PUBLICATION
Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors {alpha} and {beta} in transfected cells
- Authors
- Hanna, R., Pang, Y., Thomas, P., and Zhu, Y.
- ID
- ZDB-PUB-060816-22
- Date
- 2006
- Source
- The Journal of endocrinology 190(2): 247-260 (Journal)
- Registered Authors
- Keywords
- none
- MeSH Terms
-
- Animals
- Biotinylation
- Blotting, Western/methods
- Cell Line, Tumor
- Cell Membrane/metabolism*
- Cyclic AMP/analysis
- Female
- Flow Cytometry
- Gene Expression
- Humans
- MAP Kinase Signaling System/physiology*
- Oocytes/chemistry
- Progestins/metabolism*
- Protein Binding
- Radioligand Assay
- Receptors, Progesterone/analysis*
- Receptors, Progesterone/genetics
- Receptors, Progesterone/metabolism
- Recombinant Proteins/genetics
- Recombinant Proteins/metabolism
- Transfection/methods
- Zebrafish
- Zebrafish Proteins/genetics
- Zebrafish Proteins/metabolism
- PubMed
- 16899559 Full text @ J. Endocrinol.
Citation
Hanna, R., Pang, Y., Thomas, P., and 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):247-260.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping