High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate.

Analysis of mutations affecting the androgen receptor protein in human cells has been limited because of the low abundance and lability of these proteins in target tissues. All methods used to date have been based on the noncovalent interaction of radiolabeled androgens with the receptor's ligand binding site. We report here synthesis and use of the electrophilic affinity label dihydrotestosterone 17 beta-bromoacetate. This ligand, prepared as a radioactive compound of high specific activity, rapidly and covalently binds to a protein of 58,000 daltons in cytosol from normal genital skin fibroblasts. This protein is a high affinity, saturable specific binding site for the ligand and was not detectable in cultured cells from a subject with androgen resistance or in receptor-negative nongenital fibroblasts. The efficiency of incorporation of the covalent radiolabel into the 58-kD protein is greater than 80% based on estimates of receptor content using noncovalent ligands in intact cell assays. These studies demonstrate that dihydrotestosterone 17 beta-bromoacetate is useful for high efficiency covalent labeling of the human androgen receptor in crude cytosolic extracts from cultured cells.