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+.     

Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity

beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.     

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.     

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.     

Interaction of the beta-adrenergic receptor with Gs following delipidation. Specific lipid requirements for Gs activation and GTPase function

Preparations of beta-adrenergic receptor and Gs from turkey erythrocytes were delipidated by previously developed procedures. Three synthetic phospholipids, dioleoylglycerophosphoethanolamine, dioleoylglycerophosphocholine and dioleoylglycerophosphoserine plus an unphosphorylated lipid, were all required to restore receptor-mediated activation of Gs by GTP[gamma S]. The same lipids were necessary for the reconstitution of the isoproterenol-enhanced GTPase. The requirement for the unphosphorylated lipid could be fulfilled by 1-mono-oleoyl glycerol, alpha-tocopherol or oleic acid. Cholesterol hemisuccinate further enhanced the receptor-mediated activity of the relipidated system when present in addition to the lipids specified above. Cholesterol hemisuccinate had no effect on the basal rate of Gs activation and depressed the basal GTPase. It is therefore suggested that cholesterol hemisuccinate affects the receptor or the coupling of the receptor to Gs. In the system relipidated with the three dioleoyl phospholipids, plus alpha-tocopherol and cholesterol hemisuccinate, the initial rate of Gs activation per mole receptor appeared to be considerably higher than in the native turkey erythrocyte membrane.     

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.     

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.     

The hydrophobic tryptic core of the beta-adrenergic receptor retains Gs regulatory activity in response to agonists and thiols

The function of structural domains of the beta-adrenergic receptor were probed by studying the ability of tryptic fragments of the receptor to catalyze the binding of guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) to the GTP-binding regulatory protein, Gs. beta-Adrenergic receptor purified from turkey erythrocytes was treated with trypsin under nondenaturing conditions. Such treatment decreased beta-adrenergic ligand binding activity by only 15-25%. Active components of the limit digest were repurified by affinity chromatography on alprenolol-agarose and then reconstituted with purified Gs into unilamellar phospholipid vesicles. After reconstitution, the proteolyzed receptor was able to catalyze agonist-stimulated binding of GTP gamma S to Gs at a rate and extent equivalent to that of the nonproteolyzed receptor. The proteolyzed receptor was also partially activated upon reduction by dithiothreitol, as previously reported for the intact receptor (Pedersen, S.E., and Ross, E.M. (1985) J. Biol. Chem. 260, 14150-14157). The repurified, active tryptic digest contained two detectable peptides. One, of approximately 2 X 10(4) Da, contained either four or five of the amino-terminal membrane-spanning domains plus the intervening hydrophilic loops but not the amino-terminal extracellular, glycosylated peptide. The second, of 9,000-10,000 Da, was composed essentially of the two carboxyl-terminal membrane-spanning domains and the intervening extracellular, hydrophilic loop. These data indicate that most of the large intracellular hydrophilic loop and the hydrophilic, carboxyl-terminal region of the receptor are not necessary for the agonist-stimulated regulation of Gs.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.     

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.     

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.     

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.     

Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone-dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl-polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol-induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(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.     

Selectivity of the beta-adrenergic receptor among Gs, Gi's, and Go: assay using recombinant alpha subunits in reconstituted phospholipid vesicles.

The selective regulation of Gs (long and short forms), Gi's (1, 2, and 3), and Go by the beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta gamma subunits, and individual recombinant G-protein alpha subunits that were expressed in and purified from Escherichia coli. Receptor and beta gamma subunits were incorporated into phospholipid vesicles, and the alpha subunits bound to the vesicles stoichiometrically with respect to beta gamma. Efficient regulation of alpha subunit by receptor required the presence of beta gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP gamma S binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted alpha s (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both alpha i,1 and alpha i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha o and alpha i,2 was weak, and stimulation of alpha o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

Function of the delipidated beta-adrenergic receptor appears to require a fatty acid or a neutral lipid in addition to phospholipids

Detergent-solubilized preparations of the beta-adrenergic receptor (R) and of the guanyl nucleotide binding proteins (Gs) were extensively treated to remove phospholipids and cholesterol. Reconstitution of an R-Gs system was subsequently performed in the presence of a mixture of natural phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine or the synthetic dioleoyl derivatives of the same phospholipids. In both cases, an additional lipid was required for the agonist-dependent activation of Gs. The requirement could be fulfilled by alpha-tocopherol, or by unsaturated fatty acids such as oleic acid. Inclusion of this non-phosphorylated lipid in the reconstituted system enhanced the isoproterenol-dependent activation of Gs by guanosine 5'-O-[gamma-thio]triphosphate 16-33-fold. The rate of activation was largely dependent on the addition of the agonist. Efficient functional reconstitution of R-Gs was thus achieved in a totally defined lipid system. Additional studies of the reconstituted system and of the native membrane led to the notion that the non-phosphorylated lipid plays a role in the function of the hormone-R complex.     

Studies on nucleotide and receptor regulation of Gi proteins: effects of pertussis toxin

In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of Gi proteins are affected by PT. Experiments with the Gs-deficient Gi-regulated adenylyl cyclase of cyc- S49 cell membranes indicated that PT blocks Gi activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-Gi to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of Gi. Experiments in which Gi was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of Gi, but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes.