Recent results using
proteases suggest that
dexamethasone 21-mesylate (Dex-Mes) labeling of the rat
hepatoma tissue culture (HTC) cell
glucocorticoid receptor occurs at one or a few closely grouped
cysteine residues (Simons, S.S., Jr. (1987) J. Biol. Chem. 262, 9669-9675). In this study, a more direct approach was used both to establish that only one
cysteine is labeled by [3H]Dex-Mes and to identify the amino acid sequence containing this labeled
cysteine. Various analytical procedures did not provide the purification of the extremely hydrophobic Staphylococcus aureus V8
protease digestion fragment that is required for unique
amino acid sequencing data. Therefore, Edman degradation was performed on the limit
protease digest mixtures which appeared to contain only one 3H-labeled
peptide. These degradation experiments revealed the number of
amino acid residues between the NH2 terminus of each
peptide and the [3H]Dex-Mes-labeled
cysteine. A comparison of these
amino acid spacings with the published amino acid sequence of the HTC cell
glucocorticoid receptor (Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikstom, A-C., Gustafsson, J-A., and Yamamoto, K. R. (1986) Cell 46, 389-399) indicated that the one
cysteine labeled by [3H]Dex-Mes is Cys-656. Further analysis of the receptor sequence for the presence of the observed grouping of proteolytic cleavage sites, but without any preconditions as to which
amino acid was labeled, gave Asp-122 and Cys-656 as the only two possibilities. Potential labeling of Asp-122 could be eliminated on the basis of immunological and genetic evidence. We, therefore, conclude that the single Dex-Mes-labeled site of the HTC cell
glucocorticoid receptor has been identified as Cys-656. Since several lines of evidence indicate that [3H]Dex-Mes labeling of the receptor occurs in the
steroid binding site, Cys-656 is the first
amino acid which can be directly associated with a particular property of the
glucocorticoid receptor.