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1.
FSH interacts with a guanine nucleotide-binding protein (G-protein)-coupled receptor, which in turn modulates signal transduction via the G-protein subunit alpha s. However, it is unknown whether FSH regulates alpha-subunit gene expression and whether G-protein alpha-subunit genes other than alpha s are modulated in FSH-stimulated signal transduction. Regulation of mRNA for alpha s and alpha i-2 was studied in primary cultures of rat Sertoli cells because these proteins are linked to the control of adenylyl cyclase. In addition, mRNA for alpha i-1 and alpha i-3 were quantified because these proteins are putatively linked to ion channels but have not been well characterized in the Sertoli cell. Northern blot analyses demonstrated that FSH induced a dose-dependent increase in steady state levels of alpha i-3 mRNA. In contrast, FSH caused a dose-dependent decrease in levels of alpha i-1 and alpha i-2 mRNA. No significant effect of FSH on alpha s mRNA levels was detectable. The time course of FSH effects showed a 75% decrease in alpha i-1 mRNA levels, a 50% decrease in alpha i-2 mRNA levels and a nearly 3-fold increase in levels of alpha i-3 mRNA between 4-6 h of treatment with 100 ng/ml FSH. Steady state levels of alpha i-1 and alpha i-2 mRNA returned to pretreatment levels after 10 h FSH treatment, while alpha i-3 mRNA returned to a new steady state level approximately 50% greater than the pretreatment level.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
Expression of a G alpha s/G alpha i chimera that constitutively activates cyclic AMP synthesis 总被引:5,自引:0,他引:5
C W Woon S Soparkar L Heasley G L Johnson 《The Journal of biological chemistry》1989,264(10):5687-5693
3.
J R Hadcock M Ros D C Watkins C C Malbon 《The Journal of biological chemistry》1990,265(25):14784-14790
The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein. 相似文献
4.
BACKGROUND: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the alpha subunits of classical G(i)-type G proteins. RESULTS: To investigate the possibility that G protein betagamma subunits, rather than alpha subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein betagamma scavengers (G(t)alpha and betaARK-C(CAAX)) to sequester free Gbetagamma dimers. We demonstrated that the injection of mRNA encoding either of these Gbetagamma scavengers induced oocyte maturation. The Gbetagamma scavengers bound an endogenous, membrane-associated Gbeta subunit, indistinguishable from Xenopus Gbeta1 derived from mRNA injection. The injection of Xenopus Gbeta1 mRNA, together with bovine Ggamma2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation. CONCLUSION: An endogenous G protein betagamma dimer, likely including Xenopus Gbeta1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gbetagamma scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gbetagamma. 相似文献
5.
Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium 总被引:79,自引:0,他引:79
Biochemical studies in vertebrate olfactory tissue indicate that certain odorants stimulate adenylyl cyclase in a GTP-dependent manner. Additionally, immunochemical and toxin-labeling studies demonstrate the presence of several GTP-binding protein (G-protein) species in vertebrate olfactory epithelium. To identify the G-protein(s) responsible for olfactory signal transduction, we screened a rat olfactory cDNA library with an oligonucleotide probe and isolated 32 recombinant clones encoding five distinct types of G-protein alpha subunits. The majority of the clones encoded G alpha s, while the remaining clones encoded G alpha o, G alpha i1, G alpha i2, and a novel species, G alpha i3. Messenger RNA corresponding to each G alpha was detectable in all tissues examined; however, the levels for a given G alpha varied in a tissue-specific manner. In olfactory tissue, G alpha s was the most abundant of these messages and in combination with the biochemical studies suggests that G alpha s is the G-protein component of the olfactory signal transduction cascade. 相似文献
6.
Mutation of glycine 49 to valine in the alpha subunit of GS results in the constitutive elevation of cyclic AMP synthesis 总被引:6,自引:0,他引:6
The G-protein GS couples hormone-activated receptors with adenylyl cyclase and stimulates increased cyclic AMP synthesis. Transient expression in COS-1 cells of cDNAs coding for the GS alpha-subunit (alpha S) or alpha S cDNAs having single amino acid mutations Gly49----Val or Gly225----Thr elevated cyclic AMP levels, resulting in the activation of cyclic AMP dependent protein kinase. Stable expression in Chinese hamster ovary cells of alpha S Val49 cDNA resulted in a small constitutive elevation of cyclic AMP that was sufficient to persistently activate cyclic AMP dependent protein kinase activity 1.5-2-fold over basal activity. Stable expression of wild-type alpha S or alpha S Thr225 in Chinese hamster ovary cells was less effective in sustaining elevated cyclic AMP synthesis and kinase activation compared to alpha SVal49. 相似文献
7.
R H Paulssen E J Paulssen K M Gautvik J O Gordeladze 《European journal of biochemistry》1992,204(1):413-418
The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C. 相似文献
8.
Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes. 相似文献
9.
The Dictyostelium discoideum developmental program is initiated by starvation and its progress depends on G-protein-regulated transmembrane signaling. Disruption of the Dictyostelium G-protein alpha-subunit G alpha 3 (g alpha 3-) blocks development unless the mutant is starved in the presence of artificial cAMP pulses. The function of G alpha 3 was investigated by examining the expression of several components of the cAMP transmembrane signaling system in the g alpha 3- mutant. cAMP receptor 1 protein, cyclic nucleotide phosphodiesterase, phosphodiesterase inhibitor, and aggregation-stage adenylyl cyclase mRNA expression were absent or greatly reduced when cells were starved without exogenously applied pulses of cAMP. However, cAMP receptor 1 protein and aggregation-stage adenylyl cyclase mRNA expression were restored by starving the g alpha 3- cells in the presence of exogenous cAMP pulses. Adenylyl cyclase activity was also reduced in g alpha 3- cells starved without exogenous cAMP pulses compared with similarly treated wild-type cells but was elevated to a level twofold greater than wild-type cells in g alpha 3- cells starved in the presence of exogenous cAMP pulses. These results suggest that G alpha 3 is essential in early development because it controls the expression of components of the transmembrane signaling system. 相似文献
10.
Three serine-to-alanine mutants of the alpha subunit of the heterotrimeric G protein G(z) (alpha(z)) were examined for their signaling properties in the presence of phorbol ester treatment. All three alpha(z) mutants resembled wild-type alpha(z) in their abilities to inhibit alpha(s)-stimulated type 6 adenylyl cyclase (AC6) and phorbol ester treatment reduced their magnitudes of inhibition. Depending on the permissive condition, the betagamma-mediated stimulation of type 2 adenylyl cyclase (AC2) was differentially regulated by alpha(z) and the three mutants. Mutation of Ser(27) but not Ser(16) of alpha(z) affected the efficient release of betagamma subunits upon receptor activation and abolished the stimulation of phosphorylated but not alpha(s)-stimulated AC2. 相似文献
11.
Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium. 总被引:8,自引:3,他引:5
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cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein. 相似文献
12.
Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity 总被引:11,自引:0,他引:11
We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes. 相似文献
13.
Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation.
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S Osawa L E Heasley N Dhanasekaran S K Gupta C W Woon C Berlot G L Johnson 《Molecular and cellular biology》1990,10(6):2931-2940
G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant. 相似文献
14.
Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob) 总被引:2,自引:0,他引:2
T W Gettys V Ramkumar R J Uhing L Seger I L Taylor 《The Journal of biological chemistry》1991,266(24):15949-15955
Messenger RNA levels for the alpha subunit of G-proteins expressed in adipocytes of lean and obese (ob/ob) mice were compared with relative levels of the encoded proteins. Using both toxin labeling and Western blots, expression of Gs alpha, Gi alpha-1, and Gi alpha-3 was decreased by approximately 2-fold in adipocytes of obese mice, while levels of Gi alpha-2 did not differ between the phenotypes. The decreases in Gi alpha-1 and Gs alpha in the obese mouse were attributed to decreased mRNA levels for these proteins. Similar mRNA levels for Gi alpha-3 were noted in both phenotypes, but Gi alpha-2 message was increased 2-fold in the obese mouse. Inhibitory regulation of adipocyte adenylylcyclase through G-proteins was evaluated by comparing the ability of R-PIA to inhibit isoproterenol-stimulated responses between the phenotypes. In spite of the decrease in Gi alpha-1 and Gi alpha-3 in adipocytes from obese mice, R-PIA inhibited adenylylcyclase, cAMP-dependent protein kinase, and lipolysis in similar fashion in both phenotypes. The GTP analog, Gpp(NH)p also inhibited forskolin-stimulated adenylylcyclase in a comparable manner, but the magnitude of the inhibition was slightly less in adipocyte membranes from obese mice. In contrast, the decrease in expression of Gs alpha was translated into substantially poorer activation of isoproterenol-stimulated responses in the obese mouse. The concentration of isoproterenol producing half-maximal activation of adenylylcyclase, protein kinase, and lipolysis did not differ between the phenotypes, but the maximal responses were much lower in cells from obese mice. Similar lipolytic potential in isolated adipocytes from each phenotype and similar total forskolin-stimulated cyclase activity in adipocyte membranes from each phenotype suggest that decreased expression of Gs alpha may contribute to the characteristic alteration in mobilization of triglycerides noted in adipocytes from obese mice. 相似文献
15.
A dominant negative G alpha s mutant is rescued by secondary mutation of the alpha chain amino terminus. 总被引:6,自引:0,他引:6
The Gs protein alpha subunit, alpha s, stimulates the activity of adenylyl cyclase. The sequence 223Asp-Val-Gly-Gly-Gln227 in the alpha s polypeptide is predicted to interact with the gamma-phosphate of GTP and mediate the conformational change involved in alpha s activation. Mutation of the alpha s polypeptide within this region at Gly225----Thr had two demonstrative phenotypic effects when expressed in COS-1 cells: the mutant alpha s chain was ineffective in activating adenylyl cyclase and inhibited in a concentration-dependent manner the beta-adrenergic receptor stimulation of cAMP synthesis. Thus, the Gly225----Thr mutation alters the ability of GTP to activate the alpha s chain and when overexpressed the mutant polypeptide exerts a dominant negative phenotype. Mutation at the amino terminus which creates a constitutively active alpha s rescued the inhibited state of the Gly225----Thr mutant when both mutations were encoded in the same polypeptide. This finding defines the amino terminus as a functional regulatory domain controlling the properties of the GTP/GDP binding site of G protein alpha subunit polypeptide chains. 相似文献
16.
S K Gupta N Dhanasekaran L E Heasley G L Johnson 《Journal of cellular biochemistry》1991,47(4):359-368
The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase. 相似文献
17.
Shpakov AO Korol'kov VI Plesneva SA Kuznetsova LA Pertseva MN 《Rossi?skii fiziologicheski? zhurnal imeni I.M. Sechenova / Rossi?skaia akademiia nauk》2003,89(7):837-850
The peptide synthesised by us: 387-394-amide (10(-7)-10(-4) M), in a dose dependent manner decreases activities of adenyl cyclase and proteinkinase A evoked by serotonine and glucagon in smooth muscles of the freshwater bivalve mollusc Anodonta cygnea and that evoked by beta-agonist isoproterenol--in the rat skeletal muscles. Even in concentration 10(-7) M, the peptide completely eliminates potentiation of the hormones' stimulating effect on adenylyl cyclase activity with the non-hydrolizable analogue of guanine nucleotides (Gpp[NH]p). At the same time, the peptide does not affect stimulation of the adenylyl cyclase activity with non-hormonal agents (NaF, Gpp[NH]p and forkolin). In the presence of the peptide, inhibiting effects of the hormones on activities of adenylyl cyclase and protein kinase A will be preserved. The findings reveal the importance of the G-protein alpha s-subunit's C-terminal regional of a stimulating type for its functional coupling with receptors of a serpentine type, and elucidate the molecular mechanisms of interaction between the G-protein and the receptor. 相似文献
18.
Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein 总被引:16,自引:0,他引:16
Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase. 相似文献
19.
P J Rapiejko D C Watkins M Ros C C Malbon 《The Journal of biological chemistry》1989,264(27):16183-16189
Thyroid hormones exert "permissive effects" on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling. 相似文献
20.
N Bégin-Heick 《The Journal of biological chemistry》1985,260(10):6187-6193
In mice homozygous for the ob gene (ob/ob), the response of adipose tissue adenylate cyclase to stimulation by lipolytic hormones is abnormally low in comparison to that in lean mice (+/+). Studies on the kinetics of adenylate cyclase activation in white adipocyte membranes under a variety of conditions show the following differences between +/+ and ob/ob mice. 1) The inhibitory effects of GTP and guanyl-5'-yl imidodiphosphate, which were clearly seen in +/+ membranes, were absent in the ob/ob membranes. 2) Half-maximal activation by GTP (in the presence of isoproterenol) required at least 10 times more GTP in ob/ob than in +/+ membranes. 3) Increasing the magnesium concentration (up to 10 mM) of the assay medium facilitated the activation of cyclase by modulatory ligands proportionately more in ob/ob than in +/+ membranes; in the +/+ membranes, 10 mM Mg2+ abolished the inhibitory effects of GTP. 4) Treatment with pertussis toxin attenuated the inhibitory effects of guanine nucleotides in +/+ membranes; no effect of the treatment was seen in the ob/ob membranes. 5) Pretreatment of membranes with cholera toxin facilitated cyclase activation proportionately more in ob/ob than in +/+ membranes; in addition, this treatment led to a shift to the left of the GTP dose-response curve in the ob/ob membranes. Cholera and pertussis toxins catalyzed the incorporation of ADP-ribose into their respective substrates in both the +/+ and the ob/ob membranes, showing that the alpha subunits of the stimulatory and inhibitory proteins of the regulatory component Ns and Ni, respectively are present in both types of membranes. Taken together, the results are consistent with the hypothesis that an excess of beta subunit (either primary or secondary to an altered interaction between beta and Ni alpha or Ns alpha) is responsible for the altered sensitivity to activating ligands of the adipocyte adenylate cyclase of the ob/ob mouse. In addition to these findings, we report an effect of the ob gene on the expression of adenylate cyclase activity, since adipose tissue cyclase from heterozygous lean mice (+/ob) showed characteristics which were intermediate between those of +/+ and ob/ob membranes. 相似文献