Photoaffinity labeling of glucocorticoid receptors.
The cross-reactivity of progestins for glucocorticoid receptors was exploited to photoaffinity label glucocorticoid receptors from cultured rat hepatoma (HTC) and mouse lymphoma (S49) cell cytosol. The synthetic progestin, 17 alpha, 21-dimethyl-19-nor-pregna-4,9-diene-3,20-dione (R5020), rapidly forms covalent bonds with protein upon irradiation of either cytosol with 350 nm light. Polyacrylamide gel electrophoresis under denaturing conditions reveals a single band photolabeled by R5020 that is not observed when excess nonradioactive dexamethasone is included in the incubation. This protein band corresponds to a molecular weight of about 87,000 in both HTC and S49 cell cytosol; it is entirely absent in cytosol from glucocorticoid-resistant S49(r-) cells which lack receptor-binding activity. Another steroid-resistant mutant, S49 (nti), which exhibits normal levels of steroid-binding activity but increased binding of receptor-steroid complexes by the nucleus, yields a receptor which, when photolabeled, has an apparent molecular weight of only 39,000. These results demonstrate that glucocorticoid receptors can be photoaffinity-labeled; the data are consistent with the notion that the binding form of the receptor consists of a single polypeptide chain, Mr = 87,000, in two different species, rat and mouse, and in cells of either hepatic or lymphoid origin. The data also suggest that the lesion in the steroid-resistant S49 (nti) lymphoma cell line is a mutation of the structural gene for the glucocorticoid receptor which results in the synthesis of a truncated protein.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000378:metabolism; D009649:Norpregnadienes / Q000378:metabolism*; D010782:Photolysis; D011397:Promegestone / Q000378:metabolism* / Q000528:radiation effects; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*
D000345:Affinity Labels; D009649:Norpregnadienes; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; D011397:Promegestone