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 6 0-kDa protein that functions as a membrane receptor. The present studies we re designed to determine the P-4 binding characteristics of this protein. W estern 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 l ocalized to the plasma membrane of both granulosa cells and spontaneously i mmortalized granulosa cells (SIGCs). To determine whether this protein bind s P-4, proteins were immunoprecipitated with the C-262 antibody, electropho resed, transferred to nitrocellulose, and probed with a horseradish peroxid ase-labeled P-4 in the presence or absence of nonlabeled P-4, This study de monstrated that the 60-kDa protein specifically binds P-4. Scatchard plot a nalysis 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 bi nding was not reduced by dexamethasone, mifepristone (RU 486), or onapristo ne- (ZK98299). Further studies with SIGCs showed that P-4 inhibited apoptos is and mitogen-activated protein kinase kinase (MEK) activity, and maintain ed calcium homeostasis. These studies taken together support the concept th at the 60-kDa P-4 binding protein functions as a low-affinity, high-capacit y membrane receptor for P-4.