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1.
D R Brandt  E M Ross 《Biochemistry》1986,25(22):7036-7041
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+.  相似文献   

2.
T Asano  S E Pedersen  C W Scott  E M Ross 《Biochemistry》1984,23(23):5460-5467
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)  相似文献   

3.
T W Lee  R Seifert  X Guan  B K Kobilka 《Biochemistry》1999,38(42):13801-13809
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.  相似文献   

4.
Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity   总被引:14,自引:0,他引:14  
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.  相似文献   

5.
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.  相似文献   

6.
D C May  E M Ross 《Biochemistry》1988,27(13):4888-4893
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
T Asano  E M Ross 《Biochemistry》1984,23(23):5467-5471
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.  相似文献   

10.
GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.  相似文献   

11.
We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.  相似文献   

12.
We have previously shown that the beta-adrenergic receptor (beta-AR) stimulates activity of the ubiquitous Na-H exchanger (NHE-1) independently of changes in cAMP accumulation and independently of a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). To further investigate the potential role of a GTP-binding protein in coupling the beta-AR to NHE-1, we have used a recently available nonhydrolyzable GTP analog, "caged" guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), to study time-dependent effects of GTP on NHE-1 in intact cells. By monitoring intracellular pH (pHi) in cells loaded with the fluorescent pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, we determined NHE-1 activity in primary cultures of canine enteric endocrine cells, which express an endogenous beta-AR, and in mouse L cells stably transfected with either the wild type hamster beta 2-AR or a mutant construct of the hamster beta 2-AR containing a deletion in amino acid residues 222-229. This D(222-229)beta 2-AR is functionally uncoupled from Gs and adenylylcyclase. In all three cell types, NaF and GTP gamma S induced an increase in activity of the exchanger, determined by assessing the rate of pHi recovery from an acute intracellular acid load (dpHi/dt). This increase in pHi recovery was dependent on extracellular Na+ and sensitive to the amiloride analog ethylisopropylamiloride. GTP gamma S, but not NaF, also increased beta-adrenergic stimulation of resting NHE-1 activity. The alkalinization in response to isoproterenol was reversed by propranolol in the absence, but not the presence, of GTP gamma S and was completely blocked by GDP beta S. The ability of guanine nucleotides to regulate beta-adrenergic activation of NHE-1 in cells expressing the mutant D(222-229)beta 2-AR suggests that functional coupling of the beta-AR to NHE-1 may be mediated by a GTP-binding protein other than Gs.  相似文献   

13.
Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [K?nig, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].  相似文献   

14.
Muscarinic cholinergic receptors (mAChR) purified from chick heart were phosphorylated by protein kinase C (PKC) and reconstituted with the purified GTP-binding regulatory protein Go. The effects of PKC phosphorylation on the interaction of mAChR with Go were assessed by monitoring for agonist-stimulated guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) binding to Go, agonist-stimulated GTPase activity of Go, and the capability of Go to induce high affinity agonist binding to mAChR. Both the receptor-stimulated GTP gamma S binding and GTPase activity of Go were markedly diminished as a result of PKC-mediated phosphorylation of the mAChR, whereas the ability of Go to induce high affinity agonist binding to the receptors was unaffected. When mAChR were first reconstituted with Go and then subjected to phosphorylation with PKC, a complete inhibition of the phosphorylation of mAChR by PKC was observed. The inhibitory effect of Go on mAChR phosphorylation was concentration-dependent and was prevented by the presence of GTP gamma S in the reaction mixtures. Taken together, these results indicate that the phosphorylation of mAChR by PKC modulates receptor/G-protein interactions and that the ability of the receptors to act as substrates for PKC may be regulated by receptor/G-protein interactions.  相似文献   

15.
Subunit II (CyoA) of cytochrome bo3 oxidase, which spans the inner membrane twice in bacteria, has several unusual features in membrane biogenesis. It is synthesized with an amino-terminal cleavable signal peptide. In addition, distinct pathways are used to insert the two ends of the protein. The amino-terminal domain is inserted by the YidC pathway whereas the large carboxyl-terminal domain is translocated by the SecYEG pathway. Insertion of the protein is also proton motive force (pmf)-independent. Here we examined the topogenic sequence requirements and mechanism of insertion of CyoA in bacteria. We find that both the signal peptide and the first membrane-spanning region are required for insertion of the amino-terminal periplasmic loop. The pmf-independence of insertion of the first periplasmic loop is due to the loop's neutral net charge. We observe also that the introduction of negatively charged residues into the periplasmic loop makes insertion pmf dependent, whereas the addition of positively charged residues prevents insertion unless the pmf is abolished. Insertion of the carboxyl-terminal domain in the full-length CyoA occurs by a sequential mechanism even when the CyoA amino and carboxyl-terminal domains are swapped with other domains. However, when a long spacer peptide is added to increase the distance between the amino-terminal and carboxyl-terminal domains, insertion no longer occurs by a sequential mechanism.  相似文献   

16.
We developed a novel method to quantitatively measure GTP gamma S binding to specific G proteins in crude membranes using G-protein antibodies. The basic strategy was that the materials were initially incubated with [35S]GTP gamma S at 37 degrees C. After 4 degrees C incubation in the wells of an ELISA plate precoated with G-protein antibodies, the radioactivity of each well was counted. This method, using an anti-Gi antiserum and an anti-Gs antiserum, quantitatively and specifically detected the binding of GTP gamma S to purified Gi2 and Gs. In S49 cell membranes, GTP gamma S binding to immunoreactive Gs was observed in a time-dependent manner that obeyed first-order kinetics, and the rate constant was stimulated approximately twofold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTP gamma S binding to specific G proteins in cell membranes.  相似文献   

17.
Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form Gs alpha instead of the wild-type protein. The mutant Gs alpha protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein alpha subunit from the beta gamma subunit complex (Miller, R.T., Masters, S.B., Sullivan, K.A., Beiderman, B., and Bourne, H.R. (1988) Nature 334, 712-715). G226A Gs alpha was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) suggests that G226A Gs alpha is incapable of assuming the conformation necessary for high affinity binding of Mg2+ to the alpha subunit-GTP gamma S complex. Associated changes include the failure of Mg2+ and GTP gamma S to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of alpha from beta gamma. However, the GTPase activity of the mutant protein is near normal (at high Mg2+ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49V Gs alpha. Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A Gs alpha, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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