Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase

The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Al3+ plus F- on the catecholamine-stimulated GTPase activity of purified and reconstituted Gs

The effects of Al3+ and F- on the catecholamine-stimulated GTPase cycle were studied by using reconstituted phospholipid vesicles that contained purified beta-adrenergic receptor and the stimulatory GTP-binding protein of the adenylate cyclase system, Gs. Al3+/F- activated reconstituted Gs to levels previously reported for detergent-solubilized, purified Gs, although both activation and deactivation were faster in the reconstituted preparation. Under these conditions, Al3+/F- did not inhibit by more than 15% the beta-adrenergic agonist-stimulated GTPase activity of the vesicles nor did it significantly inhibit the rates of GTP binding, GTP hydrolysis, or GDP release. When Mg2+ (50 mM) was used instead of agonist to promote GTP hydrolysis in the receptor-Gs vesicles, Al3+/F- was found to inhibit GTP gamma S binding, GDP release, and steady-state GTPase activity to unstimulated levels. These data can be interpreted as indicating that the receptor catalyzes nucleotide exchange by Gs faster or more efficiently than does Mg2+.     

Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists

Treatment of beta-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity, and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTP gamma S binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on Gs alone. Thus, the site of action of DTT appears to be on the beta-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes.     

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.     

Catecholamine-stimulated guanosine 5'-O-(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase: kinetic analysis in reconstituted phospholipid vesicles

The stimulatory GTP-binding protein of adenylate cyclase, Gs, and beta-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasiirreversible binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gs, equivalent to Gs activation by nucleotide, was studied with respect to the stimulation of this process by beta-adrenergic agonists and Mg2+. The rate of GTP gamma S binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+ concentrations. In the absence of agonist, the apparent first-order rate constant, kapp, was 0.17-0.34 min-1 and did not vary significantly with the concentration of nucleotide. At 50 mM MgCl2, kapp increased somewhat, to 0.26-0.41 min-1, and remained invariant with the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At 10(-9) M GTP gamma S, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kapp. However, kapp measured in the presence of isoproterenol increased as an apparently saturable function of the GTP gamma S concentration, such that isoproterenol caused a 17-fold increase in kapp at 1 microM GTP gamma S. The effect of isoproterenol on kapp also appeared to saturate at high isoproterenol concentration, yielding a kapp approximately 6 min-1 at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity Gs-GTP gamma S complex to the stable activated state.     

Rapid binding of guanosine 5'-O-(3-thiotriphosphate) to an apparent complex of beta-adrenergic receptor and the GTP-binding regulatory protein Gs

When reconstituted phospholipid vesicles that contain purified beta-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the typical receptor-stimulated GTP gamma S binding reaction was preceded by an even more rapid burst of GTP gamma S binding. This burst was studied in detail at 0 degree C. The rate of the burst was second order in nucleotide and Gs [k assoc approximately 2 X 10(7) (M.min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 approximately 15 min at 0 degree C in the presence of agonist and decayed by approximately 3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at at 30 degrees C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.     

GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates

The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used to quantitate the total concentration of Gs. 1) GTPase activity was a saturable function of the concentration of GTP, with Km = 0.3 microM. MgCl2 monotonically increased the activity. The maximum observed turnover number was about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t 1/2 less than or equal to 5 s at 30 degrees C and t 1/2 approximately 1 min at 0 degrees C. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. 3) The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concentrations of either GTP or MgCl2 (above 0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was constant at 0.3-0.4. 4) The rate of dissociation of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t 1/2 of about 1 min. 5) Lubrol 12A9 potently inhibited the GTPase reaction and the dissociation of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. 6) The beta and gamma subunits of Gs markedly inhibited the dissociation of GDP from Gs in contrast to their ability to stimulate the dissociation of GTP gamma S. 7) GDP, GTP gamma S, and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively inhibited the accumulation of Gs-GDP. GTP gamma S and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and Pi did not inhibit. These data are interpretable in terms of the coexistence of two specific mechanistic pathways for the overall GTPase reaction.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Carbamylcholine inhibits beta-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the beta gamma subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free alpha s subunit. In the present study, Gs protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this beta-adrenergic receptor-coupled Gs protein function. Pretreatment of the cardiac ventricle membrane with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.     

Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes

We have recently reported (Ransn?s, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.     

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.     

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein

A membrane-bound protein cofactor (ARF) is required for the cholera toxin-dependent ADP-ribosylation of the stimulatory regulatory component (Gs) of adenylate cyclase. Improved methods for the purification of ARF from bovine brain are described. ARF has a high-affinity binding site for guanine nucleotides. Binding of GTP or GTP gamma S to ARF is necessary for the activity of the cofactor; GDP X ARF does not support ADP-ribosylation of Gs. Although the protein as purified contains stoichiometric amounts of GDP, GTPase activity of isolated ARF was not detected. Cholera toxin-dependent activation of adenylate cyclase thus requires two guanine nucleotide binding proteins.     

The stimulatory G protein of adenylyl cyclase, Gs, also stimulates dihydropyridine-sensitive Ca2+ channels. Evidence for direct regulation independent of phosphorylation by cAMP-dependent protein kinase or stimulation by a dihydropyridine agonist

We demonstrated recently that purified preparations of Gs, the stimulatory G protein of adenylyl cyclase, can stabilize Ca2+ channels in inside-out cardiac ventricle membrane patches stimulated prior to excision by the beta-adrenergic agonist isoprenaline or by the dihydropyridine agonist Bay K 8644 and that such preparations of Gs can restore activity to spontaneously inactivated cardiac Ca2+ channels incorporated into planar lipid bilayers (Yatani, A., Codina, J., Reeves, J.P., Birnbaumer, L., and Brown, A.M. (1987) Science 238, 1288-1292). To test whether these effects represented true stimulation and to further identify the G protein responsible, we incorporated skeletal muscle T-tubule membranes into lipid bilayers and studied the response of their Ca2+ channels to G proteins, specifically Gs, and manipulations known to be specific for Gs. In contrast to cardiac channels, incorporated T-tubule Ca2+ channels exhibit stable average activities over prolonged periods of time (up to 20 min at room temperature), allowing assessment of possible effects of G proteins under steady-state assay conditions. We report that exogenously added human erythrocyte GTP gamma S (guanosine 5'-O-(3-thiotriphosphate]-activated Gs (Gs) or its resolved GTP gamma S-activated alpha subunit (alpha s) stimulate T-tubule Ca2+ channels by factors of 2-3 in the presence of Bay K 8644, and of 10-20 in the absence of Bay K 8644 and that they do so in a manner that is independent of concurrent or previous phosphorylation by cAMP-dependent protein kinase. Activation of purified Gs by cholera toxin increases both its adenylyl cyclase stimulatory and its Ca2+ channel stimulatory effects. Ca2+ channels previously stimulated by the combined actions of Bay K 8644 and cAMP-dependent protein kinase still respond to Gs. We conclude that the responses seen are due to Gs rather than a contaminant, that the effect on Ca2+ channel activity is that of a true stimulation, akin to that on adenylyl cyclase, and show that a given G protein may regulate more than one effector system.     

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.     

Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.

beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.     

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.     

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.     

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.