Characterization of a putative membrane receptor for progesterone in rat granulosa cells

Progesterone (P-4) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P-4 receptor. It has been proposed that P-4 mediates ifs action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P-4 binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P-4 binding site of the nuclear P-4 receptor (C-262) confirmed that the 60kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P-4, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P-4 in the presence or absence of nonlabeled P-4, This study demonstrated that the 60-kDa protein specifically binds P-4. Scatchard plot analysis revealed that H-3-P-4 binds to a single site (i.e., single protein), which is relatively abundant 200 pmol/mg with a K-d of 360 nM. H-3-P-4 binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone- (ZK98299). Further studies with SIGCs showed that P-4 inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P-4 binding protein functions as a low-affinity, high-capacity membrane receptor for P-4.