Major advances in androgen receptor biochemistry require the isolation and sequencing of the complementary DNA (cDNA) for the androgen receptor. The goal is made possible by the recent identification from a human X chromosome library of a 15-kb DNA fragment that contains the coding sequence for the cysteine-rich DNA binding domain characteristic of steroid receptors. Its authenticity as the androgen receptor clone is strengthened by its localization between the centromere and chromosome map position q13, the region of the X chromosome shown by earlier genetic experiments to contain the androgen receptor gene. In this proposal recombinant DNA techniques will be used to complete the isolation and sequencing of androgen receptor cDNA and genomic clones until the entire coding sequence is determined. The cDNA libraries prepared in our laboratory for screening include human foreskin fibroblast, epididymis and liver, and rat testis and epididymis, all cloned in the expression vector lambda gt 11. Further proof of the identity of androgen receptor clones will come through transient expression in mammalian cells demonstrating that a cDNA codes for a specific high affinity androgen binding protein. Production of large quantities of receptor for characterization will be achieved by stable expression using a bovine papilloma virus vector or other available vectors. Co-transfection systems will allow assessment of biological activity of restricted regions of coding DNA. Androgen receptor cDNA clones will make possible studies on hormone regulation of receptor messenger RNA and serve as probes in analyzing mutations associated with the androgen insensitivity syndrome and male infertility. Selected regions of amino acid sequence deduced from DNA sequence will be used to design synthetic peptides for polyclonal antibody production. Receptor-specific antibodies will make feasible the immunocytochemical localization of receptor within and among different cell types, and western blot analysis of normal and mutant receptors. Receptor antibodies will add a new dimension to the present methods of determining receptor status in prostate cancer through the development of immunocytochemical, radioimmuno- and ELISA assays. Epitope-specific antibodies will allow mapping of receptor domains with respect to the binding of androgen, interaction with the nonsteroid binding protein components of the nontransformed receptor, and binding to specific DNA sequences. The metal ion required the role of phosphorylation in receptor transformation and DNA binding capability will be examined using receptor-specific antibodies. Continued study of the nonsteroid binding component of the nontransformed an drogen receptor will establish its relationship to the 90 kdal heat shock protein, its function in maintaining androgen binding activity, and its role in androgen regulation of gene expression.
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