Photoaffinity labeling and partial proteolysis of wild-type and variant glucocorticoid receptors

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Abstract
Glucocorticoid receptors [in rats] of wild-type lymphoid cells and of 2 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 MW 94,000 .+-. 5000 while nti-variant receptors had a MW of 40,000 .+-. 2000. Partial proteolysis of wild-type and nt- receptors with .alpha.-chymotrypsin resulted in steroid-labeled receptor fragments of MW 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 MW 29,000 as major products and some fragments of MW 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.