Non-genomic steroid receptors in the bovine ovary

Abstract

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [ 3 H ]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85 kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins ( M r, 55 and 60 kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.