SUBSTITUTION OF CYS-560 BY PHE, TRP, TYR, AND SER IN THE FIRST ZINC-FINGER OF HUMAN ANDROGEN RECEPTOR AFFECTS HORMONAL SENSITIVITY AND TRANSCRIPTIONAL ACTIVATION

We have established and characterized four human androgen receptor (AR ) mutants, AR C560F, C560W, C560Y, C560S). To assess the functional si gnificance of these substitutions, we compared the transcriptional act ivation, hormone binding affinity, receptor-DNA interaction, and the s ubcellular distribution of the hormone-receptor complexes. Binding stu dies showed that all mutants bound methyltrienolone (R1881) with wild type affinity (K-d = 0.5 nM). Transactivation efficiency, as compared to wild type AR, increased 150% with C560F and decreased to 70% with C 560W and C560Y and to 40% with mutant C560S. Subcellular receptor dist ribution showed that 85% of C560F bound with hormone was extracted fro m the nuclear fraction and 15% in the cytosol. Gel mobility shift assa ys showed that C560F expressed in CV-1 cells bound to an androgen resp onsive element (ARE) with equal efficiency as the wild type human AR. The mutants C560W and C560Y demonstrated a lower ability to bind to AR E, whereas C560S showed a significantly lower ability to interact with ARE. We propose that the change in polarity introduced into the loop structure by C560S leads to a shorter period of contact between the mu tant receptor and DNA resulting in decreased transcriptional activatio n levels.