Introduction

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Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that produce a wide variety of cellular responses (Neer, 1995). The, , and  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  subunit, resulting in dissociation of ·GTP from , each of which can transmit signals to effectors. Hydrolysis of GTP by the  subunit regulates the timing of deactivation and reassociation of  with . As intermediaries between receptors and effectors, G proteins play a crucial role in determining the specificity, nature, and degree of amplification of transmitted signals. For example, G_s mediates stimulation of adenylyl cyclase by -adrenergic receptors. However, the molecular determinants that specify receptor/G protein interactions and the mechanism by which these interactions lead to G protein activation are not well understood.

Studies of G protein function can be interpreted in the context of the x-ray crystal structures of GTPS-bound (active) (Noel et al., 1993; Coleman et al., 1994) and GDP-bound (inactive) (Lambright et al., 1994; Mixon et al., 1995) _subunits and of heterotrimers (Wall et al., 1995; Lambright et al., 1996). The  subunits consist of two domains, a GTPase domain that resembles the oncogene protein, p21^ras, and a helical domain consisting of  helices and connecting loops. Because the bound nucleotide is buried in the cleft between these domains, receptor-mediated nucleotide exchange presumably involves a conformational change that opens the cleft. Comparison of the structures of GTPS-bound and GDP-bound  subunits reveals three regions in the GTPase domain (switches I-III) that change conformation, which could be involved in the activation process.

All  subunit residues involved in associating with receptors and with , which is required for receptor-mediated activation (Fung, 1983), have not been identified. X-ray crystal structures of the complex (Wall et al., 1995; Lambright et al., 1996) showed that two  subunit regions contact the  subunit, the amino-terminal  helix and a region that includes switches I and II. The functional importance of these regions has been demonstrated using proteolysis (Navon and Fung, 1987), mutagenesis (Miller et al., 1988; Journot et al., 1991), and cross-linking (Garcia-Higuera et al., 1996). Numerous genetic and biochemical studies, reviewed by Neer (1995), have implicated the carboxyl-terminal region of the  subunit in interaction with receptors. However, the locations of these and receptor-interacting residues, which are distant from the domain cleft, have not revealed the mechanism of receptor-mediated G protein activation.

We exploited the differences in receptor specificities of _s and _i2, which are relatively divergent members of the  subunit family, sharing ~40% amino acid identity, to identify additional  subunit residues that mediate a response to receptor stimulation. Measurements of receptor-stimulated guanine nucleotide exchange in reconstituted phospholipid vesicles have demonstrated that the efficiency with which the -adrenergic receptor regulates _i2 is ~10% of that seen for _s (Rubenstein et al., 1991). The _s residues that specify interaction with the -adrenergic receptor have been localized to the carboxyl-terminal 40% of _s by means of an _i2/_s chimera (Masters et al., 1988).

By examining a panel of _s mutants in which clusters of residues were replaced by _i2 homologs or alanines, we identified four regions of sequence that are specifically required for receptor-mediated activation. Residues at the extreme carboxyl terminus are the most likely receptor contact residues. Buried residues near the guanine ring of the bound GDP, connected to the carboxyl terminus by an  helix, may transmit the signal from the receptor to modulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes switch III, are positioned to relay the receptor-initiated signal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of _s with that of _i2 in an _s/_i2/_s chimera corrects the defect in receptor-mediated activation caused by _i2 substitutions on the GTPase side of the interface. Thus, complementary interactions between residues across the domain interface seem to play a role in receptor-catalyzed activation.

	Experimental Procedures

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Materials. The expression vector pcDNA I/Amp was obtained from InVitrogen (Carlsbad, CA). Plasmids used for electroporation were prepared using Qiagen Plasmid Maxi Kits (Santa Clarita, CA). Isoproterenol, 1-methyl-3-isobutylxanthine, cAMP, and ATP were obtained from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle's medium, minimal essential medium with Earle's salts, and geneticin were obtained from GIBCO BRL (Grand Island, NY). Horse serum was obtained from Hyclone (Logan, UT). [³H]Adenine was obtained from Amersham (Little Chalfont, UK).

Construction of  subunit mutants and chimeras. The _s mutant constructs were generated from rat _s cDNA (Jones and Reed, 1987). Chimeric  subunits were constructed from rat _s cDNA and mouse _i2 cDNA (Sullivan et al., 1986). Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing. All  subunit constructs produced in this study contain an epitope, referred to as the EE epitope (Grussenmeyer et al., 1985), which was generated by mutating _s residues DYVPSD (189-194) to EYMPTE and _i2 residues SDYIPTQ (166-172) to EEYMPTE (single-letter amino acid code; mutated residues are underlined). This epitope does not affect the ability of _s to activate adenylyl cyclase in response to stimulation by the -adrenergic receptor (Wilson and Bourne, 1995).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Panel of s mutant constructs. All mutations are within the carboxyl-terminal 40% of the rat s sequence (Jones and Reed, 1987), which is depicted in two sections (residues 236-314 and 315-394). Top lines, sequence of s. Second lines, mouse i2 (Sullivan et al., 1986) in the corresponding region. Dashes, residues identical to those of s. Numbered sequences, individual mutant constructs. For each s mutant construct, mutated residues are shown by the single-letter amino acid code. Dashes, residues identical to those of s. Underlined sequences (located in regions 1-4), mutations that disrupted receptor-mediated activation of s. Elements of secondary structure, determined from the structure of s·GTPS (Sunahara et al., 1997), are indicated: a, helices; b, strands; and dashes, turns and loops. Regions that switch conformation between the GDP-bound and GTPS-bound forms of t (Lambright et al., 1994) and i1 (Mixon et al., 1995) (switches II and III) are indicated. The alignment shown, which is based on the recently solved structure of s·GTPS (Sunahara et al., 1997), differs from our previous alignment (Berlot and Bourne, 1992) in the location of an insertion of s sequence relative to that of i2. In the previous alignment, s residues 324-336 were inserted between i2 residues 299 and 300.

cAMP accumulation assay. Transient transfections were performed using a subclone of cyc S49 lymphoma cells (Bourne et al., 1975) that stably expresses Simian virus 40 large T antigen. These cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated horse serum and 0.6 mg/ml geneticin. Transient transfection of cells expressing TAg with vectors containing a Simian virus 40 origin of replication has been shown to maximize expression levels (Clipstone and Crabtree, 1992). Therefore, we used the expression vector, pcDNA I/Amp, which contains a Simian virus 40 origin of replication, as well as the cytomegalovirus promoter, to electroporate the cyc cells.

	Results

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Panel of _s mutant constructs for studying receptor-mediated activation. The 159-residue carboxyl-terminal segment of _s (residues 236-394), which specifies interaction with the -adrenergic receptor (Masters et al., 1988), contains 59 amino acids that are identical in the sequence of _i2 and therefore do not specify interaction with this receptor. We previously demonstrated that mutations of residues in three adjacent regions of the  subunit structure, the 2/4, 3/5, and 4/6 loops (Noel et al., 1993), disrupt the ability of _s to activate adenylyl cyclase (Berlot and Bourne, 1992). Because the current study of receptor-mediated activation of _s mutants used a cAMP accumulation assay, we did not test 16 residues in these loops. Of the remaining 84 nonidentical residues, 61 were changed in small clusters to _i2 homologs or to alanines using 15 _s mutant constructs (Fig. 1).

Transient transfection assay for receptor-mediated activation of _s. To test the abilities of mutant _s proteins to be activated by the -adrenergic receptor, we measured receptor-dependent stimulation of cAMP synthesis after transient transfection of cyc S49 lymphoma cells (Bourne et al., 1975), which lack endogenous _s (Harris et al., 1985). Basal cAMP levels in cells transfected with 10-90 µg of vector containing _s varied linearly in proportion to the plasmid dose (Fig. 2A). Stimulation of these _s-transfected cells with the -adrenergic agonist isoproterenol produced increased cAMP levels that also exhibited a linear relationship to the amount of transfected plasmid (Fig. 2A). We also determined receptor-independent cAMP accumulation by measuring basal cAMP levels in cells transfected with versions of the _s mutants in which Arg201 is mutated to cysteine. The _s containing this mutation, _sRC, exhibits constitutive activation due to inhibited GTPase activity (Landis et al., 1989). As with _s-transfected cells, basal cAMP levels in cyc cells transfected with 10-90 µg of vector containing _sRC varied linearly in proportion to the plasmid dose (Fig. 2B).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Transient transfection assay for receptor-mediated activation of s. A, cAMP accumulation in cyc cells electroporated with the indicated doses of vector containing s. cAMP levels were measured in the presence and absence of 0.1 mM isoproterenol. B, Receptor-independent cAMP accumulation in cyc cells electroporated with the indicated doses of vector containing sRC. For the 0-µg points, 30 µg of vector was used. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean ± standard error of three independent experiments.

View larger version (55K): [in this window] [in a new window]   Fig. 3.   Receptor-mediated activation of mutant s proteins. A, cAMP accumulation in cyc cells containing the indicated mutants in the s context. Cells were electroporated with 20 µg of vector containing s (2), 30 µg of vector alone, 30 µg of vector containing s and s (3 and 10-15), 60 µg of vector containing s (5), 90 µg of vector containing s (1, 4, 7, and 8), 120 µg of vector containing s (9), and 180 µg of vector containing s (6). Dark gray, cAMP values from unstimulated cells. Light gray, cAMP values from cells stimulated with 0.1 mM isoproterenol. B, Receptor-independent cAMP accumulation in cyc cells containing the indicated mutants in the sRC context. For each mutant, the same amount of plasmid was used as is indicated in A for the corresponding s mutant. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean ± standard error of at least three independent experiments.

Receptor-mediated activation of _s mutant constructs. Receptor-stimulated cAMP accumulation due to 9 of the 15 _s mutant constructs was similar to that of _s (Fig. 3A). The other 6 constructs produced reduced receptor-dependent increases in cAMP levels and delineated four regions of sequence containing seven or fewer _s mutations that disrupt the ability of _s to be activated by the -adrenergic receptor (Fig. 1). Region 1, defined by _s(1) and _s(2), contains V247, S250, S252, N254, M255, I257, and R258. Region 2, defined by _s(6), contains G304, K305, and K307-Y311. Region 3, defined by _s(11) and _s(12), contains V367, E370, and I372-R374. Region 4, defined by _s(15), contains R389-E392 and L394.

Mapping of mutations that block receptor-mediated activation onto the structure of a heterotrimeric G protein. Because receptors interact with heterotrimers, we mapped the _s residues in which mutations disrupted activation by the -adrenergic receptor onto the x-ray crystal structure of an _t/_i1 chimera complexed with _tt (Lambright et al., 1996) to visualize their positions in three dimensions (Fig. 4). The recently solved structure of _s·GTPS (Sunahara et al., 1997) is very similar to the structures of _t·GTPS (Noel et al., 1993) and _i1·GTPS (Coleman et al., 1994), indicating that the structure of _t/_i1tt is a good model for the G_s heterotrimer. Structural features unique to _s that are relevant to the mutations that blocked receptor-mediated activation are discussed. Some of the mutations that blocked receptor-mediated activation map onto solvent-exposed residues that could potentially interact directly with the receptor, whereas others map onto residues that are buried and are more likely to mediate nucleotide exchange by propagating conformational changes within _s.

View larger version (83K): [in this window] [in a new window]   Fig. 4.   Mapping of s mutations that disrupt receptor-mediated activation onto the structure of an heterotrimer. A, View of the  subunit. The subunits have been omitted for clarity. Left of GDP (yellow), helical domain. Right, GTPase domain. Blue spheres, residues mutated in s(1) in region 1. Red spheres, residues mutated in s(2) in region 1. Green spheres, residues mutated in s(6) in region 2. Orange spheres, buried residues in region 3 mutated in s(16). Residues mutated in s(15) in region 4 are not seen in this structure, but the most carboxyl-terminal t/i1 residue visualized (residue 343 at the end of 5) is a magenta sphere. The amino-terminal portion of the sis chimera, consisting of s residues (light blue), extends from the amino terminus to the end of 1. The middle portion of the chimera, consisting of i2 residues (pink), extends from the s/i2 junction (s/i) to the i2/s junction (i/s) at the end of 2. The carboxyl-terminal portion of the chimera consists of s residues (light blue). Gold, switches I-III. Numbers on the spheres, s residue numbers. B, View of the heterotrimer. The model is rotated 180 degrees around the vertical axis relative to the view in A. Gold, strands in the  subunit. Yellow, amino-terminal  helix and the connecting loops. White, subunit. Other colors, as in A. X-ray coordinates of an t/i1 chimera complexed with tt are from Lambright et al. (1996). These figures were drawn using MidasPlus, developed by the Computer Graphics Laboratory at UCSF.

Mutations of buried, but not surface-exposed, residues in region 3 disrupt receptor-mediated activation. To determine the role of the buried and surface-exposed residues in region 3, we mutated separately each class of residues. We found that substitution of the buried residues with the homologous _i2 residues in _s(16) specifically reduced receptor-mediated increases in cAMP production, whereas substitution of the surface-exposed residues with alanine residues in _s(17) had no effect (Fig. 5). Therefore, this region does not seem to be a receptor contact site but instead probably is important for transmitting the receptor signal to the bound GDP. Of the two residues mutated in _s(16), V367 is located in the 6/5 loop, presumably in contact with the GDP, whereas I372 is near the beginning of 5. Because each of these residues was tested separately in _s(11) and _s(12), both regions seem to be involved in responding to receptor stimulation.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Substitution of buried but not solvent-exposed residues in region 3 impairs receptor-mediated activation. A, Representation of s mutants as in Fig. 1. B, cAMP accumulation in cyc cells containing the indicated mutants in the s context. Cells were electroporated with 30 µg of vector alone and of vector containing s and s(16) and with 45 µg of vector containing s(17). Dark gray, cAMP values from unstimulated cells. Light gray, cAMP values from cells stimulated with 0.1 mM isoproterenol. C, Receptor-independent cAMP accumulation in cyc cells containing the indicated mutants in the sRC context. For each mutant, the same amount of plasmid was used as is indicated in B for the corresponding s mutant. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean ± standard error of at least three independent experiments.

Defects caused by _i2 substitutions in regions 1 and 2 can be complemented by replacing the helical domain with _i2 residues. Based on the locations of the region 1 and 2 residues at the interface between the GTPase and helical domains, we hypothesized that they mediate receptor-dependent activation via interactions with helical domain residues. Mismatches across the domain interface between _i2 residues in the GTPase domain and _s residues in the helical domain thus would be the cause of the reduced abilities of the region 1 and 2 _s mutant constructs to be activated by receptor stimulation. According to this hypothesis, replacing the helical domain of _s with that of _i2 would be expected to reverse the defect in receptor-mediated activation caused by the _i2 for _s substitutions in regions 1 and 2. To test this prediction, we produced an  subunit chimera, _sis, in which _i2 homologs are substituted for _s residues 62-235, extending from the end of 1 to the end of 2 (see Fig. 4). The helical domain of this chimera is composed of _i2 residues, and the GTPase domain contains _s residues.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Receptor-mediated activation of sis. A, cAMP accumulation in cyc cells electroporated with the indicated doses of vector containing sis. cAMP levels were measured in the presence and absence of 0.1 mM isoproterenol. B, Receptor-independent cAMP accumulation in cyc cells electroporated with the indicated doses of vector containing sisRC. For the 0-µg points, 30 µg of vector was used. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. Each value represents the mean ± standard error of three independent experiments.

View larger version (41K): [in this window] [in a new window]   Fig. 7.   Receptor-mediated activation of sis constructs containing i2 substitutions at the domain interface. A, cAMP accumulation in cyc cells containing the indicated clusters of mutations in the s or sis context. Numbers in parentheses, construct numbers in Fig. 1. Cells were electroporated with 20 µg of vector containing sis(2), 30 µg of vector alone and of vector containing s and sis, 40 µg of vector containing sis(2 + 6), 60 µg of vector containing sis(1) and sis(6), and 180 µg of vector containing s(2 + 6). Dark gray, cAMP values from unstimulated cells. Light gray, cAMP values from cells stimulated with 0.1 mM isoproterenol. B, Receptor-independent cAMP accumulation in cyc cells containing the indicated clusters of mutations in the sRC or sisRC context. For each mutant, the same amount of plasmid was used as is indicated in A for the corresponding s or sis mutant. cAMP levels in [3H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean ± standard error of at least three independent experiments.

	Discussion

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In the analysis of _s mutants presented here, we identified four regions of sequence that mediate activation by the -adrenergic receptor. Two of these regions, the extreme carboxyl terminus (region 4) and the 6/5 loop (region 3), have been implicated previously in receptor/G protein interactions. However, current models of receptor-mediated activation based on these regions have not addressed the issue of how the bound GDP is released from its buried position between the GTPase and helical domains. The effects of mutations in the two other _s regions identified in the current study (regions 1 and 2), located in the 4/3 and G/4 loops at the interface between these domains, suggest that interactions between residues across the domain interface may play a role in the response to receptor stimulation.

Region 4 (the extreme carboxyl terminus) is the most likely candidate for being a receptor contact site. This region was not visualized in the _t/_i1tt structure (Lambright et al., 1996) and occupies different positions in the structures of _t·GTPS (Noel et al., 1993), _s·GTPS (Sunahara et al., 1997), _i1·GDP·AlF₄·RGS4 (Tesmer et al., 1997), and _i112 (Wall et al., 1995), which could indicate a high degree of mobility. In the structure of _t·GTPS (Noel et al., 1993), this region contacts the 2/4 loop, which interacts with the  subunit in the heterotrimer structures (Wall et al., 1995; Lambright et al., 1996). Because receptors interact only with  subunits that are associated with (Fung, 1983), the carboxyl terminus of the  subunit may be located near the interface between 2/4 and the  subunit in the heterotrimer/receptor complex, so that receptors interact with both  and  in this region.

Residues at the extreme carboxyl termini of  subunits are sometimes, but not always, sufficient to confer receptor specificity (Conklin et al., 1993, 1996; Lee et al., 1995). For instance, although both the V₂ vasopressin and ₂-adrenergic receptors couple to _s, only the V₂ vasopressin receptor can stimulate a chimeric  subunit in which the last five residues of _q are replaced with the corresponding residues of _s (Conklin et al., 1996). Differences in the specificities of  subunits and receptors may dictate whether carboxyl-terminal swaps between  subunits will switch receptor specificities. Also, other regions of _s, not identified in the current study, probably are receptor contact sites. For example, synthetic peptides corresponding to _s residues 354-372 and 384-394, which extend from the 4/6 loop to the beginning of 5 and from the end of 5 to the  subunit carboxyl terminus, respectively, can mimic the effects of _s on the ₂-adrenergic receptor (Rasenick et al., 1994). Our study of _s, using homologous substitutions, does not rule out these regions as being receptor contact sites because residues that are identical in _s and _i2 were not changed. Substitutions of surface-exposed _s residues with alanine residues would be required to determine whether they are receptor contact sites. Furthermore, evaluation of receptor-mediated activation of _s mutants with substitutions in regions important for adenylyl cyclase activation, which includes residues in the 4/6 loop (Berlot and Bourne, 1992), would require an assay that is independent of adenylyl cyclase activation.

The results of our study suggest that the role of region 3 (the 6/5 loop and the amino terminal portion of the 5 helix) in _s is to regulate GDP affinity because substitutions of buried residues near the GDP, but not of adjacent solvent-exposed residues, disrupt receptor-mediated activation. GDP release by _s constructs with these substitutions is not entirely blocked because receptor-independent activation of adenylyl cyclase by the GTPase-inhibited versions of the constructs is normal. Of the two region 3 residues in which substitutions decreased receptor-mediated activation, V367, which contacts the guanine ring of the GDP, also is near the helical domain. However, the importance of this proximity to the helical domain is unclear because the helical domain residues closest to V367 are conserved among  subunits and are superimposable on the structures of _s, _i1, and _t. Substitutions of other buried residues in this region have been shown to affect guanine nucleotide exchange in several  subunits. The replacement of serine by alanine at position 366 of _s causes constitutive activation by increasing GDP release (Iiri et al., 1994). Also, the substitution of alanine for cysteine at position 325 of _o decreases affinity for GDP (Thomas et al., 1993). A mutagenesis study of _t (Onrust et al., 1997) identified two residues in the 6/5 loop in which mutations reduced receptor-mediated activation, of which one, T323, corresponds to V367 in _s.

Receptors activate G proteins by promoting GTP binding, which involves both accelerating GDP release and increasing the relative affinity for GTP compared with GDP (Florio and Sternweis, 1989). Our results suggest that the role of regions 1 and 2 in this process may be to relay conformational changes initiated by receptor/G protein binding across the domain interface rather than to specify interaction with the -adrenergic receptor. Altered contacts between the two domains might facilitate nucleotide exchange by opening the cleft in which GDP is buried. This hypothesis is based on our observation that replacing _s residues with _i2 homologs on the GTPase side of the domain interface impairs receptor-mediated activation of _s, but not of a chimera, _sis, in which the helical domain consists of _i2 residues. Thus, matching _i2 residues on both sides of the domain interface of _s seems to restore receptor-initiated activation. As is sometimes the case with second-site suppressors of mutations, substitution of the helical domain of _i2 for that of _s corrects the defects of the region 1 and 2 mutations but does not, on its own, cause a defect in receptor-mediated activation.

In support of the concept of communication between regions 1 and 2 and the helical domain, comparison of the structures of GTPS-bound and GDP-bound  subunits (Noel et al., 1993; Coleman et al., 1994; Lambright et al., 1994; Mixon et al., 1995) shows that conformational changes in switch III (located in region 1) are associated with changes in the positions of helical domain residues in the D/E loop, which it contacts in the GTPS-bound form. Although it is not clear how receptor/G protein interaction might lead to conformational changes in switch III, communication between switches II and III, which are closer together in the structures of GTPS-bound versus GDP-bound  subunits, could be involved. In the structure of _s·GTPS (Sunahara et al., 1997), the side chain of the switch III residue E259 is hydrogen-bonded to the backbone nitrogen of the switch II residue R228, and the side chain of the switch II residue R231 is hydrogen-bonded to the side chain of E268 in the 3 helix, adjacent to switch III. Substitution of a histidine for R231 in switch II of _s leads to a decrease in receptor-mediated activation (Iiri et al., 1997).

Our results also suggest that interdomain interactions may regulate the basal activity of _s, because the basal activity of _sis is elevated. From the experiments presented here, we cannot determine whether the increased basal activity of _sis is due to increased nucleotide exchange or decreased GTPase activity. However, because the helical domain of _s, when expressed on its own and reconstituted with the GTPase domain of _s, stimulates GTPase activity and promotes binding of GTPS (Markby et al., 1993), interactions across the interface probably are important for both aspects of guanine nucleotide handling.

Other studies support the idea that interdomain communication plays a role in receptor-catalyzed G protein activation. A salt bridge interaction between D173 in the helical domain and K293 in the GTPase domain of _s is required for receptor-mediated activation (Codina and Birnbaumer, 1994). A mutation within region 1 that substitutes tryptophan for arginine at position 258 in _s, R258W, was found in a patient with pseudopseudohypoparathyroidism, and biochemical analysis revealed that although the mutant activated adenylyl cyclase normally in response to GTPS, it exhibited attenuated responses to AlF₄ and the -adrenergic receptor (Warner DR and Weinstein LS, personal communication). Regions 1 and 2 may be important for receptor-mediated activation of other  subunits as well because a segment of 16 (residues 220-240), which overlaps with regions 1 and 2, contributes to the specificity of activation by the C5a receptor (Lee et al., 1995).

Our study suggests that complementary interactions between  subunit residues across the interface between the GTPase and helical domains play a role in facilitating receptor-mediated activation. Biochemical studies using purified _s mutant proteins with substitutions in these regions and cell lines that stably express these _s mutants will be required to more fully understand the nature of these intramolecular interactions and the mechanism by which receptor-catalyzed conformational changes lead to G protein activation.