Limited proteolysis of covalently labeled glucocorticoid receptors as a probe of receptor structure.
[3H]Dexamethasone 21-mesylate affinity-labeled glucocorticoid receptors were subjected to controlled proteolysis by trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on denaturing constant percentage or gradient polyacrylamide gels. The molecular weights (Mr congruent to 98 000) and cleavage patterns for rat liver and HTC cell receptors indicated extensive homology between the glucocorticoid receptors from normal rat liver and a transformed rat liver cell line. The major DNA-binding species generated by chymotrypsin treatment was found to be a 42K fragment that was accompanied by several unresolved, slightly lower molecular weight fragments. The meroreceptors obtained after trypsinization were comprised of two species of Mr 30 000 and 28 000. Each of the three proteases, despite their differing specificities, generated fragments with molecular weights close to 42 500, 30 500, and 27 000. Nevertheless, each of the three proteases gave rise to a distinctive "ladder" of labeled fragments. No differences could be detected in the digestion patterns of unactivated and activated HTC cell complexes for all three proteases. Also, native and denatured receptor-steroid complexes yielded surprisingly similar digestion patterns with each enzyme. Digestion of denatured complexes readily generated large amounts of a fragment of Mr congruent to 15 000 that was much smaller than the protease-resistant meroreceptors formed from native complexes. The presence of these approximately 15K fragments suggested that the [3H]dexamethasone 21-mesylate labeling of the steroid-binding cavity is restricted to a relatively small segment of the receptor.
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D008099:Liver / Q000032:analysis; D008114:Liver Neoplasms, Experimental / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011987:Receptors, Steroid / Q000378:metabolism*; D012697:Serine Endopeptidases
D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D003907:Dexamethasone; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; C031710:dexamethasone 21-methanesulfonate