Mutations at the Domain Interface of Gsalpha Impair Receptor-mediated Activation by Altering Receptor and Guanine Nucleotide Binding*

Galina Grishina and Catherine H. BerlotDagger

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

G protein alpha  subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous alpha i2 residues for four alpha s residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of alpha s to be activated by the beta -adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of alpha s for the beta -adrenergic receptor, based on an increased amount of high affinity binding of the beta -adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the beta -adrenergic receptor to increase the apparent affinity of alpha s for the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous alpha i2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heterotrimeric G proteins transmit signals from cell surface receptors to effector proteins that modulate a wide variety of cellular processes (1, 2). The alpha  and beta gamma subunits of G proteins are associated in the inactive GDP-bound form. Receptors activate G proteins by catalyzing replacement of GDP by GTP on the alpha  subunit. Receptor-catalyzed nucleotide exchange is thought to involve an "opening" of the guanine nucleotide binding pocket that facilitates GDP release and increases the relative affinity for GTP compared with GDP (3, 4). The transient empty state of the G protein has a high affinity for the hormone-receptor complex. However, this state is short-lived due to the high intracellular concentration of GTP. Binding of GTP leads to dissociation of the receptor from alpha ·GTP and beta gamma , each of which can transmit signals to effectors. Hydrolysis of GTP by the alpha  subunit regulates the timing of deactivation and reassociation of alpha  with beta gamma .

alpha subunit structures consist of two domains, a GTPase domain that resembles the oncogene protein p21ras and a helical domain consisting of alpha  helices and connecting loops. The bound GDP is buried between the two alpha  subunit domains, suggesting that the helical domain may present a barrier to GDP release. Three regions in the GTPase domain (switches I-III) assume different conformations in the structures of GTPgamma S1-bound versus GDP-bound alpha  subunits (5-8). Switches I and II correspond to conformational switch regions in the structures of both p21ras and EF-Tu. Like the helical domain, switch III, which is located at the interface of the two domains, is unique to the structures of heterotrimeric G protein alpha  subunits. The conformational switch regions are important for the interaction of alpha  subunits with effectors (9), beta gamma (10, 11), and RGS (regulators of G protein signaling) regulators of G proteins (12). Most likely, they play a role in receptor-mediated activation as well.

We previously identified a cluster of four switch III residues2 in alpha s at the interface between the GTPase and helical domains in which substitutions with alpha i2 homologs in the mutant construct N254D/M255L/I257L/R258Aalpha s decreased receptor-mediated activation of adenylyl cyclase in transiently transfected cells (13). The activation defect caused by substituting alpha i2 residues for these alpha s residues was corrected by replacing the helical domain of alpha s with that of alpha i2 in a chimera, alpha sis, in which alpha i2 homologs were substituted for alpha s residues 62-235, extending from the end of the alpha 1 helix to the end of the alpha 2 helix (13). Thus, matching alpha i2 residues on both sides of the domain interface of alpha s restored receptor-initiated activation.

We now report a detailed analysis of the activation defects caused by these switch III substitutions and of a mutation that replaces a nearby helical domain residue, Asn167, with the homologous alpha i2 residue, arginine. Measurements in stably transfected cells of isoproterenol binding to the beta -adrenergic receptor, and the time course and dose-dependence of adenylyl cyclase stimulation by the hydrolysis-resistant GTP analog, GTPgamma S, in the presence and absence of isoproterenol indicate that the mutations increase the affinity of alpha s for the beta -adrenergic receptor, decrease the rate of receptor-mediated activation, and block receptor-stimulated increases in GTPgamma S affinity. Additional mutational analysis refines the nature of the interdomain interactions that play a role in receptor-mediated activation by demonstrating that of the switch III substitutions, R258A alone causes a defect in receptor-mediated activation, that this defect is corrected when the helical domain of alpha s is replaced with that of alpha i2, and that the defect caused by the N167R substitution is corrected when combined with the N254D substitution but not the R258A substitution. These results suggest that interdomain interactions are involved in the transmission of signals between the receptor and the guanine nucleotide binding pocket.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of alpha  Subunit Mutants-- alpha s mutant constructs were generated from rat alpha s cDNA (14). Chimeric alpha  subunits were constructed from rat alpha s cDNA and mouse alpha i2 cDNA (15). Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing. All alpha  subunit constructs produced in this study contain an epitope, referred to as the EE epitope (16), which was generated by mutating alpha s residues DYVPSD (189-194) to EYMPTE and alpha i2 residues SDYIPTQ (166-172) to EEYMPTE (single letter amino acid code; mutated residues are underlined). This epitope does not affect the ability of alpha s to activate adenylyl cyclase in response to stimulation by the beta -adrenergic receptor (17).

The alpha s cDNA was subcloned into the expression vector, pcDNA I/Amp (Invitrogen) as a HindIII fragment. N167Ralpha s, N167Aalpha s, N254Dalpha s, M255Lalpha s, I257Lalpha s, R258Aalpha s, and N254D/M255L/I257L/R258Aalpha s were produced by oligonucleotide-directed in vitro mutagenesis (18) using the Bio-Rad Muta-Gene kit. N167R/N254Dalpha s and N167R/R258Aalpha s were produced by ligating Alwn I fragments containing either the N254D or the R258A mutations into N167Ralpha s in place of the analogous fragment to produce an alpha s cDNA containing both mutations. Construction of the alpha sis chimera, in which alpha s residues 62-235 are replaced by the homologous alpha i2 residues, has been described elsewhere (13). R258Aalpha sis was produced by ligating a BamHI fragment containing the R258A mutation into alpha sis in place of the analogous fragment.

Receptor-independent cAMP accumulation in transiently transfected cells was measured after introducing a second mutation (RC) that substitutes cysteine for the arginine at position 201 and causes constitutive activation by decreasing GTPase activity (19). alpha sRC versions of N254Dalpha s, M255Lalpha s, I257Lalpha s, and R258Aalpha s were produced by ligating BamHI fragments containing the mutations into alpha sRC in place of the analogous fragment. alpha sRC versions of N167Ralpha s and N167Aalpha s were produced by ligating Alwn I fragments containing the RC mutation into N167Ralpha s and N167Aalpha s, respectively, in place of the analogous fragment. alpha sRC versions of N167R/N254Dalpha s and N167R/R258Aalpha s were produced by ligating Alwn I fragments containing the RC mutation and either the N254D or the R258A mutation into N167Ralpha s in place of the analogous fragment. The alpha sisRC version of R258Aalpha sis was produced by ligating a BamHI fragment containing the R258A mutation into alpha sisRC in place of the analogous fragment.

Preparation of Stable Cell Lines-- alpha s constructs were subcloned as HindIII fragments into the retroviral vector pMV7 (20) and then stably expressed as described (21), in a subclone of cyc- S49 lymphoma cells, cyc-kin- (22), in which cAMP-dependent protein kinase is inactivated. Single colonies containing the pMV7 vector were obtained using limiting dilution in microtiter wells and selection in G418 (1 mg/ml). Clones expressing alpha s constructs were identified by immunoblotting with the anti-EE monoclonal antibody. Cell membranes were prepared after nitrogen cavitation as described (23).

Immunoblots-- 25 µg of membrane proteins were resolved by SDS-polyacrylamide electrophoresis (10%), transferred to nitrocellulose, and probed with a monoclonal antibody to the EE epitope (16). The antigen-antibody complexes were detected using an anti-mouse horseradish peroxidase-linked antibody according to the ECL Western blotting protocol (Amersham Pharmacia Biotech).

Adenylyl Cyclase Assay-- Conversion of [alpha 32P]ATP to [32P]cAMP in the presence of various activators was measured as described (23). Membranes were incubated at 30 °C. Reactions shown in Figs. 1 and 4 were preincubated for 5 min in the absence of [alpha 32P]ATP and then incubated for 30 min. For the time courses shown in Fig. 3, membranes were preincubated in the absence of [alpha 32P]ATP and activators for 6 min. At time = 0, [alpha 32P]ATP and either GTPgamma S or GTPgamma S and isoproterenol were added and aliquots were removed at the indicated times for cAMP determination. To determine EC50 values for stimulation of adenylyl cyclase by GTPgamma S shown in Fig. 4, the observed adenylyl cyclase activity was fitted to the equation,
Y=b+<FR><NU>a−b</NU><DE>1+(X/c)<SUP>d</SUP></DE></FR> (Eq. 1)
where X is the concentration of GTPgamma S, Y is the observed adenylyl cyclase activity, a is the adenylyl cyclase activity observed in the absence of GTPgamma S, b is the maximum observed adenylyl cyclase activity, c is the half-maximal effective concentration (EC50) of GTPgamma S, and d is the slope factor.

Receptor Binding Assay-- Membranes were incubated with 75 pM [125I]ICYP in competition with a range of concentrations of isoproterenol (10-11 to 10-3 M) in the presence or absence of 300 µM GTP for 1 h at 30 °C as described (24). At the end of this time, the membranes were diluted and washed on Whatman GF/C filters, and bound [125I]ICYP was measured. The experimental data were analyzed for competition at two sites by nonlinear least-squares curve fitting as described (24). KL and KH, the low and high affinity dissociation constants, respectively, were assumed to be the same in the presence and absence of GTP. When KL and KH were allowed to vary in the two conditions, improved fits to the data were obtained. Therefore, the two-state model may be an oversimplification of receptor behavior, as has been suggested (25).

cAMP Accumulation Assay in Transiently Transfected cyc- S49 Lymphoma Cells-- alpha subunit constructs were introduced by electroporation into a subclone of cyc- S49 lymphoma cells (26) that stably expresses Simian virus 40 large T antigen, and cAMP accumulation was measured after labeling with [3H]adenine as described (13). Nucleotides were separated on ion-exchange columns (27), and cAMP accumulation was expressed as [3H]cAMP/([3H]ATP + [3H]cAMP) × 1000. Receptor-independent cAMP accumulation was determined by measuring basal cAMP levels in cells transfected with the alpha sRC versions of the mutant constructs.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutations at the Domain Interface of alpha s Decrease Activation by the beta -Adrenergic Receptor and by Aluminum Fluoride-- The ability of N254D/M255L/I257L/R258Aalpha s to be activated by the beta -adrenergic receptor and by AlF4-, which mimics the gamma -phosphate of GTP, was measured after expression in cyc- S49 lymphoma cells (26), which lack endogenous alpha s (28). At equal expression levels (Fig. 1A), adenylyl cyclase activity stimulated by both isoproterenol and by AlF4- was reduced by 80% in membranes of cells expressing N254D/M255L/I257L/R258Aalpha s compared with membranes of alpha s-expressing cells (Fig. 1B). Stimulation by the hydrolysis-resistant GTP analog, GTPgamma S, not only was intact, but increased by almost 2-fold.


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Fig. 1.   N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibit impaired activation by receptors and AlF4-. A, expression of alpha s, N254D/M255L/I257L/R258Aalpha s, and N167Ralpha s in membranes of stably transfected cyc- cells. Membranes were prepared and immunoblotted as described under "Experimental Procedures." B, adenylyl cyclase activities in membranes of cyc- cells stably transfected with the indicated constructs. Activator concentrations were as follows: 100 µM GTP, 100 µM isoproterenol (Iso), 100 µM GTPgamma S, and 10 µM AlCl3 + 10 mM NaF (AlF4-). Values represent means ± S.D. for triplicate determinations in a single experiment. Two additional experiments gave similar results. Similar results for each construct were obtained using a second independent clone of stably transfected cyc- cells.

Because the activation defect of N254D/M255L/I257L/R258Aalpha s, as assessed in transiently transfected cells, was suppressed when the helical domain consisted of alpha i2 residues (13), we analyzed the x-ray crystal structures of alpha  subunits to identify nearby helical domain residues that might be responsible for the conditional defect of these switch III substitutions. Comparison of the GTPgamma S- and GDP-bound alpha  subunit structures (5-8) reveals slight changes in the positions of helical domain residues in the alpha D/alpha E loop, which is in contact with switch III in the GTPgamma S-bound form. In alpha s, the only residue in this loop that is close to any of the four residues and differs in the sequences of alpha s and alpha i2 is Asn167. Mutation of Asn167 to the homologous alpha i2 residue to produce N167Ralpha s decreased both isoproterenol-stimulated and AlF4--stimulated adenylyl cyclase activity by 60% in membranes of cells expressing equal amounts of protein compared with membranes of alpha s-expressing cells (Fig. 1). As with N254D/M255L/I257L/R258Aalpha s, stimulation by GTPgamma S was increased ~2-fold (Fig. 1).

Mutations at the Domain Interface of alpha s Increase the Apparent Affinity of alpha s for the beta -Adrenergic Receptor-- Because N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibited decreased receptor-mediated activation, we used a competitive binding assay to determine whether these mutant alpha  subunits exhibit alterations in binding to the beta -adrenergic receptor. This assay measures an alpha s-dependent increase in the affinity of the beta -adrenergic receptor for the agonist, isoproterenol (24, 29), which occurs in the absence of bound guanine nucleotide. The high affinity hormone binding state of the receptor is thought to reflect its interaction with Gs in the nucleotide-free state. In the presence of GTP, receptors in membranes of alpha s-expressing cells were predominantly in the low affinity state (Fig. 2A). In the absence of GTP, alpha s caused the appearance of high affinity binding sites for isoproterenol on the receptor (Fig. 2A). Like alpha s, both N254D/M255L/I257L/R258Aalpha s and N167Ralpha s increased the affinity of the beta -adrenergic receptor for isoproterenol in the absence of GTP compared with in its presence (Fig. 2, B and C). However, in membranes of cells expressing these constructs, the affinity of the receptor for isoproterenol in both the presence and absence of GTP was greater than in membranes from alpha s-expressing cells, due to decreases in KL and KH, the low and high affinity dissociation constants, respectively, as well as increases in the percentage of receptors in the high affinity form, % RH. This increase in hormone-receptor binding was particularly striking in cells expressing N167Ralpha s, in which 50% of the receptors were in the high affinity form in the presence of GTP. The simplest explanation of the increased amount of hormone-receptor binding observed in the presence of N254D/M255L/I257L/R258Aalpha s and N167Ralpha s is that these mutant alpha  subunits increase the affinity of Gs for the receptor in both the nucleotide-bound and -free states.


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Fig. 2.   Competition between isoproterenol and [125I]iodocyanopindolol for binding to the beta -adrenergic receptor. Membranes of cyc- cells stably expressing alpha s (A), N254D/M255L/I257L/R258Aalpha s (B), or N167Ralpha s (C) were incubated with [125I]ICYP (75 pM) and the indicated concentrations of isoproterenol, in the presence (filled symbols) or absence (open symbols) of 300 µM GTP. Values represent the means of duplicate determinations in a single experiment, which is representative of two such experiments. The solid lines represent a nonlinear least-squares fit to the data according to the two-state model for receptor activation (40). KL and KH are the low and high affinity dissociation constants, respectively, and % RH is the percentage of receptors in the high affinity form. KL and KH were assumed to be the same in the presence and absence of GTP. In B and C, the binding curves for membranes from alpha s-expressing cells, from A, are redrawn as dotted lines. Similar results for each construct were obtained in two additional experiments using a second independent clone of stably transfected cyc- cells. The binding assay and data analysis were performed as described (24).

Mutations at the Domain Interface of alpha s Decrease the Rate of Activation by the beta -Adrenergic Receptor-- Because dissociation of Gs from the activated receptor must precede adenylyl cyclase activation, we investigated whether N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibited altered rates of receptor-mediated activation. To estimate relative rates of receptor-mediated activation, we determined the effects of isoproterenol on the time courses of adenylyl cyclase activation by GTPgamma S. In membranes of cells expressing alpha s, GTPgamma S activated adenylyl cyclase with a time lag that was greatly reduced by isoproterenol (Fig. 3A). This decreased time lag reflects receptor-stimulated increases in the rates of GDP dissociation and GTPgamma S binding. In the absence of isoproterenol, GTPgamma S activated adenylyl cyclase in membranes containing N254D/M255L/I257L/R258Aalpha s or N167Ralpha s with somewhat shorter time lags than in alpha s membranes (Fig. 3, B and C). Isoproterenol increased the activation rates of these mutants, but not to the same extent as for alpha s. Thus, in the presence of isoproterenol, the time lags of the mutants were longer than that of alpha s (Fig. 3, B and C). N167Ralpha s, which caused the appearance of the greatest amount of high affinity binding to the receptor (Fig. 2C), exhibited the longest time lag in the presence of isoproterenol. Thus, N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibit decreased rates of receptor-mediated activation, which could reflect decreased rates of GTP-dependent dissociation from receptors. Alternatively, or in addition, decreased rates of receptor-mediated activation could be due to defects in receptor-stimulated GTP binding, which we investigated as described below.


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Fig. 3.   N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibit increased time lags for activation by isoproterenol. Membranes of cyc- cells stably expressing alpha s (A), N254D/M255L/I257L/R258Aalpha s (B), or N167Ralpha s (C) were preincubated in reaction mix at 30 °C for 6 min. At time 0, [gamma 32P]ATP and either 100 µM GTPgamma S (triangles) or 100 µM each of isoproterenol (Iso) and GTPgamma S (circles) were added. Aliquots were removed at the indicated times for determination of adenylyl cyclase activity. Data points represent the means of duplicate determinations in a single experiment. Similar results were obtained in four additional experiments.

Mutations at the Domain Interface of alpha s Disrupt the Ability of the beta -Adrenergic Receptor to Promote Binding of GTPgamma S-- Receptors stimulate guanine nucleotide exchange on G proteins by increasing the rate of GDP release and by causing a preference for GTP compared with GDP (3, 4). For alpha s, this results in an approximately 8-fold decrease in the half-maximal effective concentration (EC50) for GTPgamma S stimulation of adenylyl cyclase in the presence of isoproterenol compared with in its absence (Fig. 4A). In the absence of isoproterenol, N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibited EC50 values for GTPgamma S stimulation of adenylyl cyclase that were slightly lower than that of alpha s (Fig. 4, B and C). However, these EC50 values were unchanged by isoproterenol (Fig. 4, B and C) so that in the presence of isoproterenol, their apparent affinities for GTPgamma S were less than that of alpha s. Thus, although isoproterenol increases the rate at which these mutant alpha  subunits exchange nucleotide (Fig. 3), it does not increase their apparent affinities for GTP.


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Fig. 4.   Isoproterenol does not decrease the EC50 for activation of N254D/M255L/I257L/R258Aalpha s and N167Ralpha s by GTPgamma S. Adenylyl cyclase activities in membranes of cyc- cells stably expressing alpha s (A), N254D/M255L/I257L/R258Aalpha s (B), or N167Ralpha s (C) were determined in the presence of the indicated concentrations of GTPgamma S, in the presence (filled symbols) or absence (open symbols) of 100 µM isoproterenol (Iso). Data points represent the means from three independent experiments and are expressed as the percentage of the maximum observed adenylyl cyclase activity. EC50 values were calculated as described under "Experimental Procedures."

Localization of a Single Switch III Residue on the GTPase Side of the Domain Interface That Is Important for Receptor-mediated Activation of alpha s-- We individually tested each of the four switch III residues that were mutated in N254D/M255L/I257L/R258Aalpha s to determine their roles in receptor-mediated activation. Receptor-dependent stimulation of cAMP synthesis was measured in transiently transfected cyc- S49 lymphoma cells. The only substitution that decreased receptor-mediated activation was R258A (Fig. 5A). Receptor-independent cAMP accumulation was also measured after introducing a second mutation (the RC mutation) that substitutes cysteine for the arginine at position 201 (19). alpha sRC has decreased GTPase activity and is constitutively activated. R258Aalpha sRC produced receptor-independent cAMP accumulation similar to that of alpha sRC, indicating that, as is the case for N254D/M255L/I257L/R258Aalpha s, R258Aalpha s can activate adenylyl cyclase when it is in the GTP-bound form.


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Fig. 5.   Receptor-mediated activation of mutant alpha s proteins. cAMP accumulation was measured in cyc- cells transiently expressing the indicated alpha s constructs. cAMP values from unstimulated cells and from cells stimulated with 0.1 mM isoproterenol are dark gray and light gray, respectively. Cells were electroporated with 30 µg of each construct, with the following exceptions: N254Dalpha s, 10 µg; N167R/N254Dalpha s, 10 µg; N254D/M255L/I257L/R258Aalpha s, 20 µg; and N167R/R258Aalpha s, 60 µg. At these plasmid doses, equivalent amounts of receptor-independent cAMP accumulation (mean, 7.3; S.D., 1.5) were produced by versions of the constructs in which arginine 201 was replaced by cysteine, which causes constitutive activation by decreasing GTPase activity (19). cAMP levels in [3H]adenine-labeled cells were determined as described under "Experimental Procedures." Within each panel, the values for alpha s and the vector alone represent the activities obtained on the days that the indicated mutants were tested. Day-to-day variation in cAMP accumulation in transiently transfected cells accounts for the differences in these values in between A, B, and C. All values represent the means ± S.E. of at least three independent experiments.

Complementation of the Activation Defect of R258Aalpha s-- Because the activation defect of N254D/M255L/I257L/R258Aalpha s was corrected by replacing the helical domain of alpha s with that of alpha i2 in a chimera, alpha sis, in which alpha i2 homologs were substituted for alpha s residues 62-235, (Fig. 6) (13), we investigated whether introducing the single homolog substitution (R258A) responsible for the defect of N254D/M255L/I257L/R258Aalpha s into alpha sis would result in normal activation properties. R258Aalpha sis exhibited activation properties similar to those of alpha s rather than those of R258Aalpha s (Fig. 5A). Thus, the defect produced by the R258A substitution appears to be due to an alteration in interactions with alpha s residue(s) in the helical domain.


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Fig. 6.   Mapping of alpha s residues at the domain interface that are important for receptor-mediated activation onto the structure of an alpha beta gamma heterotrimer. View of the alpha  subunit based on the x-ray crystal structure of an alpha t/alpha i1 chimera complexed with beta tgamma t (10). The beta gamma subunits have been omitted for clarity. The helical domain is to the left of the GDP, which is yellow. The GTPase domain is to the right. The spheres are centered on the alpha -carbons of the corresponding residues and are numbered according to the alpha s sequence. Asn167 and Arg258, in which substitutions with alpha i2 homologs disrupt receptor-mediated activation, are red spheres. Met255 and Ile257, which can be mutated without impairing receptor-mediated activation, are green spheres. Substitution of the homologous alpha i2 residue for Asn254 (magenta sphere) does not impair receptor-mediated activation, but corrects the defect caused by replacing Asn167 with the homologous alpha i2 residue. Asp173 and Lys293 (dark blue spheres) form a salt bridge that is required for receptor-mediated activation (31). The amino-terminal portion of the alpha sis chimera, consisting of alpha s residues (light blue), extends from the amino terminus to the end of alpha 1. The middle portion of the chimera, consisting of alpha i2 residues (pink), extends from the alpha s/alpha i2 junction (labeled s/i) to the alpha i2/alpha s junction (labeled i/s) at the end of alpha 2. The carboxyl-terminal portion of the chimera consists of alpha s residues (light blue). Switches (Sw) I-III are gold. This figure was drawn using MidasPlus, developed by the Computer Graphics Laboratory at the University of California, San Francisco.

Combining the N167R and R258A Substitutions Results in an Additive Defect in Receptor-mediated Activation-- We investigated the effect of combining the substitutions on each side of the domain interface, N167R and R258A, that caused significant decreases in receptor-mediated activation. Although Asn167 and Arg258 are both at the domain interface, they are not close enough to make contact in the x-ray crystal structures of alpha  subunits (see Fig. 6). N167R/R258Aalpha s exhibits a more severe activation defect (Fig. 5B) than either N167Ralpha s (Fig. 5C) or R258Aalpha s (Fig. 5A) does, although receptor-independent activation by N167R/R258Aalpha sRC is normal. Because the defects of the N167R and R258A substitutions are additive, the contributions of these substitutions to defects in receptor-mediated activation are independent. Furthermore, some other residue(s) in the helical domain other than the alpha i2 homolog of Asn167 must be responsible for the suppression of the R258A defect in R258Aalpha sis.

Combining the N167R and N254D Substitutions Corrects the Defect of the N167R Substitution-- According to the x-ray crystal structures of alpha  subunits, Asn167 is close to Asn254 (see Fig. 6). Therefore, we hypothesized that the N167R substitution might cause a conditional defect, depending on the identity of the residue at position 254. According to this hypothesis, replacing Asn254 with aspartate should correct the defect caused by the N167R mutation. Indeed, we found that an alpha  subunit with both substitutions, N167R/N254Dalpha s, exhibits activation properties similar to those of alpha s (Fig. 5B). Thus, the N254D substitution, which on its own does not disrupt receptor-mediated activation, corrects the activation defect caused by the N167R substitution.

Substitution of Asn167 by Alanine Does Not Cause a Defect in Receptor-mediated Activation of alpha s-- To further investigate the mechanism by which the N167R substitution causes a defect in receptor-mediated activation, we determined the effect of mutating Asn167 to alanine. Alanine substitutions eliminate the side chain beyond the beta  carbon but generally do not alter the main chain conformation or impose significant electrostatic or steric effects (30). Therefore, if the activation defect resulting from the N167R substitution is due to a steric or electrostatic incompatibility with Asn254, then alanine substitution might not cause an activation defect. However, if the N167R substitution removes a favorable interaction between Asn167 and Asn254, then alanine substitution should also cause a defect. Because N167Aalpha s exhibited normal receptor-stimulated cAMP production (Fig. 5C), the activation defect of N167Ralpha s appears to be due to a steric or electrostatic incompatibility that is reversed by the N254D substitution.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our analysis of alpha s mutants with substitutions at the interface of the GTPase and helical domains suggests that interdomain interactions play a role in the bi-directional transmission of signals between receptors and the nucleotide binding site. Interaction between activated receptors and G proteins promotes GTP binding by accelerating GDP release and increasing the relative affinity for GTP compared with GDP (3, 4). Conversely, nucleotide binding decreases the affinity of G proteins for receptors (24, 29). Substitution of the homologous alpha i2 residues for four alpha s residues (Asn254, Met255, Ile257, and Arg258) in switch III of the GTPase domain or of one nearby helical domain residue (Asn167) in the alpha D/alpha E loop causes defects in both directions of this communication process. Signal transmission from the receptor to the nucleotide binding site is defective in that the affinities of these alpha s mutants for GTPgamma S are unchanged by isoproterenol. Conversely, altered communication between the guanine nucleotide binding pocket and the receptor binding site(s) is demonstrated by high affinity hormone-receptor binding in the presence of 300 µM GTP.

Contacts between the alpha D/alpha E loop in the helical domain and three regions of the GTPase domain have been implicated as playing a role in receptor-mediated activation. In the heterotrimer-based alpha  subunit model shown in Fig. 6, the helical domain side of the interface "above" the GDP consists of the alpha D/alpha E loop. Moving up from the GDP toward the top of the alpha  subunit, the corresponding GTPase side of the interface consists of the beta 5/alpha G, beta 4/alpha 3, and alpha G/alpha 4 loops. Closest to the GDP, a salt bridge interaction between Asp173 in the carboxyl-terminal portion of the alpha D/alpha E loop and Lys293 in the beta 5/alpha G loop (Fig. 6, dark blue) is required for activation by the beta -adrenergic receptor and by AlF4-, but not by GTPgamma S (31). These residues are highly conserved among alpha  subunits, and Lys293 is located in the NKXD motif, which is important for GTP binding by monomeric GTPases. Mutation of Asp173 increases GTP affinity, consistent with the idea that the mutation "frees" Lys293 from Asp173 to interact with GTP, whereas mutation of Lys293 decreases GTP affinity. Because Asp173 and Lys293 are adjacent to the bound guanine nucleotide, they are more directly involved in regulating guanine nucleotide binding than the residues mutated in our study are. The effects of mutating Asp173 and Lys293 on receptor affinity and receptor-dependent changes in GTP affinity have not been determined.

Arg258 (Fig. 6, red) is located further up, in the beta 4/alpha 3 loop, which includes switch III. Interestingly, a mutation that substitutes tryptophan for Arg258 was found in a patient with Albright hereditary osteodystrophy, and R258Walpha s exhibited more severe defects than did R258Aalpha s.3 The defect caused by the R258A substitution is suppressed (Fig. 5A) by substituting the entire helical domain of alpha i2 for that of alpha s in the alpha sis chimera (Fig. 6), but we have not identified the helical domain residue(s) responsible. In addition to Asn167, there are two other alpha s residues in the alpha D/alpha E loop, Ile172 and Cys174, that differ in the sequences of alpha s and alpha i2. Cys174 is not close to Arg258 when modeled onto the heterotrimeric G protein structures (10, 11) or in the structure of alpha s·GTPgamma S (32). However, in the latter structure, the side chains of Ile172 and Arg258 are within ~4 Å of each other (see Fig. 7).


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Fig. 7.   Close-up view of the interface between switch III and the alpha D/alpha E loop. The interface of alpha s from the x-ray crystal structure of alpha s·GTPgamma S complexed with the catalytic domains of adenylyl cyclase (9) is on the right and that of alpha i1 from the x-ray crystal structure of alpha i1·GTPgamma S (6) is on the left. The pink and cyan tubes trace the overall fold of the helical and GTPase domains, respectively, at this interface, as in Fig. 6. The sequences of alpha i1 and alpha i2 are identical in the regions shown. The side-chains of residues that differ in the sequences of alpha s and alpha i2 are shown. Oxygens are red, nitrogens are blue, and sulfurs are yellow. The dotted yellow line indicates the hydrogen bond between Asn167 and Asn254 in alpha s and between the corresponding residues in alpha i2, Arg145 and Asp232. This figure was drawn using MidasPlus.

Asn167 (Fig. 6, red) is located further away from the GDP in the alpha D/alpha E loop. The activation defect caused by replacing Asn167 with its alpha i2 homolog (arginine) is corrected (Fig. 5B) by simultaneously replacing Asn254 (Fig. 6, magenta) in the beta 4/alpha 3 loop with its alpha i2 homolog (aspartate). In the alpha  subunit structures, the corresponding residues are hydrogen bonded to each other via the side chain of the residue corresponding to Asn167 and either the side chain (in alpha t (5, 7) and an alpha t/alpha i1 chimera complexed with beta tgamma t (10)) or the backbone carbonyl (in alpha s (9, 32) and alpha i1 (6)) of the residue corresponding to Asn254 (see Fig. 7). The N254D substitution in alpha s might correct the defect caused by the N167R substitution via a charge neutralization mechanism. This idea is supported by the fact that all alpha  subunits with an arginine at the position corresponding to Asn167 (alpha q, alpha 11, alpha 14, alpha 15, alpha 16, and alpha o) have an aspartate at the position corresponding to Asn254 and by the observation (Fig. 5C) that the N167A substitution in alpha s leaves receptor-mediated activation intact. It is not surprising that the activation defect of N167R/R258Aalpha s is worse than those of N167Ralpha s and R258Aalpha s (Fig. 5), because these residues are not within contact distance. Because the two mutations cause additive defects, the two residues also do not appear to influence each other through electrostatic or steric effects (33).

We previously found that substitution of alpha i2 residues for alpha s residues 304, 305, and 307-311 in the alpha G/alpha 4 loop (furthest from the nucleotide on the GTPase side of the interface in Fig. 6) disrupts receptor-mediated activation in the context of alpha s but not alpha sis (13). Of the mutated alpha s residues, only Lys305 and Tyr311 are close to the interface in the structure of alpha s·GTPgamma S (32).

Although interactions between residues in switch III and the alpha D/alpha E loop are important for receptor-mediated activation, the known receptor binding sites of alpha s, the carboxyl terminus of alpha 5 (13, 21, 34) and possibly the alpha 4/beta 6 loop (34), are not near this interface. alpha  subunits bind to beta gamma , which is required for receptor-mediated activation, via switches I and II and the amino terminus (10, 11), which are also not near this interface. Thus, receptors initiate activating signals at a significant distance from the domain interface, possibly via an interaction between switches II and III.

N254D/M255L/I257L/R258Aalpha s and N167Ralpha s exhibit two characteristics in the absence of receptor stimulation that are not normal and that resemble those of wild-type alpha s upon activation by hormone-bound receptors. They exhibit slightly elevated basal rates of activation (Fig. 3) and somewhat increased basal affinities for GTPgamma S (Fig. 4). The basal activation rates of these alpha s mutants, which reflect basal nucleotide exchange rates, are not nearly as elevated as in an alpha s mutant, A366Salpha s, which is both thermolabile and constitutive activated (35). However, increased rates of basal GDP dissociation in N254D/M255L/I257L/R258Aalpha s and N167Ralpha s would account for their observed defects in activation by aluminum fluoride, which requires the presence of bound GDP.

The defects in guanine nucleotide and receptor binding of N254D/M255L/I257L/R258Aalpha s and N167Ralpha s may be interrelated. For instance, decreased receptor-stimulated GTP binding would stabilize the high affinity hormone-receptor-G protein complex, which forms when the G protein is in the nucleotide-free state. Conversely, higher affinity receptor-G protein interactions could decrease nucleotide binding, because activated receptors can cause dissociation of both GDP and GTP analogs (36, 37). However, although there are other reported alpha s mutants with defects in GTP binding and receptor-mediated activation, there is no precedent for an associated increase in receptor affinity. R231Halpha s, containing a mutation in the alpha 2 helix (38), and S54Nalpha s, containing a substitution in the alpha 1 helix (39), exhibit impaired activation by receptors and AlF4- but can be activated by GTPgamma S. The affinities of these alpha s mutants for GTP are decreased upon receptor stimulation. The affinity of R231Halpha s for the receptor is normal, and the affinities of S54Nalpha s and of A366Salpha s, which exhibits accelerated GDP release (35), were not determined. The defect of S54Nalpha s appears to be due to altered interactions with the bound Mg2+, with which Ser54 interacts. Thus, the activation defects of these alpha s mutants are distinct from those of N254D/M255L/I257L/R258Aalpha s and N167Ralpha s.

Further studies will be required to elucidate how the alpha  subunit domain interface mediates communication between the residues responsible for receptor binding and the guanine nucleotide binding pocket. This type of regulation appears to be unique to heterotrimeric G proteins, which interact with seven-transmembrane-spanning receptors, compared with monomeric GTPases, which lack switch III and the helical domain and which utilize different exchange factors.

    ACKNOWLEDGEMENTS

We thank David Lambright and Paul Sigler for the coordinates of the alpha t/alpha i1 chimera complexed with beta tgamma t, Stephen Sprang for the coordinates of alpha s·GTPgamma S complexed with the catalytic domains of adenylyl cyclase, Dennis Warner and Lee Weinstein for sharing data prior to publication, and Thomas Hynes for helpful discussions and critical reading of the text.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM50369 (to C. H. B.).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 Established Investigator of the American Heart Association. To whom correspondence should be addressed. Tel.: 203-785-3202; Fax: 203-785-4951; E-mail: cathy_berlot{at}qm.yale.edu.

1 The abbreviation used is: GTPgamma S, guanosine 5'-O-(3-thiotriphosphate).

2 Residue numbering throughout is according to the long splice variant of alpha s.

3 D. R. Warner and L. S. Weinstein, personal communication.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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