Molecular cloning and functional characterization of the canine androgen receptor.

ABSTRACT

Sex steroids, including testosterone, play a major role in determining peak bone mass in mammals and the subsequent loss of total bone mass with advancing age. Testosterone, and its active metabolite dihydrotestosterone (DHT), bind with high affinity to the androgen receptor (AR), a member of the nuclear hormone receptor superfamily. These receptors function as transcription factors, binding together with accessory proteins to specific DNA response elements in the promoters of androgen responsive genes. To further characterize AR function in a model species of relevance to bone and pharmaceutical research, we cloned a partial canine AR from a canine kidney cDNA library and then cloned the remaining 5' segment by PCR from canine ventral prostate cDNA. The complete sequence obtained was 3577 bp. This sequence contained a single open reading frame of 2721 bp, potentially encoding a protein of 907 amino acids with a predicted molecular weight of 98.7 kD. Sequence analysis of the protein encoded by this open reading frame reveals that the modular domains providing the DNA binding and ligand binding functions are identical to those reported for eight other mammalian ARs. Northern analysis of poly-A+ RNA from ventral prostate revealed three very low abundance transcripts of approximately 9 kb and RT-PCR analysis showed relatively high expression of AR in canine ventral prostate, testis, and kidney, with lower levels detectable in spleen, skeletal muscle, heart, and liver. Competition binding studies using 3H-DHT as ligand demonstrated specific displacement by DHT, testosterone, and the anabolic steroid stanozolol, with IC50 values of 1.3, 2.5 and 3.8 nM, respectively. Binding of DHT also resulted in the stimulation of an androgen responsive-luciferase reporter following cotransfection with the canine AR into 293 cells. Immunohistochemistry using an antibody directed to the C-terminal 19 amino acids of the human AR showed strong staining of the secretory epithelial cells in canine ventral prostate. Together, these data indicate that we have cloned the canine AR and that its functional DNA binding and ligand binding domains are absolutely conserved with those reported for eight other species.