Evidence for receptor‐regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes

The activity of the adenylate cyclase inhibitory guanine‐nucleotide‐binding regulatory protein (G i ), measured as inhibition of forskolin‐stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory α 2 ‐adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside‐triphosphate‐regenerating system, GTP (0.1–10 μM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5′‐[β‐thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside‐triphosphate‐regenerating system, which by itself had no influence on control forskolin‐stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 μM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the G i protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the G i protein in the presence of GTP. Furthermore, the nucleoside‐triphosphate‐regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine‐5′‐[β‐thio]triphosphate (10 nM). which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog. The action of the nucleoside‐triphosphate‐regenerating system was mimicked by NaCl, which primarily, at low concentrations, suppressed the inhibitory effect of GTP alone, thereby leading to an apparent requirement of NaCl for observing the epinephrine‐induced inhibition, and which, finally, at high concentrations, also reduced and abolished the action of hormone plus GTP. The data presented, thus, indicate that the adenylate cyclase inhibitory G protein can apparently be activated by GTP, locally formed from GDP by the action of a membrane‐associated nucleoside diphosphokinase, and suggest that this conversion can be stimulated by an agonist‐activated inhibitory receptor. The data obtained with GTP and its analog, guanosine‐5′‐[β‐thio]triphosphate, furthermore, suggest that G i ‐protein activation by these guanine nucleotides may involve additional, so far uncharacterized, phosphate‐transfer reactions. Finally, the data indicate that the use of a nucleoside‐triphosphate‐regenerating system, which is usually included in adenylate cyclase assays, can mask important regulatory aspects of this multicomponent signal‐transduction system.