ACTIVATION OF FSH-RESPONSIVE ADENYLATE-CYCLASE BY STAUROSPORINE - ROLE FOR PROTEIN-PHOSPHORYLATION IN GONADOTROPIN RECEPTOR DESENSITIZATION

Prolonged stimulation of gonadotropin receptors in granulosa cells lea ds to desensitization of the cellular response to gonadotropic hormone s which is evident by decrease in cAMP formation. In order to explore the mechanism of desensitization and to examine whether protein phosph orylation may play a role in this phenomenon, we have studied the effe ct of various stimulators and inhibitors of protein phosphorylation on FSH-induced cAMP formation in the FSH-responsive cell line, GFSHR-17, recently established in our laboratory. Both ovine and human FSH acti vated the hormone sensitive adenylate cyclase in a dose-dependent mann er with an ED(50) of 0.5 nM. This stimulation was followed by a sharp decrease in cAMP formation after 30 min incubation of the cell with th e hormone. When cells were preincubated for 60 min with staurosporine, cAMP accumulation during 20 min of FSH stimulation was elevated about 500%, compared to cells stimulated by FSH alone, Staurosporine alone showed a negligible effect on cAMP accumulation in these cells. In cel ls stimulated with forskolin, a non-specific activator of adenylate cy clase, or with cholera toxin (CT), an inhibitor of GTPase activity ass ociated with Gs of adenylate cyclase, preincubation with staurosporine increased cAMP formation in these cells by only 50-70 or 80-120%, res pectively. Preincubation of cells with the protein kinase C (PKC) inhi bitors chelerythrine and GF109203X increased FSK-stimulated accumulati on of cAMP by 50 and 30%, respectively. These drugs exhibit a similar effect on forskolin-stimulated cells. Preincubation of cells for 60 mi n with a PKC stimulator; TPA, suppressed FSH-mediated cAMP response in these cells by 40%. Tyrosine kinase inhibitors such as AG18, AG33 and genistein exhibit a modest inhibitory effect of up to 20% on FSH-stim ulated cAMP accumulation. All the above results were obtained both in the presence and absence of IBMX, a potent inhibitor of the cellular p hosphodiesterases. Upon prolonged incubation with FSH (3 h) cells pret reated with staurosporine exhibited a much slower rate of decline in i ntracellular cAMP levels. Moreover, in desensitized cells, following 1 or 2 h of continuous stimulation with FSH, staurosporine could marked ly enhance cAMP formation in the presence of FSH. Our data suggest tha t staurosporine-sensitive phosphorylation of serine or threonine in th e FSH receptor cyclase system may be responsible for desensitization o f the FSH coupled activation of cAMP formation, while reactivation of the system can be achieved by protein dephosphorylation at these speci fic sites. Because specific inhibition of PKC could not mimic the stau rosporine effect on FSH-stimulated cAMP formation, nor could activatio n of kinase C antagonize it, it is suggested that a specific staurospo rine-sensitive receptor kinase may be responsible for modulation of th e coupling between the gonadotropin receptor and the adenylate cyclase system.