University of Groningen Pertussis toxin inhibits cAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum Snaar-Jagalska, B. Ewa; van Haastert, Petrus

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation. The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent, These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process. Abbreviations used: ATPyS Adenosine 5'-0-(3-Thiotriphosphate), GTP~(S Guanosine 5'-0-(3-thiotriphosphate), (Sp)-cAMPS Adenosine 3',5'-monophosphorothioate-Sp-isomer, dcAMP 2'-deoxyadenosine 3',5'-monophosphate, Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, D T T Dithiothreitol, buffer A 10mM KH2POa/Na2HPO4, pH6.5, buffer B 40mM Hepes/NaOH, 0.5mM EDTA, 250 mM sucrose, pH 7.7