Photoaffinity labeling and partial proteolysis of wild-type and variant glucocorticoid receptors.
Glucocorticoid receptors of wild-type lymphoid cells and of two classes of glucocorticoid-resistant variants of "nuclear transfer deficient" (nt-) and "increased nuclear transfer" (nti) phenotypes, respectively, were investigated. Photoaffinity labeling of receptor complexes with a radiolabeled glucocorticoid of high affinity was used to analyze these receptor types by electrophoresis in sodium dodecyl sulfate containing gels. Wild-type and nt- -variant receptors yielded radiolabeled polypeptide bands of Mr 94 000 +/- 5000 while nti-variant receptors had a molecular weight of 40 000 +/- 2000. Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin resulted in steroid-labeled receptor fragments of Mr 37 000-38 000 while nti-variant receptors remained unchanged. In the case of wild-type receptors, the chymotryptic fragment had increased affinity for DNA indistinguishable from that of native nti-variant receptors. Depending on the nt- cell clone, the chymotryptic receptor fragments containing the steroid binding site had either the same low affinity for DNA as the undigested receptors or a slightly increased affinity. Partial proteolysis with trypsin of wild-type, nt-, and nti receptors resulted in steroid-labeled fragments of Mr 29 000 as major products and some fragments of Mr 27 000. These tryptic receptor fragments were devoid of DNA binding ability regardless of the original receptor types. With a lysine-specific protease, similar fragments were obtained from wild-type, nt-, and nti receptors. In contrast, a protease specific for arginine residues did not produce receptor fragments detectable by our techniques. A model of the wild-type receptor is discussed.
D000818:Animals; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000031:analogs & derivatives; D014644:Genetic Variation; D006801:Humans; D007945:Leukemia, Lymphoid / Q000378:metabolism; D008223:Lymphoma / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009374:Neoplasms, Experimental / Q000378:metabolism; D010447:Peptide Hydrolases; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics*; D014222:Triamcinolone Acetonide / Q000378:metabolism
C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D002482:Cellulose; D004247:DNA; D010447:Peptide Hydrolases; D014222:Triamcinolone Acetonide