The objective is to understand the mechanism of androgen receptor regulation of gene expression. The immediate goals are to [1] purify androgen receptor and 8S androgen receptor-promoting factor (8S-PF) for preparation of monoclonal antibodies, [2] elucidate the mechanism of androgen receptor transformation to the active nuclear binding form, and [3] identify those components of the nuclear matrix required for receptor binding. Crucial toward progress in understanding the mechanism involved in androgen receptor activation of genes will be receptor purification and preparation of monoclonal antibodies. Methods for androgen receptor purification include affinity chromatography on steroid matrixes and an 8S-PF Sepharose affinity column. Through the use of 8S receptor stabilizing agents such as sodium molybdate and zinc, the nonactivated 8S receptor will be purified. Covalent coupling of a labeled androgen ([3H]7Alpha,17Alpha-dimethyl-19-nor-testosterone) to receptor will enable purification of receptor by electrophoresis on denaturing gels. Monoclonal antibodies to receptor and 8S-PF will be prepared which should permit precise immunocytochemical studies on cellular localization and provide a method for analysis of the steroid-free receptor. With regard to androgen receptor transformation and nuclear binding, a working model is proposed that incorporates the recent observations of this laboratory that zinc potentiates receptor binding to nuclear matrix whereas 8S-PF inhibits receptor binding. The effects of Zn2+, and other divalent cations, in particular Ca2+ and Mg2+, on the formation and dissociation of the receptor-8S-PF complex and on nuclear matrix binding will be investigated. The possible role of calmodulin in Ca2+-induced receptor transformation will be established. In addition, it will be determined if receptor phosphorylation effects receptor transformation and/or matrix binding. If a small RNA is found to be a component of the 8-9S receptor, its role in receptor transformation will be investigated. Receptor transformation will be monitored by sucrose gradient centrifugation and by binding to nuclear matrix and DNA-Sepharose. Finally, efforts will be made to identify, isolate and establish the androgen dependence of the acceptor component of nuclear matrix responsible for receptor binding.
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