range of duplicate determinations. This experiment was representative of three experiments. Maximum [35S]GTPgamma S in the presence of 5 mM EDTA was 0.5 pmol for Gsalpha -WT and 0.3 pmol for Gsalpha -E259D, and it was ~2.5 pmol for both in the presence of Mg2+.

In contrast to Gsalpha -E259D, there is a slow rate of dissociation of GTPgamma S from Gsalpha -R231H in the presence of high Mg2+ concentrations (33). Another Gsalpha mutant (Gsalpha -G226A) also displays an abnormally high apparent Kd for Mg2+ to prevent GTPgamma S dissociation (34). Similar to Gsalpha -E259D, GTPgamma S dissociates from Gsalpha -G226A in the presence of micromolar concentrations of Mg2+. There is also considerable dissociation of GTPgamma S from Gsalpha -G226A even in the presence of maximal (millimolar) concentrations of Mg2+, because this mutant cannot attain the active conformation that stabilizes the Mg2+-GTPgamma S complex. The ability of Gsalpha -E259D to irreversibly bind GTPgamma S in the presence of 1 mM Mg2+ suggests that this mutant can attain the active conformation necessary to stabilize Mg2+-GTPgamma S, consistent with the results obtained in the adenylyl cyclase and trypsin protection assays (Table I and Fig. 1).

Gsalpha -E259Q, E259A, and E259D, but not Gsalpha -E259V, Maintain Normal Overall Conformation and Gbeta gamma Interaction-- We examined the ability of each Gsalpha -E259 mutant to interact with beta gamma by subjecting in vitro translates to sucrose density gradient centrifugation in the presence or absence of purified bovine brain beta gamma . We previously showed that Gsalpha has a sedimentation coefficient of ~3.7 S (15, 19). When in vitro translates of each Gsalpha -E259 mutant was held on ice, the gradient profiles of all mutants were virtually the same as Gsalpha -WT and consistent with the overall proper conformation (sedimentation coefficient, ~3.7 S) (Fig. 5A). When preincubated on ice with purified bovine brain beta gamma , Gsalpha -WT, -E259Q, -E259A, and -E259D formed heterotrimers, as demonstrated by significant shifting of the peak toward the bottom of the gradient (Fig. 5B). In contrast, beta gamma had no effect on the sedimentation profile of Gsalpha -E259V, indicating that this mutant does not interact with beta gamma . After preincubation at 30 °C, gradient profiles demonstrate that all mutants except Gsalpha -E259V maintain an the normal 3.7 S conformation, whereas for Gsalpha -E259V, the majority of the protein is a higher S value material and is presumably denatured (19). Therefore, the valine substitution probably alters the overall conformation and stability of the protein due to nonspecific steric effects of its bulky hydrophobic side chain. In contrast, the activation defect in Gsalpha -E259A, E259Q, and E259D is not secondary to defects in thermostability or beta gamma binding.


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Fig. 5.   Sucrose density gradient centrifugation of Gsalpha in vitro translation products. A, [35S]methionine-labeled in vitro translates of both Gsalpha -WT and -E259 mutants were preincubated for 1 h at 0 °C (open circle ) or 30 °C (bullet ) and subjected to sucrose density gradient centrifugation as described under "Experimental Procedures." Fractions (6 µl each) were collected, and odd-numbered fractions were analyzed by SDS-polyacrylamide gel electrophoresis and phosphorimaging (15, 19). The data are expressed as the percentage of total Gsalpha present in each fraction. Fraction 1 represents the top of the gradient. The position and S value of standard proteins are indicated at the top of the Gsalpha -WT gradients. B, sucrose density gradient profiles of Gsalpha -WT (bullet ), Gsalpha -E259A (black-square),Gsalpha -E259Q (black-triangle),Gsalpha -E259D (), and Gsalpha -E259V (triangle ) after preincubation for 1 h at 0 °C in the presence of purified bovine brain Gbeta gamma (20 µg/ml). The profile for Gsalpha -WT in the absence of Gbeta gamma is also shown (open circle ). All Gsalpha -E259 (except Gsalpha -E259V) mutants held at 0 °C in the absence of Gbeta gamma had sucrose density gradient profiles similar to that of Gsalpha -WT (data not shown). Gsalpha -E259V had a somewhat broader peak at 0 °C that was unaltered in the presence of Gbeta gamma . Conditions were modified to optimize separation between free alpha -subunit and heterotrimer as outlined under "Experimental Procedures." Similar results were obtained with the detergent octyl-beta -glucoside (0.3% w/v).


    DISCUSSION

We previously reported that substitution of the Gsalpha switch 3 residue Arg258 leads to impaired activation in the presence of AlF4- or activated receptor (isoproterenol + GTP) but normal activation in the presence of GTPgamma S (15). The impaired activation by AlF4- was reversible in the presence of excess GDP, and further characterization demonstrated a defect in GDP binding, presumably due to loss of direct contact between Arg258 and a residue(s) in the helical domain that would open the cleft through which guanine nucleotide must exit. In this study, we examined the effect of substituting the adjacent switch 3 residue (Glu259) on Gsalpha function for the following reasons: 1) this residue is strictly conserved among G protein alpha -subunits and therefore might have an important role in the biochemical function of these proteins; 2) upon activation, the Glu259 side chain interacts with several residues in the switch 2 region (7, 12) and therefore substitutions of this residue might be predicted to directly impair G protein activation; 3) this Gsalpha residue is mutated to a valine in a patient with Albright hereditary osteodystrophy (16), a human disorder associated with heterozygous inactivating mutations within the Gsalpha gene (27, 28).

Substitution of Gsalpha Glu259 to valine had a marked effect on the conformation and stability of the protein. This mutant was unable to interact with beta gamma , even though Glu259 is not within the beta gamma interaction site (11). This mutant was also more thermolabile. Presumably, the presence of a bulky and branched side chain provided by valine introduces nonspecific steric effects that severely affect the conformation and stability of the protein. We would predict that the primary biochemical defect in the patient harboring this mutation is lack of expression of Gsalpha -E259V in the membrane at physiological temperatures, similar to what is observed in other patients with mutants encoding unstable forms of Gsalpha protein (15, 19, 32).

In order to determine whether residue Glu259 is critical in maintaining either the basal or activated state, we generated mutants with more subtle alterations of Glu259 side chain. The most subtle mutation was Gsalpha -E259D, in which the charge of the residue is maintained but the length of the side chain is shortened by one methylene group. We also made two mutants in which the side chain was either removed (Gsalpha -E259A) or converted from an acidic to neutral amino acid (Gsalpha -E259Q). In all three of these mutants, the overall conformation and stability, as well as the ability to interact with beta gamma , was maintained, as determined by sucrose density gradient experiments. Based upon adenylyl cyclase and trypsin protection assays, activation of Gsalpha -E259D by GTPgamma S was normal, demonstrating that this mutant has not lost its intrinsic ability to attain the active conformation and activate adenylyl cyclase. However, this mutant had decreased ability of to be activated by AlF4- or receptor. Gsalpha -E259Q and -E259A showed a more severe phenotype, with decreased activation in the presence of all agents. In all three mutants, GTPgamma S was the most efficient activator whereas AlF4- was the least efficient. Mutation of the analogous residue in transducin (Glu232) to leucine had no effect on the ability of the G protein to interact with beta gamma or its receptor (rhodopsin), but it did appear to decrease the ability of GTPgamma S to mediate trypsin protection and effector activation (13).

One possible mechanism for impaired activation by AlF4- is decreased ability to maintain the GDP-bound state, because binding of GDP is a prerequisite for AlF4- binding and activation. This is the primary mechanism by which substitutions of Gsalpha Arg258 lead to impaired activation by AlF4- (15). However, the ability of Gsalpha -E259 mutants to maintain the GDP-bound state was similar to that of Gsalpha -WT, as demonstrated by both Gsalpha -E259A and -E259D having a rate of GDP release that was similar to Gsalpha -WT, as well as an inability for excess GDP to significantly reverse the AlF4--induced activation defect. Consistent with normal guanine nucleotide binding, both Gsalpha -E259A and -E259D were thermostable. Binding of AlF4- to the GDP-bound alpha -subunit results in formation of a stable and activated GDP-AlF4--protein complex that mimics the transition state of the GTPase reaction and will slow the rate of GTPgamma S binding, probably by inhibiting GDP release (8). The ability of AlF4- to inhibit the rate and extent of GTPgamma S binding to both Gsalpha -E259A and -E259D was significantly reduced, suggesting that in these mutants the activation defect in response to AlF4- is due at least in part to impaired AlF4- binding. The fact that the activation defect is greater for AlF4- than GTPgamma S suggests that mutation of Glu259 has a more dramatic effect on stabilizing the transition (AlF4--bound) state than the activated (GTPgamma S-bound) state.

It is of interest that the biochemical phenotype of our Gsalpha -Glu259 mutants is quite similar to that previously described for another Gsalpha mutant present in a patient with Albright hereditary osteodystrophy, in which the switch 2 residue Arg231 is mutated to histidine (Gsalpha -R231H) (33, 35). Similar to the Gsalpha -Glu259 mutants, this mutation leads to normal GTPgamma S-mediated but decreased AlF4-- and receptor-mediated activation. Moreover, similar to the Gsalpha -R231H mutant, the AlF4--induced activation defect in Gsalpha -E259D was more pronounced at low Mg2+ concentrations (33). This is not surprising, based upon mutual interactions between Glu259 and Arg231 present in the active (GTPgamma S-bound) conformation of Gsalpha (Fig. 6). Upon activation, interactions between switches 2 and 3 stabilize the GTP-bound form of the G protein. Specifically, Arg231 in switch 2 interacts with Glu259 in switch 3 through a water molecule and directly with Glu268 in the alpha 3 helix (Fig. 6). Conversely, Glu259 interacts with two basic switch 2 residues, Arg228 and Arg231. Both Arg231 and Glu259 interact with Gly226, a residue that is critical for both AlF4- binding (36) and conformational switching of switch 2 upon binding of GTP or AlF4- (29). Therefore, the impaired activation and AlF4- binding observed in Gsalpha -Glu259 mutants might be the direct result of loss of contacts with Gly226. Loss of these contacts may also result in the apparent decreased affinity of Gsalpha -Glu259 and R231H mutants for Mg2+ because mutation of Gly226 to alanine also lowers the apparent affinity of Gsalpha for Mg2+ (34).


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Fig. 6.   Crystal structure of Gsalpha -GTPgamma S. Detailed view of interactions between Glu259 in switch 3, Glu268 in alpha 3, and residues in switch 2 and between Gly226 and the gamma  phosphate of GTPgamma S. Hydrogen bonds are shown as dotted lines. The atom coloring scheme is as follows: black, carbon; red, oxygen; blue, nitrogen; and yellow, sulfur. Mg2+ and water molecules are shown as magenta and cyan spheres, respectively. The figure was generated with MOLSCRIPT (39) and rendered with RASTER3D (37) using coordinates for the short form of bovine Gsalpha -GTPgamma S (Protein Data Bank accession code 1AZT (12)), although the numbering on the figure corresponds to the long form of Gsalpha (17). This view is similar to that previously shown for transducin (6).

Mutation of Glu259 leads to a subtle defect in receptor-mediated activation (at least when compared with activation by GTPgamma S). Gsalpha -E259 mutants are able to bind beta gamma , and mutation of the analogous residue in transducin (Glu232) has no effect on interactions with beta gamma or receptor (13). It has been proposed that decreased receptor activation of Gsalpha -R231H is due to a conditional defect in GTP binding, which is more pronounced in states in which guanine nucleotide binding is destabilized (such as interaction with activated receptor (33)). Our results are consistent with those observed with Gsalpha -R231H and support this hypothesis.

In conclusion, this study provides further evidence for the role of switch 3 in the activation mechanism and demonstrates the importance of interactions between Glu259 and switch 2 residues. Taken together with the prior studies on Arg258 mutants (15, 38), the present results demonstrate the importance of switch 3 in maintaining both the basal and active states.

    ACKNOWLEDGEMENTS

We thank J. Nagle for performing DNA sequencing analysis, A. G. Gilman and R. K. Sunahara for providing plasmid pQE60-alpha s-H6 and helpful technical advice on the expression and purification of Gsalpha from E. coli, S. Sprang for providing the coordinates for the crystal structure of Gsalpha , V. Rao and J. Hurley for assistance with Fig. 6, and P. Fishman for helpful advice.

    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: Bldg. 49, Rm. 2A28, National Institutes of Health, Bethesda, MD 20892-4440. Tel.: 301-496-2007; Fax: 301-496-8244; E-mail: dwarner{at}helix.nih.gov.

    ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide-binding protein; Gs, stimulatory G protein; Gsalpha , Gs alpha -subunit; Gsalpha -E259D, -E259A, -E259Q, and -E259V, Gsalpha mutant with Glu259 substituted to aspartate, alanine, glutamine, and valine, respectively; AlF4-, mixture of 10 µM AlCl3 and 10 mM NaF; GTPgamma S, guanosine-5'-O-(3-thiotriphosphate); WT, wild type.

2 All numbering is based on the Gsalpha -1 sequence reported by Kozasa et al. (17).

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