During the last year we have continued to employ both biochemical and genetic approaches to the study of the acquisition of steroid resistance in human leukemic cells. In particular we have concentrated on the analysis of the normal glucocorticoid receptors of the human lymphoid cell line IM-9 in order to provide a basis for the ultimate analysis of glucocorticoid receptor mutants in the clonal glucocorticoid-resistant human leukemic cell lines we have previously isolated. Using high-resolution 2-dimensional gel electrophoresis of affinity labeled, immunopurified IM-9 receptors we have identified two isoforms of the 92 kilodalton human glucocorticoid receptor. Analysis of tryptic and chymotryptic digests of these isoforms has demonstrated that the charge heterogeneity resides in the 24 kilodalton tryptic fragment of the receptor which contains the steroid binding site. In addition, we have determined: (1) that there is no covalent charge modification of the 92 kilodalton steroid binding protein during activation of the steroid-receptor complex to its DNA binding form; and (2) that only the more basic of the two 92 kilodalton isoforms can bind to DNA. These results suggest that activation of the steroid-receptor complex does not involve dephosphorylation of the steroid binding protein. Our anti-human glucocorticoid receptor antibodies have also been used to obtain a partial cDNA clone of IM-9 glucocorticoid receptor mRNA. This clone was obtained after immunological screening of a lambda gt11 cDNA expression library. The availability of this clone will allow direct analysis of mutant receptor mRNA's and genes as well as provide a powerful reagent for the study of glucocorticoid receptor gene organization and regulation. Thus, we are in an advantageous position to study both the protein chemistry of mutant glucocorticoid receptors and the mutated genes which encode them. (D)
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