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Activation of adenylate cyclase in human platelet membranes by guanosine 5'-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

ABSTRACT: Incubation of platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate causes a slow increase in GS (stimulatory GTP-binding protein) activation of adenylate cyclase. Mg2+ is necessary for this slow activation. This process is inhibited in the presence of ATP, and inhibition is greater if cyclic AMP is also included in the incubation. Adenosine 5'-[beta gamma-imido]triphosphate instead of ATP in the incubation facilitates the slow activation in the presence of cyclic AMP, and incubation of membranes with cyclic-AMP-dependent protein kinase inhibitor decreased inhibition of the slow activation of adenylate cyclase by ATP and cyclic AMP. A protein of 45 kDa in platelet membranes is phosphorylated in a cyclic-AMP-dependent manner. The transition from a reversibly activated form of GS to an irreversibly activated form is substantially slower in the presence of ATP and cyclic AMP. We propose that guanosine 5'-[beta gamma-imido]triphosphate-activated GS may exist in phosphorylated or non-phosphorylated forms, and that the non-phosphorylated form is the more active of the two species. The non-phosphorylated form of GS may correspond to the irreversibly activated state.

SUBMITTER: Farndale RW

PROVIDER: S-EPMC1147759 | biostudies-other | 1987 Mar

REPOSITORIES: biostudies-other

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Project description:1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

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Activation of adenylate cyclase in human platelet membranes by guanosine 5'-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

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