Participation of critical residues from the extreme C-terminal end of the human androgen receptor in the ligand binding function

A Short C-terminal end is present at the end of the human androgen receptor (hAR) similar to that of other steroid receptors. It is located directly a fter helix 12 of the ligand binding domain and has never been described as being part of the hydrophobic binding pocket. Although some fragmentary dat a have indicated the involvement of this region in ligand binding, its prec ise function still remains unclear. To gain deeper insight into the role of the hAR extreme C-terminal end, an extensive mutational analysis was carri ed out by using site-directed mutagenesis and alanine scanning over the 13- residue C-terminal end region. Both ligand binding and transcriptional acti vity were tested with each mutant. Our study demonstrates the participation of almost all of the amino acids in this region for the ligand binding fun ction and consequently for the transcriptional activity. A conformational s tudy by limited proteolysis was performed with the mutants that most affect ed the affinity of the receptor. It was remarkable that the mutants with a low binding affinity adopted an inactive conformation and were either less or not able to undergo a following conformational change to provide the act ive form of the receptor. Our results demonstrate the importance of hydroph obicity for the function of the C-terminal end with residues located at ver y precise positions. Especially, both hydrophobicity and aromaticity on pos ition 916 are critical for providing the correct ligand binding conformatio n of the receptor. Furthermore, this study highlights essential criteria re garding the C-terminal amino acids which could be applied to other steroid receptors.