GTP analogues stimulate inositol trisphosphate formation transiently in Dictyostelium

Abstract

Permeabilization of amoebae of Dictyostelium discoideum with saponin was found not to uncouple the chemotactic cell surface cyclic AMP receptors from inositol trisphosphate (IP3) formation, and stimulation of permeabilized amoebae with 50 nM-cyclic AMP produced peaks of IP3 at 5, 15 and 30 s in a manner comparable to that seen previously in non-permeabilized cells. The possible involvement of a GTP-binding protein (G-protein) in this IP3 signal transduction pathway was investigated by studying the effects on such permeabilized amoebae of added GTP and non-hydrolysable GTP analogues. While GDP produced only very minor effects, stimulation of the amoebae (in the absence of added cyclic AMP) with GTP or the non-hydrolysable GTP analogues GTP gamma S (guanosine 5'-O-(3-thio-triphosphate] and Gpp(NH)p (5'-guanylylimidodiphosphate) induced transient formation of IP3 in an oscillatory manner, with peaks similar in magnitude and timing to those elicited by cyclic AMP. A dose-response curve for GTP gamma S indicated a concentration for half-maximal stimulation of approximately 8 microM. When tested at 300 s after addition of GTP gamma S, the basal level of IP3 was found to be twofold elevated with shallow (presumably asynchronous) oscillations still just discernible. The significance of the IP3 oscillations elicited by GTP and its analogues is discussed in relation to the mechanism of signal adaptation and the presumed role of G-proteins.