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Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells.
J Endocrinol 2006; 190(2):247-60JE

Abstract

Recently, a unique family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRalpha and mPRbeta isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). The zebrafish mPRalpha and a previously uncharacterized mPRbeta 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 mPRalpha- or mPRbeta-transfected cells showed high affinity (mPRalpha, K(d) 7 nM; mPRbeta, K(d) 12 nM), saturable, displaceable, single-binding sites specific for 17,20beta-DHP, whereas negligible specific 17,20beta-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 mPRalpha and mPRbeta was the same (17,20beta-DHP>progesterone >4-pregnen-17,20beta,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20beta-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 mPRalpha- and mPRbeta-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRalpha was blocked by pertussis toxin, implying activation of a G(i) protein, while sensitivity to pertussis or cholera toxin was not shown with mPRbeta-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRalpha and mPRbeta signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

Authors+Show Affiliations

Department of Biology, East Carolina University, 1000 E. 5th Street, Greenville, North Carolina 27858-4553, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16899559

Citation

Hanna, Richard, et al. "Cell-surface Expression, Progestin Binding, and Rapid Nongenomic Signaling of Zebrafish Membrane Progestin Receptors Alpha and Beta in Transfected Cells." The Journal of Endocrinology, vol. 190, no. 2, 2006, pp. 247-60.
Hanna R, Pang Y, Thomas P, et al. Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells. J Endocrinol. 2006;190(2):247-60.
Hanna, R., Pang, Y., Thomas, P., & Zhu, Y. (2006). Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells. The Journal of Endocrinology, 190(2), pp. 247-60.
Hanna R, et al. Cell-surface Expression, Progestin Binding, and Rapid Nongenomic Signaling of Zebrafish Membrane Progestin Receptors Alpha and Beta in Transfected Cells. J Endocrinol. 2006;190(2):247-60. PubMed PMID: 16899559.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells. AU - Hanna,Richard, AU - Pang,Yefei, AU - Thomas,Peter, AU - Zhu,Yong, PY - 2006/8/11/pubmed PY - 2006/11/9/medline PY - 2006/8/11/entrez SP - 247 EP - 60 JF - The Journal of endocrinology JO - J. Endocrinol. VL - 190 IS - 2 N2 - Recently, a unique family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRalpha and mPRbeta isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). The zebrafish mPRalpha and a previously uncharacterized mPRbeta 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 mPRalpha- or mPRbeta-transfected cells showed high affinity (mPRalpha, K(d) 7 nM; mPRbeta, K(d) 12 nM), saturable, displaceable, single-binding sites specific for 17,20beta-DHP, whereas negligible specific 17,20beta-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 mPRalpha and mPRbeta was the same (17,20beta-DHP>progesterone >4-pregnen-17,20beta,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20beta-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 mPRalpha- and mPRbeta-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRalpha was blocked by pertussis toxin, implying activation of a G(i) protein, while sensitivity to pertussis or cholera toxin was not shown with mPRbeta-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRalpha and mPRbeta signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity. SN - 0022-0795 UR - https://www.unboundmedicine.com/medline/citation/16899559/Cell_surface_expression_progestin_binding_and_rapid_nongenomic_signaling_of_zebrafish_membrane_progestin_receptors_alpha_and_beta_in_transfected_cells_ L2 - https://joe.bioscientifica.com/doi/10.1677/joe.1.06694 DB - PRIME DP - Unbound Medicine ER -