Progesterone (P4) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P4 receptor. It has been proposed that P4 mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P4 binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P4 binding site of the nuclear P4 receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P4, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P4 in the presence or absence of nonlabeled P4. This study demonstrated that the 60-kDa protein specifically binds P4. Scatchard plot analysis revealed that 3H-P4 binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a Kd of 360 nM. 3H-P4 binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P4 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 P4 binding protein functions as a low-affinity, high-capacity membrane receptor for P4.
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