The purpose of these proposed studies is the development of new tools for the analysis of the structure and function of the human androgen receptor protein. The specific goals of the proposal are: 1) the development of high affinity monoclonal antibodies capable of recognizing the human androgen receptor, 2) the application of photoaffinity labeling techniques to produce a radiolabeled steroid-receptor complex covalently linked at the steroid binding site, and 3) the further elucidation of the process by which androgen-receptor complexes acquire DNA-binding capacity. These tools will be applied to the study of the clinical syndromes of androgen resistance, a diverse clinical spectrum of disorders ranging from complete failure of male phenotypic development to the occurrence of unexplained oligospermia in otherwise normal men. The availability of the tools and techniques described will open new avenues for the elucidation of the molecular defects responsible for androgen resistance. The production of monoclonal antibodies that recognize the human androgen receptor will be approached by two routes. A conventional approach involving partial purification of the androgen receptor protein by affinity chromatography coupled with DNA-cellulose chromatography will be used to provide androgen receptor for immunization and production of monoclonal antibodies. In addition, anti-idiotypic antibodies against a purified high affinity monoclonal anti-dihydro-testosterone antibody will be produced and tested for their ability to recognize the androgen binding site of the authentic receptor. The production of these antibodies will be pursued using both conventional immunizations as well as newer in vitro techniques of immunization. In a separate series of experiments covalent linkage of photoreactive ligands to partially purified androgen receptors will be attempted; the photolabeled products will be analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Finally, the role of partial proteolysis in the generation of DNA-binding androgen receptor complexes, and in the lability of some androgen receptor complexes under transforming conditions will be evaluated using a DNA-cellulose binding assay coupled with the use of assays for cytosolic proteases using synthetic fluorogenic substrates. The availability of these tools and techniques will permit the initiation of a number of direct structural and functional studies of normal and mutant androgen receptor proteins, such as gel electrophoresis followed by blotting with antibody, electrophoresis of affinity labeled receptors or analyses of the DNA-binding capacity of partially proteolyzed receptors.
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| Kovacs, W J; Bell, B W; Turney, M K et al. (1988) Monoclonal antibodies to rat androgen-binding protein recognize both of its subunits and cross-react with rabbit and human testosterone-binding globulin. Endocrinology 122:2639-47 |
| Kovacs, W J; Turney, M K (1988) High efficiency covalent radiolabeling of the human androgen receptor. Studies in cultured fibroblasts using dihydrotestosterone 17 beta-bromoacetate. J Clin Invest 81:342-8 |
| Kovacs, W J; Olsen, N J (1987) Androgen receptors in human thymocytes. J Immunol 139:490-3 |
