Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.     

Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.     

Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity

We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.     

Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.     

Cholera toxin induces cAMP-independent degradation of Gs

Cholera toxin stimulates adenylyl cyclase by catalyzing ADP-ribosylation of the alpha chain (alpha s) of Gs, a guanine nucleotide binding regulatory protein. In a rat pituitary cell line, GH3, the toxin-induced increase in GTP-dependent adenylyl cyclase activity is maximal at 1 h; adenylyl cyclase remains elevated for at least 32 h. Surprisingly, cholera toxin also induces a 74-95% decrease in the amount of immunoreactive alpha s in the same cells, as assessed on immunoblots probed with either of two antisera directed against separate alpha s peptide sequences. The decrease in immunoreactive alpha s, which begins after 1 h of toxin treatment and is complete by 8 h, is accompanied by a comparable decrease in the amount of biochemically active alpha s, as assessed by its ability to complement the biochemical defect of alpha s-deficient S49 cyc- membranes. Cholera toxin induces similar decreases in alpha s in wild type S49 lymphoma cells, in S49 kin- mutants, which lack cAMP-dependent protein kinase, and in S49 H21 a mutants, in which alpha s is unable to assume an active conformation upon binding GTP. The toxin-induced decrease in alpha s is somewhat temperature-dependent, but is not blocked by agents that increase lysosomal pH or by colchicine, which promotes breakdown of microtubules. alpha s in detergent-solubilized GH3 membranes is susceptible to proteolysis by an endogenous protease; this susceptibility is markedly increased in membranes from cells previously exposed to cholera toxin for 1 h. Taken together, these results suggest that cholera toxin-induced covalent modification of alpha s marks the protein for accelerated degradation. In addition, the persistence of elevated GTP-dependent adenylyl cyclase activity despite loss of a substantial fraction of alpha s suggests that the amount of alpha s membranes is greater than the amount necessary for maximal activation of cAMP synthesis by cholera toxin.     

Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein

Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.     

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc- variants are prevented by islet-activating protein

cyc--Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc- cells with islet-activating protein (IAP), which causes ADP-ribosylation of an Mr 40 000 polypeptide in cyc- membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTP gamma S. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.     

Activating mutations in the NH2- and COOH-terminal moieties of the Gs alpha subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation.

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.     

Clonal variation of adenylyl cyclase activity in a rat tumor cell line caused by change in G protein-catalytic unit interaction

Two subclones of the rat XC cell line characterized by different morphology exhibited quite different adenylyl cyclase responses upon various stimulations. Upon treatment with cholera toxin, clone RK1 accumulated a high level of intracellular cAMP thereby changing its polygonal morphology to an elongated morphology, while the other clone, LK1, with a fibroblastic morphology, failed to increase the intracellular cAMP and remained morphologically unchanged. When membrane fractions derived from these two clones were stimulated with 10 microM forskolin, 10 microM GTP gamma S, or 10 mM NaF, five- to 20-fold more cAMP was accumulated in RK1-derived membranes than in LK1-derived membranes. With the same membrane fractions, upon treatment with Mn(+)+, which directly stimulates the catalytic unit, a high level of cAMP was accumulated both in RK1 and LK1, indicating that the catalytic function inducible by Mn(+)+ was similar in both clones. There was no significant difference in the level of expression of G protein alpha 2, alpha i (at least alpha i1 and alpha i2), and beta subunits between LK1 and RK1. Cholate extracts of the membrane proteins of LK1 and RK1 reconstituted the adenylyl cyclase activity of the cyc- variant of S49 lymphoma cells to the same level. Therefore, it is inferred that the defect in LK1 resides in the interaction of stimulatory G proteins and the actual catalyst.     

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.     

Abnormal Gs function in mitral valve prolapse dysautonomia is not associated with abnormal alpha S cDNA sequence.

We have previously shown that a subset of patients with mitral valve prolapse and hyperadrenergic symptoms has enhanced isoprenaline-stimulated beta-adrenergic receptor high-affinity state formation (supercoupling) and increased adenylyl cyclase activity due to abnormal signal transduction by the stimulatory guanine nucleotide regulatory protein (Gs). In this study we looked for an alteration of the nucleotide coding sequence of the gene for alpha s, the subunit of Gs that is directly responsible for formation of the high affinity state and adenylyl cyclase activation, by cloning and sequencing the alpha s cDNA from neutrophils of 4 symptomatic patients and 1 control. No difference was observed between patients and control in the alpha s cDNA sequence. The splice variant concentrations in the fully expressed protein were also grossly unchanged in five patients and four controls. These data show that a primary alteration of the alpha s gene coding sequence is not responsible for defective Gs-associated signal transduction in dysautonomic MVP patients, and suggest that the molecular lesion could be an abnormal posttranslational modification of alpha s, a defect in the beta or gamma subunits of Gs, or an unusual interaction between the subunits in the Gs of these patients.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-adrenergic receptor: a potential mechanism of G protein activation

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.     

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.     

A dominant negative G alpha s mutant is rescued by secondary mutation of the alpha chain amino terminus.

The Gs protein alpha subunit, alpha s, stimulates the activity of adenylyl cyclase. The sequence 223Asp-Val-Gly-Gly-Gln227 in the alpha s polypeptide is predicted to interact with the gamma-phosphate of GTP and mediate the conformational change involved in alpha s activation. Mutation of the alpha s polypeptide within this region at Gly225----Thr had two demonstrative phenotypic effects when expressed in COS-1 cells: the mutant alpha s chain was ineffective in activating adenylyl cyclase and inhibited in a concentration-dependent manner the beta-adrenergic receptor stimulation of cAMP synthesis. Thus, the Gly225----Thr mutation alters the ability of GTP to activate the alpha s chain and when overexpressed the mutant polypeptide exerts a dominant negative phenotype. Mutation at the amino terminus which creates a constitutively active alpha s rescued the inhibited state of the Gly225----Thr mutant when both mutations were encoded in the same polypeptide. This finding defines the amino terminus as a functional regulatory domain controlling the properties of the GTP/GDP binding site of G protein alpha subunit polypeptide chains.