Modulation of FSH receptor phosphorylation correlates with hormone-induced coupling to the adenylate cyclase system

The authors have recently demonstrated that an inhibitor of protein phosphorylation, staurosporine (SSP), can dramatically enhance follicle-stimulating hormone (FSH) stimulated cyclic adenosine monophosphate (cAMP) accumulation in rat granulosa cell line (GFSHR-17) overexpressing about 20-fold FSH receptor than primary granulosa cells. Moreover, incubation with SSP can partially release the cells from FSH-induced desensitization. In this work, it was examined whether coupling of FSH receptor to the adenylate cyclase is correlated with the degree of receptor phosphorylation. Immunoprecipitation of FSH receptor after metabolic labeling of the cells with 32P-orthophosphate revealed that preincubation of the cells with SSP resulted in pronounced reduction in FSH receptor phosphorylation compared to control cells, concomitantly with a dramatic increase in FSH-stimulated cAMP accumulation. In contrast, incubation of the cells with saturating dose of FSH, which leads to uncoupling between the receptor and the adenylate cyclase, resulted in enhanced receptor phosphorylation. Moreover, cells preincubated with FSH could be released from desensitization by further incubation with SSP and a significant reduction in FSH receptor phosphorylation. Immunostaining of the cells with FSH receptor antibody reveal a homogenous distribution of the receptor on the surface of SSP-treated cells. Some aggregation of the receptor was evident in control cells that were not treated with SSP. In contrast, massive clustering and capping of the receptor molecules were observed on the surface of FSH-stimulated cells. The current data suggest that phosphorylation-dephosphorylation of the receptor molecules play an important role in the degree of coupling between the receptor and the adenylate cyclase system. Moreover, desensitization to FSH stimulation that is implicated with high degree of receptor phosphorylation may lead to aggregation of the receptor molecules on the cell surface.