The actions of dihydrotestosterone, a hormone of central importance in controlling the function and growth of male reproductive tissues, such as the prostate and seminal vesicle, appear to be mediated interaction with an intracellular protein, the androgen receptor. Although much is known about the nature of this protein, little is known about the gene which codes for its mRNA or the regulation of the gene during development and following hormonal stimulation. Our initial efforts have been to purify and characterize the androgen receptor from several target tissues. The focus of the current proposal is to obtain cDNA probes which can be used to elucidate the molecular properties of the androgen receptor gene. In order to accomplish this goal, a monospecific antibody will be raised against the purified androgen receptor. The antibody will be characterized with respect to its specificity and affinity for the androgen receptor binding activity. A Lambdagt11 library will be screened for the expression of androgen receptors with the use of the anti-receptor antibodies, and a full-length cDNA clone will be isolated and sequenced. A number of properties of the cDNA will be examined in order to verify that it codes for the androgen receptor. First, we will examine the tissue distribution and content of the mRNA recognized by the cDNA by Northern dot blot analysis and compare these levels with receptor binding activity in order to demonstrate that there is a correlation between the two. Both normal and testicular feminized (Tfm) rats, and mice which have defective receptors, will be used in these studies. Second, we will determine if the gene coding for the cDNA is located on the X chromosome. Third, we will utilize fusion protein analysis and amino acid sequencing to confirm that cDNA is indeed coding for the receptor. Fourth, we will produce mRNA from plasmids containing a bacteriophage SP6 promoter and translate the message in vitro to determine if it codes for the androgen receptor. The in vitro product will be analyzed by peptide fragmentation, steroid binding and DNA binding analyses in order to compare it with the purified receptor. These studies should yield new information regarding the molecular properties of the androgen receptor. Availability of such probes will significantly enhance our ability to study androgen receptors at the molecular level and may help us understand androgen-related diseases such as benign prostate hyperplasia.
