A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs

Abstract

To identify guanine nucleotide binding proteins (G-proteins) in sea urchin eggs and to investigate their role in signal transduction at fertilization, we used cholera toxin (CTX) and pertussis toxin (PTX), which catalyze the specific ADP-ribosylation of G-proteins. Cell surface complex, consisting of plasma membranes and adhering cortical vesicles, was prepared from eggs of Lytechinus variegatus and incubated with 32P-labeled NAD in the presence of CTX or PTX. CTX catalyzed the ADP-ribosylation of a 47-kDa polypeptide, whereas PTX catalyzed the ADP-ribosylation of a 40-kDa polypeptide. Microinjection of approximately 30 micrograms/ml whole CTX or approximately 20 micrograms/ml CTX subunit A into intact eggs caused exocytosis of cortical vesicles. However, if the eggs were first injected with EGTA (0.6-1.4 mM), injection of CTX did not cause exocytosis. Eggs injected with 0.8-2.8 mM cAMP or 1.0-4.0 mM adenosine 3':5'-monophosphotioate cyclic Sp-isomer (cAMP-S), a hydrolysis-resistant analog of cAMP, did not undergo exocytosis. These results suggest that a CTX-sensitive G-protein is involved in regulating Ca2+ release and exocytosis of cortical vesicles in sea urchin eggs.