Characterization of a Putative Membrane Receptor for Progesterone in Rat Granulosa Cells1

Abstract

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 its 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 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 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 (3)H-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. (3)H-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).