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 (mPRa, mPRb, and mPRg) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRa and mPRb isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, . The zebrafish mPRa and a previously uncharacterized mPRb 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 mPRa-or mPRb-transfected cells showed high affinity (mPRa, K d 7 nM; mPRb, K d 12 nM), saturable, displaceable, single-binding sites specific for 17,20b-DHP, whereas negligible specific 17,20b-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 mPRa and mPRb was the same (17,20b,. Interestingly, the MIS in zebrafish, 17,20b-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 mPRa-and mPRb-transfected cells in a dose-responsive and timedependent manner. In addition, signaling of the zebrafish mPRa was blocked by pertussis toxin, implying activation of a G i protein, while sensitivity to pertussis or cholera toxin was not shown with mPRb-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRa and mPRb signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

Internalisation of membrane progesterone receptor-α after treatment with progesterone: Potential involvement of a clathrin-dependent pathway

“…in agreement with these results, membrane localisation of mPrs has been demonstrated by several research groups in a variety of cell types (1,10,(18)(19)(20)(21)(22). other researchers have reported mPrs to be localised intracellularlly in the endoplasmic reticulum (23)(24)(25).…”

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1

“…Moreover, in zebrafish with asynchronous ovarian development, it has been postulated earlier that relatively high level of endogenous estrogen is required to promote vitelloegenesis and to support follicular growth (Pang & Thomas 2009), which could be sufficiently inhibitory for the daily spawning of this species. While studies in zebrafish have revealed participation of mPRa in MIS-induced OM that involves rapid activation of Ga i and decrease in cAMP (Hanna et al 2006, Hanna & Zhu 2011, we observed involvement of PI3K/Akt signalling pathway is essential for insulininduced OM in this species (Das et al 2013). Moreover, hCG (analogue of Lh) stimulation of meiosis resumption in zebrafish ovarian follicles in vitro has been shown to require synthesis of maturational steroid, DHP, as well as Igf ligands, mainly Igf3 (Nelson & Van Der Kraak 2010a, Li et al 2011.…”

“…While participation of Gbg subunit of heterotrimeric G-protein has been implicated in DHP stimulation of PI3K/Akt and MAPK activation (Hanna et al 2006), down regulation of cAMP/PKA pathway involves mPRa/Gai -mediated attenuation of Ac (Zhu et al 2003). On the other hand, Igf1 action at oocyte surface requires participation of Igf1r, and triggers rapid activation of Akt-mediated signalling cascade in denuded oocytes in vitro.…”

New Insight into Progesterone-dependent Signalization

“…Taking into consideration a broad distribution and significance of non-classical pathways and membrane receptors, the aspect of receptor stimulation or inhibition could be useful in treating multiple pathologies, e.g. some forms of infertility [1,46]. Plenty of aspects regarding membrane receptors and non-classical pathways are still uncovered or controversial.…”

“…mPR, especially mPRα, is directly coupled with G-proteins and typically activates pertussis toxin-sensitive inhibitory Gi protein, ultimately leading to adenylate cyclase down-regulation and decrease in cAMP concentration [26]. However, the analysis of the mPRβ signal path revealed that the receptor is not sensitive to pertussis and cholera toxins, what suggests that it activates another G-protein, insusceptible to the aforementioned toxins [45,46]. mRNA for mPRs is widely distributed in both, reproductive and non-reproductive tissues of vertebrates, what indicates multipotent properties of membrane receptors [26].…”