Photoaffinity labeling of the androgen receptor from human skin fibroblasts

Abstract

The reproducible photolabeling of the androgen receptor from human skin fibroblasts, using [3H]methyltrienolone (R-1881) as ligand is described. Crude nuclei were irradiated for 2 min using a UV lamp with an emission line at 352 nm and a CuSO4 filter. After KCl extraction, proteins were precipitated with trichloroacetic acid, washed with ether and assayed for radioactivity. Specific binding was determined as the difference in bound radioactivity between cells incubated with [3H]R-1881 +/- a 200-fold excess of unlabeled dihydrotestosterone (DHT). The photolabeled proteins were analyzed on SDS-polyacrylamide gel electrophoresis yielding one peak of 90 kDa and in several cases, one of 43 kDa. These peaks comprised 60 +/- 20% of the saturable binding recovered on the gels. The overall efficiency of photolabeling was between 1 and 5%. The amount of covalently bound radioactivity was proportional to the number of cells used. The labeling was inhibited by R-1881, DHT, the anti-androgens hydroxyflutamide and cyproterone acetate and to a lesser extent by estradiol and progesterone. No covalent attachment of R-1881 to any protein was observed when nuclei from patients with androgen insensitivity were irradiated, whether or not the cells were receptor positive or negative. In conclusion the androgen receptor from human skin fibroblast can be efficiently photolabeled and could be used as a marker to follow receptor purification. The absence of photolabeling of nuclear extracts from receptor-positive androgen-insensitive patients may reflect some abnormality of the receptor.