Stimulatory GTP-binding Protein Gs Research Articles

Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs.

Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (Gs) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (Sf9) cells. Enhancement by hydroxylamine of immunoaffinity purified Gs was still 1.5-fold, while that of Gs purified according to the multiple-step procedure by Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (Proc. Natl Acad. Sci. USA 78, 6516-6520, 1980) was close to unity. The alpha-subunit of the stimulatory GTP-binding protein expressed in Escherichia coli (r(alpha)s), likewise, failed to show an effect by hydroxylamine. Surprisingly, guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-liganded r(alpha)s, treated with palmitoyl-CoA for 14 h at 4 degrees C resulted in sixfold enhancement of reconstitutive activity. In contrast, that of the GDP-liganded r(alpha)s(beta)gamma heterotrimer was not improved by palmitoylation and consecutive activation with GTP[S], although incorporation of [3H]palmitate into momomer and heterotrimer was identical. While adenylyl cyclase type-V activity reconstituted by r(alpha)s x GTP[S] was not influenced by betagamma-subunits, that activated by palmitoylated r(alpha)s x GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated r(alpha)s for betagamma-subunits. On treatment of either form with the proteinase Lys-C, less than 25% of the label was found in a stable M(r) 38000 fragment with intact C terminus, but lacking the N-terminal portion. Absence of the latter did not affect activation by r(alpha)s, but caused a >90% loss of extra-activation by palmitoylated r(alpha)s. The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of alpha(s) occurs predominantly on Cys 3.

Sporadic cardiac myxomas and tumors from patients with Carney complex are not associated with activating mutations of the Gs alpha gene.

Cardiac myxomas are rare tumors that may be encountered sporadically or in the context of the Carney complex. The molecular basis for the development of cardiac myxomas and Carney complex tumors is unclear. Pathological myocardial function and myocardial hypertrophy have been associated with alterations in the heterotrimeric GTP-binding proteins. The postulated proto-oncogenic character of the gene encoding the alpha sub-unit of the stimulatory GTP-binding protein Gs alpha (gsp) in pituitary and thyroid tumors, the finding of identical somatic gsp mutations in the myocardium of patients with McCune-Albright syndrome, and the associated endocrine anomalies of the Carney complex prompted us to investigate the occurrence of activating missense mutations in the Gs alpha gene in 10 sporadically occurring atrial myxomas and in 8 tumors from 7 patients with Carney complex. No gsp mutations could be demonstrated by using the polymerase chain reaction and denaturing gradient gel electrophoresis complemented by direct DNA sequencing. Thus, activating Gs alpha mutations neither are associated with the development of atrial myxomas, nor can be demonstrated in other tumors from patients with Carney complex. The significance of these mutations in the myocardium of asymptomatic patients with McCune-Albright syndrome remains to be determined.

Gonadotrophin receptors

Precocious puberty may be gonadotrophin-dependent, due to premature activation of the hypothalamic GnRH pulse generator, or gonadotrophin-independent wherein serum LH and FSH levels are undetectable despite near-adult levels of gonadal steroidogenesis. In one form of gonadotrophin-independent familial male precocious puberty (FMPP) activating mutations of the LH receptor cause constitutive activation of the cAMP signal transduction cascade in Leydig cells with attendant testosterone biosynthesis. The LH receptor is a member of the seven transmembrane group of receptors coupled to signal transduction through GTP binding intermediate proteins. In FMPP the initial mutations were described in the sixth membrane spanning domain close to the third intracellular loop which is known to be important for coupling to the subunit of the stimulatory GTP binding protein Gs. Subsequently, activating mutations at several other sites in the LH receptor have been described. Activating mutations in the second step of the signalling cascade, the GTP binding protein Gs, have been described in sporadic somatotrophinomas in the pituitary and solitary thyroid adenomas. The McCune-Albright syndrome may also be rarely associated with pituitary and thyroid adenomas, in addition to the more common polyostotic fibrosus dysplasia and gonadotrophin-independent precocious puberty. Thus, in the luteinized ovarian tissue of patients with this condition activating mutations in Gs alpha subunit have been described at codon 201. These mutations are somatic, not germ line, and only one allele needs to be affected as they are dominant. In an uncommon form of male pseudohermaphroditism associated with hypergonadotrophic hypogonadism and Leydig cell hypoplasia, inactivating mutations of the LH receptor are described. These, too, are in the sixth transmembrane domain and both alleles must be mutant as the condition is autosomal recessive. These 'experiments of nature' have provided invaluable insights into the structure-function relations of the LH receptor, and the mutations described have been recreated and studied in vitro, where the predicted activation or failure to stimulate steroidogenesis confirm their pathogenic nature. As yet no such activating or inhibiting mutations in the FSH receptor gene have been described, but it is likely these will be forthcoming in future.

Cyclic AMP regulation of messenger RNA level of the stimulatory GTP-binding protein Gs alpha. Isoproterenol, forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate increase the level of Gs alpha mRNA in cultured astroglial cells.

The existence of a cAMP-dependent regulation of the expression of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs) in a well characterized astroglial cells culture was established. The culture of astroglial cells for 3-6 h with isoproterenol (10 microM) or forskolin (10 microM) (a cAMP-inducing agent) increased (200-400%) the response of adenylylcyclase to agents which bypass the receptor; GTP, GTP[S] or forskolin. For prolonged exposure times (15 h or more) to isoproterenol or forskolin, the adenylycyclase activity decreased to the value observed in control cells. The same biphasic response of adenylylcyclase to isoproterenol (10 microM) plus GTP (10 microM) occurred in membrane fractions from cells cultured with forskolin, whereas a diminished response to isoproterenol was observed in isoproterenol-treated cells, indicating that the beta-adrenergic receptor was desensitized. To understand the molecular mechanism of these phenomena, we measured the levels of the alpha subunits of the guanine-nucleotide binding protein (Gs and Gi) by Western-blot analysis. The culture of astroglial cells with isoproterenol or forskolin (3-24 h) resulted in a transient increase of both the Gs alpha and the Gi alpha protein levels, while the level of G beta subunits was unaffected. We also identified Gs alpha protein (about 40% of the total cellular protein) in the supernatant fraction of astroglial cells but its level was not modified by the stimulation of cells by forskolin. The level of Gs alpha mRNA measured by Northern-blot analysis was transiently increased (200%) after stimulation of astroglial cells with isoproterenol or forskolin for an incubation period of 6-9 h, then returned to that of control cells for longer period of time. In addition, the Gs alpha mRNA level was threefold increased when cells were cultured for 2-6 h with 8-bromoadenosine 3':5'-cyclic monophosphate (10 microM), a permeant analogue of cAMP. These results indicate that cAMP induces a time-dependent increase of Gs alpha mRNA. The half-life of Gs alpha protein and Gs alpha mRNA were determined. Pulse-chase studies revealed that the decay of Gs alpha protein was clearly biphasic with an early phase (5-6 h) and a slower second phase (20-25 h) but the treatment of cells with forskolin did not accelerate or slow down the turnover of Gs alpha protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylylcyclase: Characterization in the Heart and Its Regulation in Heart Failure.

It is generally acknowledged that heart failure is characterized by several disorders in cardiac autonomic properties, including sympathetic, parasympathetic and baroreceptor responses. Part of the mechanism of altered cardiovascular control and reduced catecholamine responsiveness in heart failure involves changes in end-organ responses mediated via beta adrenergic receptors. We have now determined the changes that occur in these components during the inception of heart failure induced by cardiac pacing. First, we quantitiated the stoichiometry and function of each of the components of the beta-adrenergic signaling pathway, including the receptor itself, Gs and the adenylylcyclase catalyst. Two key abnormalities occur: (1) the receptor uncouples from Gs, as indicated by loss of high affinity agonist binding sites without a change in receptor density; (2) there is an associated loss adenylylcyclase catalytic activity without any change in the concentration or function of the stimulatory GTP-binding protein Gs. To understand, therefore, what factors underlie the loss in adenylylcyclase catalytic activity; i.e., whether these are the results of a reduced concentration of the catalyst or due to its post-translational modification, we first cloned the cardiac isoforms of adenylylcyclase. Two novel adenylylcyclase cDNAs, types V and VI, were identified that share the motif of tandem six-transmembrane spans separated by a large hydrophilic cytoplasmic loop. Our data suggest that a decrease in the content of the adenylylcyclase catalyst itself contributes to impaired cyclic AMP production in heart failure and may represent a fundamental defect contributing to a progressive decline in LV function. (Hypertens Res 1993; 16: 79-83)

Influence of pregnancy on G-protein coupling to adenylate cyclase activation in guinea-pig myometrium

The regulatory factors controlling uterine activity during pregnancy remain unclear in many species. Since myometrial relaxants raise intracellular cyclic AMP, modulation of signalling pathways coupling cell-surface receptors to adenylate cyclase activation could be an important site for control. To assess the functional activity of the stimulatory GTP-binding protein Gs we have measured adenylate cyclase activation by GTP, its non-hydrolysable analogue guanosine 5'-(beta-gamma-imido)triphosphate (Gpp(NH)p), fluoride, forskolin and manganese in a 50,000 g membrane fraction prepared from the myometrium of non-pregnant, mid-pregnant (30-32 days) and late-pregnant (62-66 days) guinea-pigs (full term 67 +/- 2 days). While forskolin- and manganese-dependent enzyme activation was unaltered by pregnancy, maximal stimulation by Gpp(NH)p and fluoride was enhanced by up to 200%. Recovery of adenylate cyclase activity in the 50,000 g fraction was essentially constant at 20-24% of the total activity throughout pregnancy, and thus cannot explain the increases observed. Since guanine nucleotides and fluoride stimulate adenylate cyclase through activating Gs, and forskolin and manganese act at the level of the catalytic unit, these data are consistent with a pregnancy-related increase in Gs functional coupling while adenylate cyclase activity is unaltered. These observations suggest a physiological regulation of myometrial Gs activity during pregnancy which could facilitate hormonal stimulation of adenylate cyclase and contribute to uterine quiescence by increasing uterine sensitivity to relaxants.

Reversal and inhibition of cholera toxin‐induced secretion in isolated rabbit ileum.

1. Cholera toxin (1 microgram/ml) abolished net fluid absorption by everted sacs of rabbit ileum, leading to net fluid secretion. This action occurred via the toxin-catalysed ADP ribosylation of the stimulatory GTP-binding protein Gs which is linked to adenylate cyclase. Nicotinamide (10 mM), a reaction product of ADP ribosylation, reversed cholera toxin-induced secretion, restoring absorption. Lower concentrations of nicotinamide induced partial reversal. 2. Nicotinamide (1 mM) blocked the secretory action of cholera toxin applied to ileal sacs. This inhibitory action was more effective in the presence of methionine (1 mM). 3. Other inhibitors of ADP ribosylation, benzamide and adenine, blocked the secretory action of cholera toxin. Hypoxanthine, an analogue and metabolite of adenine, was similarly effective. 4. Nicotinamide was not, however, effective in blocking or reversing the secretory action of theophylline (10 mM) or of heat-stable E. coli enterotoxin STa. This indicates that nicotinamide has a highly specific action against ADP ribosylating toxins. 5. It is proposed that nicotinamide reverses the secretory action of cholera toxin by reversing ADP ribosylation, simply by the law of mass action. This counters the established idea that the effects of cholera and other ADP-ribosylating toxins are irreversible under physiological conditions.

Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells

We investigated the mechanism for lithium-induced inhibition of vasopressin-stimulated adensoine 3',5'-cyclic monophosphate (cAMP) production in the renal epithelial cell line LLC-PK1. In LLC-PK1 membranes lithium caused direct inhibition of hormone-stimulated adenylate cyclase activity by competing with magnesium. Fifty percent inhibition occurred at 20 mM lithium. The maximum transport activity (Vmax) but not the activation constant (Ka) for activation by vasopressin was altered. Activation by GTP and its nonhydrolyzable analogues was also inhibited by lithium. Furthermore, kinetic studies revealed that the lag phase in the activation of adenylate cyclase by 5'-guanylimi-dotriphosphate [Gpp(NH)p] was prolonged from 1 to 3 min, suggesting an effect of lithium on magnesium-dependent activation of the stimulatory GTP binding protein Gs. The function of the corresponding inhibitory GTP-binding protein Gi, as assessed by GTP inhibition of vasopressin-stimulated adenylate cyclase activity in the presence and absence of pertussis toxin pretreatment, was unaffected. Intact LLC-PK1 cells incubated in 10 mM lithium (approximate urinary concentration in lithium-treated patients) attained an intracellular lithium concentration of 17 mM, which led to a 40% reduction in cAMP formation. Magnesium loading of intact cells with the ionophore A23187 reversed the inhibitory effect of lithium. It is concluded that lithium directly inhibits the activation of vasopressin-sensitive adenylate cyclase in renal epithelia by competing with magnesium for activation of Gs. This direct effect on Gs activation accounts for the inhibitory effect of lithium on cAMP production in the intact cell.