Phosphorylated sites within the functional domains of the .apprx.100-kDa steroid-binding subunit of glucocorticoid receptors

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Abstract
The steroid-binding subunit of the glucocorticoid receptor is known to be a .apprx.100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. When isolated from WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel et al., 1987). To identify the domains that contain these phosphorylated sites, we have analyzed the phosphate content of selected proteolytic fragments of the .apprx.100-kDa steroid-binding protein from nonactivated and activated receptors. The .apprx.100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).1 Chymotrypsin digestion of this protein yields a .apprx.45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. Trypsin digestion of the protein yields a .apprx.29-kDa fragment encompassing the steroid-binding domain but not the DNA-binding domain of the .apprx.100-kDa protein, which also contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups. Since the entire tryptic fragment is contained within the chymotryptic fragment, we conclude that this phosphoryl group is in the steroid-binding domain of the .apprx.100-kDa protein and, therefore, that the DNA-binding domain is not phosphorylated. To determine more directly the phosphate content of the DNA-binding domain of the .apprx.100-kDa proteins, we isolated a .apprx.16-kDa tryptic fragment from cytosol of WEHI-7 cells grown in the presence of [32P]orthophosphate and [35S]cysteine. This fragment contains the BuGR2 epitope and has DNA-binding, but not steroid-binding, activity. Comparison of the 32P/35S ratio of this fragment to that of the intact protein indicated that it contains less than 0.2 mol of phosphoserine/mol of protein. This is insufficient to account for a single phosphorylated residue within the DNA-binding domain, a result that is consistent with our previous conclusion. These data, in conjunction with our phosphoamino acid determination, indicate that there is one phosphoserine residue within the steroid-binding domain of the .apprx.100-kDa subunit of the glucocorticoid receptor in WEHI-7 cells, none in the DNA-binding domain, and therefore, probably one or two in the immunogenic domain.