The androgen receptor (AR) plays a central role in sexual differentiation. Until recently it has been difficult, if not impossible, to study many of the functional and physiological properties of this protein. Our laboratory has recently cloned and sequenced the full-length human and mouse androgen receptor cDNA's. Mutations associated with androgen insensitivity were identified in both mouse and human. The receptor protein was expressed both in vitro with a reticulocyte lysate system and in vivo in E. coli as a fusion protein. The expressed receptor was recognized by an androgen receptor-specific monoclonal antibody and exhibited both androgen-specific and DNA-binding activities. The current grant proposes to identify amino acids which are critical for the functional activity of the androgen receptor by defining natural mutations in both human (androgen insensitive patients) and mouse (testicular feminization mutations). Recombinant androgen receptor protein will be expressed in the eukaryotic baculovirus expression system. The recombinant protein will be purified and characterized wit respect to its steroid- and DNA-binding domains. Both deletion- and point-mutations will be characterized by polymerase chain reactions and Southern blot analyses. By combining current knowledge from (i) other steroid receptors, (ii) the androgen receptor's primary structure (DNA and amino acid sequences), and (iii) mutations found in human and mouse AR, critical amino acids will be assessed. The androgen receptor gene will be mutagenized and the mutated receptor will be analyzed by transfection studies with regards to steroid binding, DNA binding and transactivation properties. Although the androgen receptor mRNA has been shown to be down-regulated by its own ligand in reproductive tissues, little is known about the regulatory mechanism at the molecular level. We have isolated a 10 Kbp fragment from the 5'-flanking region of the gene. Several complementary approaches will be used to identify and characterize putative negative androgen response elements and other cis regulatory elements which may be important for control of AR gene expression. These will include: 1) gene transfer experiments, 2) filter binding assays, 3) in vitro DNA-protein interaction assays (e.g., DNase 1 foot printing, band shifting, methylation interference, etc.) and 4) mutational analysis. Subsequently, transacting factor(s) bound to the putative prostate-specific cis acting elements will be identified and characterized biochemically. These studies should lead to a better understanding of the molecular properties of the androgen receptor and its role in mediating androgen action in reproductive organs.
