Identification of a steroid receptor on the surface of Xenopus oocytes by photoaffinity labeling.
The synthetic progestin, 17,21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione (R5020), was used to photoaffinity label a steroid-binding protein on the Xenopus oocyte plasma membrane. R5020 effectively induced oocyte maturation with half-maximal response at a concentration of 1.4 X 10(-6) M. Polyacrylamide gel electrophoretic analysis of plasma membranes following photolysis with tritiated R5020 resulted in the identification of a single labeled protein with a Mr = 110,000. The specificity of this steroid receptor interaction for R5020 was demonstrated by the competitive inhibition of R5020 photolabeling with nonradioactive R5020 and the lack of inhibition by 17 beta-estradiol. Covalent labeling of the 110,000-dalton protein was saturable with both time of photolysis and concentration of R5020 with the maximum number of photolabeled binding sites equal to 0.7 pmol/oocyte, and kinetic analysis of the photolabeling of the 110,000-dalton receptor protein yielded an apparent KD of 1 X 10(-6) M R5020. Progesterone had a biphasic effect on the kinetics of photolabeling with concentrations of progesterone below 5 microM increasing photolabeling by elevating Vmax up to 5-fold and higher concentrations of progesterone reducing the rate of photolabeling. Membrane-associated adenylate cyclase measured in the presence of guanyl-5'-yl imidodiphosphate was inhibited up to 70% after photolysis with [3H]R5020. Inhibition was proportional to the level of [3H]R5020 covalently bound to the 110,000-dalton protein, and significant inhibition was observed at 1 X 10(-6) M R5020.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D009865:Oocytes / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014981:Xenopus
D011980:Receptors, Progesterone; D011374:Progesterone; D011397:Promegestone; D000262:Adenylyl Cyclases