Receptor-Mediated Activation of Gsa: Evidence for Intramolecular Signal Transduction

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of as (residues 236–394) to identify residues that play a role in receptormediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc S49 lymphoma cells, which lack endogenous as. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an a helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding, 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 as with that of ai2 in an as/ai2/as chimera corrects the defect in receptor-mediated activation caused by ai2 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. 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). Thea, b, and g 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 a subunit, resulting in dissociation of azGTP from bg, each of which can transmit signals to effectors. Hydrolysis of GTP by the a subunit regulates the timing of deactivation and reassociation of a with bg. 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, Gs mediates stimulation of adenylyl cyclase by b-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 GTPgS-bound (active) (Noel et al., 1993; Coleman et al., 1994) and GDP-bound (inactive) (Lambright et al., 1994; Mixon et al., 1995) asubunits and of abg heterotrimers (Wall et al., 1995; Lambright et al., 1996). The a subunits consist of two domains, a GTPase domain that resembles the oncogene protein, p21, and a helical domain consisting of a 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 GTPgS-bound and GDP-bound a subunits reveals three regions in the GTPase domain (switches I–III) that change conformation, which could be involved in the activation process. All a subunit residues involved in associating with receptors and with bg, which is required for receptor-mediated activation (Fung, 1983), have not been identified. X-ray crystal structures of the abg complex (Wall et al., 1995; Lambright et al., 1996) showed that two a subunit regions contact the b subunit, the amino-terminal a 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 a subunit in interaction with receptors. However, the locations of these bg and receptor-interacting residues, which are disThis research was supported by National Institutes of Health Grant GM50369 to C.H.B. ABBREVIATIONS: GTPgS, guanosine-59-O-(3-thio)triphosphate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEK, human embryonic kidney. 0026-895X/98/060981-10$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:981–990 (1998). 981 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from tant from the domain cleft, have not revealed the mechanism of receptor-mediated G protein activation. We exploited the differences in receptor specificities of as and ai2, which are relatively divergent members of the a subunit family, sharing ;40% amino acid identity, to identify additional a 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 b-adrenergic receptor regulates ai2 is ;10% of that seen for as (Rubenstein et al., 1991). The as residues that specify interaction with the b-adrenergic receptor have been localized to the carboxyl-terminal 40% of as by means of an ai2/as chimera (Masters et al., 1988). By examining a panel of as mutants in which clusters of residues were replaced by ai2 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 a 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 as with that of ai2 in an as/ai2/as chimera corrects the defect in receptor-mediated activation caused by ai2 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 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). [H]Adenine was obtained from Amersham (Little Chalfont, UK). Construction of a subunit mutants and chimeras. The as mutant constructs were generated from rat as cDNA (Jones and Reed, 1987). Chimeric a subunits were constructed from rat as cDNA and mouse ai2 cDNA (Sullivan et al., 1986). Subcloning and mutagenesis procedures were verified by restriction enzyme analysis and DNA sequencing. All a subunit constructs produced in this study contain an epitope, referred to as the EE epitope (Grussenmeyer et al., 1985), which was generated by mutating as residues DYVPSD (189–194) to EYMPTE and ai2 residues SDYIPTQ (166–172) to EEYMPTE (single-letter amino acid code; mutated residues are underlined). This epitope does not affect the ability of as to activate adenylyl cyclase in response to stimulation by the b-adrenergic receptor (Wilson and Bourne, 1995). The amino acid substitutions in the as mutant constructs produced in this study are shown in Figs. 1 and 5. To generate these constructs, the as cDNA was subcloned into the expression vector pcDNA I/Amp as a HindIII fragment. The mutations in as(1), as(2), as(10), as(11), as(12), as(13), as(14), as(15), as(16), and as(17) were introduced into the as cDNA by oligonucleotide-directed in vitro mutagenesis (Kunkel et al., 1987) using the BioRad Muta-Gene kit (Hercules, CA). The mutations in as(3), as(4), as(5), as(6), as(7), as(8), and as(9) were introduced into the as cDNA by ligating BamHI fragments from previously generated constructs (Berlot and Bourne, 1992) that contained these mutations into as in place of the analogous fragment. To produce as(2 1 6), as(6) was digested with BglII and EcoRV to yield a fragment containing the as(6) mutations, which was ligated into as(2) in place of the analogous fragment to produce an as cDNA containing both the as(2) and as(6) mutations. Because receptor-dependent stimulation of cAMP synthesis was used to measure receptor-mediated activation of the mutant as constructs, any effects the mutations might have on receptor-independent cAMP synthesis were controlled for by measuring basal cAMP accumulation in response to parallel constructs (the asRC versions), in which substitution of cysteine for the arginine at position 201 (Landis et al., 1989) inhibits GTPase activity and causes constitutive activation. asRC versions of the constructs were produced by ligating BamHI fragments containing the mutations into asRC in place of the analogous fragment. An asis chimera, in which as residues 62–235 are replaced by the homologous ai2 residues, was produced from as and an ais chimera, in which as residues 1–235 are replaced by the homologous ai2 residues. The ai2 cDNA was subcloned into pcDNA I/Amp as an EcoRI fragment. To generate ais, the as cDNA was digested with BamHI and the fragment encoding as residues 236–394 and the 39 untranslated region of as was ligated into ai2 in place of the analogous fragment. Then, asis was generated using polymerase chain reactions that produced DNA fragments with overlapping ends that were combined subsequently in a fusion polymerase chain reaction (Horton et al., 1989). An RC version of asis, asisRC, was produced by substituting cysteine for Arg179, which causes constitutive activation of ai2 by inhibiting GTPase activity (Wong et al., 1991). asisRC was derived from aisRC, which was generated by ligating the BamHI as fragment encoding as residues 236–394 and the 39 untranslated region of as into ai2RC in place of the analogous fragment. To produce asisRC, ai2RC was digested with DraIII to yield a fragment containing the RC mutation, which was ligated into asis in place of the analogous fragment. To introduce the as(1), as(2), as(6), and as(2 1 6) mutations into asisRC and asis, these mutations were first subcloned as BamHI fragments into ai2RC and ai2 in place of the analogous fragments. Digestion of these ai2RC and ai2 constructs with DraIII yielded fragments containing the as(1), as(2), as(6), and as(2 1 6) mutations with or without the RC mutation, respectively, which were ligated into asispcDNA I/Amp in place of the analogous fragments to produce asisRC and asis constructs, respectively, containing the desired mutations. 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. The a subunit constructs were introduced into cyc cells (2 3 10 cells in 1.0 ml of 20 mM HEPES-buffered minimal essential medium with Earle’s salts without bicarbonate) by electroporation at room temperature using a GIBCO BRL Cell-Porator (capacitance setting, 1600 mF; voltage setting, 250 V; Grand Island, NY). After electroporation, the cells were added to 4.0 ml of Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated horse serum in 60-mm tissue culture dishes. At 24 hr after electroporation, the cells were labeled with 12 mCi/ml [H]adenine. Then, 24 hr later, cAMP accumulation was measured. The cells first were washed in assay me982 Marsh et al. at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from dium (20 mM HEPES-buffered Dulbecco’s modified Eagle’s medium without bicarbonate). The cells were transferred to 24-well plates and incubated at 37° for 30 min in the same medium containing 1 mM concentration of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine, with or without the addition of 0.1 mM isoproterenol (a saturating stimulus). During this incubation, the cells attached to the wells. Reactions were terminated by aspiration and the immediate addition of 5% trichloroacetic acid plus 1 mM concentration each of ATP and cAMP. Nucleotides were separated on ion exchange columns (Salomon et al., 1974). cAMP accumulation was expressed as [H]cAMP/([H]ATP 1 [H]cAMP) 3 1000. Results Panel of as mutant constructs for studying receptormediated activation. The 159-residue carboxyl-terminal segment of as (residues 236–394), which specifies interaction with the b-adrenergic receptor (Masters et al., 1988), contains 59 amino acids that are identical in the sequence of ai2 and therefore do not specify interaction with this receptor. We previously demonstrated that mutations of residues in three adjacent regions of the a subunit structure, the a2/b4, Fig. 1. Panel of as mutant constructs. All mutations are within the carboxyl-terminal 40% of the rat as sequence (Jones and Reed, 1987), which is depicted in two sections (residues 236–314 and 315–394). Top lines, sequence of as. Second lines, mouse ai2 (Sullivan et al., 1986) in the corresponding region. Dashes, residues identical to those of as. Numbered sequences, individual mutant constructs. For each as mutant construct, mutated residues are shown by the single-letter amino acid code. Dashes, residues identical to those of as. Underlined sequences (located in regions 1–4), mutations that disrupted receptor-mediated activation of as. Elements of secondary structure, determined from the structure of aszGTPgS (Sunahara et al., 1997), are indicated: a, ahelices; b, bstrands; and dashes, turns and loops. Regions that switch conformation between the GDP-bound and GTPgS-bound forms of at (Lambright et al., 1994) and ai1 (Mixon et al., 1995) (switches II and III) are indicated. The alignment shown, which is based on the recently solved structure of aszGTPgS (Sunahara et al., 1997), differs from our previous alignment (Berlot and Bourne, 1992) in the location of an insertion of as sequence relative to that of ai2. In the previous alignment, as residues 324–336 were inserted between ai2 residues 299 and 300. Receptor-Mediated Activation of Gsa 983 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from a3/b5, and a4/b6 loops (Noel et al., 1993), disrupt the ability of as to activate adenylyl cyclase (Berlot and Bourne, 1992). Because the current study of receptor-mediated activation of as 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 ai2 homologs or to alanines using 15 as mutant constructs (Fig. 1). Transient transfection assay for receptor-mediated activation of as. To test the abilities of mutant as proteins to be activated by the b-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 as (Harris et al., 1985). Basal cAMP levels in cells transfected with 10–90 mg of vector containing as varied linearly in proportion to the plasmid dose (Fig. 2A). Stimulation of these as-transfected cells with the b-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 as mutants in which Arg201 is mutated to cysteine. The as containing this mutation, asRC, exhibits constitutive activation due to inhibited GTPase activity (Landis et al., 1989). As with as-transfected cells, basal cAMP levels in cyc cells transfected with 10–90 mg of vector containing asRC varied linearly in proportion to the plasmid dose (Fig. 2B). We initially measured receptor-independent cAMP accumulation due to the asRC mutants using 30 mg of plasmid. At this plasmid dose, the activities of some of the asRC mutants were reduced compared with that of asRC. The expression levels of transiently expressed as proteins in cyc 2 cells were not high enough to be detected using an immunoblot but could be determined in transiently transfected HEK 293 cells. asRC mutants with reduced activities in cyc 2 cells had similarly reduced activities in HEK 293 cells. The activities of these asRC mutants directly correlated with their expression levels as determined by immunoblotting of HEK 293 cell membranes (data not shown). Because the activities of both as and asRC in cyc 2 cells were directly proportional to the amount of transfected plasmid (Fig. 2), it was possible to normalize the expression levels of these as and asRC mutant constructs to that of as and asRC by transfecting with increased amounts of plasmid. To compare receptor-dependent activation of as mutants with that of as, we identified plasmid doses for which the activities of the asRC mutants were similar to those for 30 mg of the asRC-containing plasmid (Fig. 3B). At these plasmid doses, we compared receptor-dependent cAMP accumulation due to the corresponding as mutants with that for 30 mg of the as-containing plasmid (Fig. 3A). An assumption underlying this normalization procedure is that the substitutions in the mutant constructs have similar effects on the expression levels of as and asRC. This assumption is supported by the observation that the basal activity of asRC is ;10-fold greater than that of as (Figs. 2 and 3) and the basal activities of each of the asRC mutants also are ;10-fold greater than those of the corresponding as mutants (Fig. 3). Of the four regions of sequence in which mutations disrupted receptor-mediated activation (see below), all except one of them (region 2) included at least one cluster of residues that did not decrease expression level. Studies using stably transfected cells confirmed that receptor-mediated activation of the region 2 mutant was decreased (see below). Thus, although a 9-fold range of plasmid doses was used for the transient transfection assay, our conclusions do not depend on the activities in this assay of the constructs with low expression levels. Receptor-mediated activation of as mutant constructs. Receptor-stimulated cAMP accumulation due to 9 of the 15 as mutant constructs was similar to that of as (Fig. 3A). The other 6 constructs produced reduced receptor-dependent increases in cAMP levels and delineated four regions of sequence containing seven or fewer as mutations that disrupt the ability of as to be activated by the b-adrenergic receptor (Fig. 1). Region 1, defined by as(1) and as(2), contains V247, S250, S252, N254, M255, I257, and R258. Region 2, defined by as(6), contains G304, K305, and K307-Y311. Region 3, defined by as(11) and as(12), contains V367, E370, and I372-R374. Region 4, defined by as(15), contains R389E392 and L394. Because as(6) in region 2 was poorly expressed in transiently transfected cells, we established lines of cyc cells Fig. 2. Transient transfection assay for receptor-mediated activation of as. A, cAMP accumulation in cyc 2 cells electroporated with the indicated doses of vector containing as. 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 asRC. For the 0-mg points, 30 mg of vector was used. cAMP levels in [H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean 6 standard error of three independent experiments. 984 Marsh et al. at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from stably transfected with this as mutant construct to investigate further the role of region 2 in receptor-mediated activation. As expected from the results of the transient transfection assay, the expression levels obtained in as(6)-expressing lines, as determined by immunoblotting, were lower than those in as-expressing lines. In addition, a defect in receptormediated activation was seen in that isoproterenol-stimulated adenylyl cyclase activity was reduced relative to that stimulated by GTPgS (Grishina G and Berlot CH, unpublished observations). Thus, the mutations in region 2 impair receptor-dependent activation but also seem to decrease the stability of as. Mapping of mutations that block receptor-mediated activation onto the structure of a heterotrimeric G protein. Because receptors interact with abg heterotrimers, we mapped the as residues in which mutations disrupted activation by the b-adrenergic receptor onto the x-ray crystal structure of an at/ai1 chimera complexed with btgt (Lambright et al., 1996) to visualize their positions in three dimensions (Fig. 4). The recently solved structure of aszGTPgS (Sunahara et al., 1997) is very similar to the structures of atzGTPgS (Noel et al., 1993) and ai1zGTPgS (Coleman et al., 1994), indicating that the structure of at/ai1btgt is a good model for the Gs heterotrimer. Structural features unique to as that are relevant to the mutations that blocked receptormediated activation are discussed. Some of the mutations that blocked receptor-mediated activation map onto solventexposed 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 as. The residues in regions 1 and 2 are located in the GTPase domain at the interface between the GTPase and helical domains. Region 1 extends from the middle of b4 to the middle of the b4/a3 loop and overlaps with switch III, which assumes different conformations in the structures of GTPgSbound and GDP-bound a subunits (Noel et al., 1993; Coleman et al., 1994; Lambright et al., 1994; Mixon et al., 1995). Region 2 is located in the adjacent aG/a4 loop. Residues in the amino-terminal part of region 1, defined by as(1), are buried within the interior of the molecule, making contacts with other residues in the GTPase domain. In the carboxylterminal part of region 1, the side chains of N254, M255, and R258 in as(2) are in close proximity to residues in the helical domain. The residues in region 2 immediately precede a 12-residue insertion of sequence in as relative to at and ai. However, comparison of the structures of aszGTPgS (Sunahara et al., 1997), atzGTPgS (Noel et al., 1993), and ai1zGTPgS (Coleman et al., 1994) reveals that the orientation of region 2 with respect to the helical domain is the same in all of the a subunits. In the structure of aszGTPgS (Sunahara et al., 1997), the 12-residue insertion is located farther from the interface than the location of region 2 (Fig. 4A, to the right of Region 2). Of the region 2 residues, K305 and Y311 are closest to the interface, and all of the residues except for I308 are surface-exposed. Region 3 is located near the guanine nucleotide binding pocket and includes residues in the b6/a5 loop and the beginning of a5. Three of the residues, E370, R373, and R374, are solvent-exposed, whereas two, V367 and I372 (shown in Fig. 4), are buried. V367 contacts the guanine ring of the bound nucleotide (Sunahara et al., 1997). The residues in region 4, located at the extreme carboxyl terminus, were not visualized in the at/ai1btgt structure (Lambright et al., 1996) and occupy different positions in the structures in which they were visualized. In the structure of atzGTPgS (Noel et al., 1993), this region contacts the a2/b4 loop, whereas in the structures of aszGTPgS (Sunahara et al., 1997), ai1zGDPzAlF4 zRGS4 (Tesmer et al., 1997), and ai1b1g2 (Wall et al., 1995), the extreme carboxyl terminus is distant from the rest of the a subunit. Region 4 is linked to region 3 by the a5 helix. 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 ai2 residues in as(16) specifically reduced receptor-mediated increases in cAMP production, whereas substitution of the surface-exposed residues with alanine residues in as(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 as(16), V367 is located in the b6/a5 loop, presumably in contact with the GDP, whereas I372 is near the beginning of Fig. 3. Receptor-mediated activation of mutant as proteins. A, cAMP accumulation in cyc cells containing the indicated mutants in the as context. Cells were electroporated with 20 mg of vector containing as (2), 30 mg of vector alone, 30 mg of vector containing as and as (3 and 10–15), 60 mg of vector containing as (5), 90 mg of vector containing as (1, 4, 7, and 8), 120 mg of vector containing as (9), and 180 mg of vector containing as (6). Dark gray, cAMP values from unstimulated cells. Light gray, cAMP values from cells stimulated with 0.1 mM isoproterenol. B, Receptorindependent cAMP accumulation in cyc cells containing the indicated mutants in the asRC context. For each mutant, the same amount of plasmid was used as is indicated in A for the corresponding as mutant. cAMP levels in [H]adenine-labeled cells were determined as described in Experimental Procedures. All values represent the mean 6 standard error of at least three independent experiments. Receptor-Mediated Activation of Gsa 985 at A PE T Jornals on M ay 9, 2017 m oharm .aspeurnals.org D ow nladed from