Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors α and β in transfected cells

Abstract

Recently, a unique family of membrane progestin receptors (mPRα, mPRβ, and mPRγ) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRα and mPRβ isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20β-diol-3-one (17,20β-DHP). The zebrafish mPRα and a previously uncharacterized mPRβ isoform were stably expressed in nuclear progesterone receptor-deficient mammalian breast cancer cells, MDA-MB-231. Expression and surface localization of the receptors were verified by flow cytometry, biotin surface labeling, and Western blotting. Plasma membrane proteins from mPRα- or mPRβ-transfected cells showed high affinity (mPRα, Kd 7 nM; mPRβ, Kd 12 nM), saturable, displaceable, single-binding sites specific for 17,20β-DHP, whereas negligible specific 17,20β-DHP binding was observed in nontransfected cells. Progestin treatment caused significant activation of mitogen-activated protein kinase (MAPK) within 5 min in cells transfected with either of the receptors as measured by western blotting and flow cytometry. The rank order of the potencies of several progestins in activating MAPK via mPRα and mPRβ was the same (17,20β-DHP>progesterone >4-pregnen-17,20β,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20β-DHP, was also the most potent inhibitor, among the progestins tested, of adenylyl cyclase activity in cells transfected with either of the receptors. This progestin significantly decreased cAMP levels in both mPRα- and mPRβ-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRα was blocked by pertussis toxin, implying activation of a Gi protein, while sensitivity to pertussis or cholera toxin was not shown with mPRβ-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRα and mPRβ signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.