The androgen receptor (AR) is a ligand activated transcriptional regulatory protein that mediates androgen induced male sexual development and function. Sequence analysis of AR cDNA confirmed it as one in the family of steroid hormone receptor transcription factors who share a structural arrangement of the major functional domains. The approach of site directed mutagenesis and transient expression will be used to further delineate and characterize specific functional domains of the AR protein. AR phosphorylation sites will be established by mutagenesis of consensus kinase recognition sequences and by direct phosphoamino acid analysis of AR protein. Effects of phosphorylation on AR function will be determined by coexpression of mutant and wild type AR expression vectors in the luciferase reporter gene assay using a variety of cell types and promoter sequences. The role of phosphorylation in AR activation, hormone binding, specific DNA binding and gene activation will be determined. Results with expressed recombinant AR will be corroborated and extended by studies on endogenous AR in a hormone responsive highly differentiated Leydig cell tumor cell line, MA10 cells, that contain a high level of AR. The essential residues of the AR nuclear targeting signal will be delineated by testing for the existence of a bipartite nuclear targeting signal that spans portions of exons C and D encoding the DNA binding and hinge regions. Cell factors required for AR nuclear transport will be investigated using a permeabilized cell system in vitro. We will further define the AR N-terminal and hinge region sequences required for transactivation, and pursue the hypothesis that a leucine zipper motif in the N-terminal domain is involved in protein/protein interactions that facilitate transactivation by AR. The role of phosphoamino acids in modulating these actions will also be investigated. AR homo- and/or heterodimerization will be studied with a focus on two proteins that interact with AR to enhance DNA binding (AR binding proteins l and 2). The requirement for AR oligomerization in DNA binding and in gene activation will be determined. Finally, an in vitro transcription system will be established in which the molecular requirements for AR initiation of gene transcription will be investigated. These studies will be facilitated by our recent success in high level expression of AR from a baculovirus vector in the Trichoplusia ni cabbage looper system for AR isolation. Studies outlined in this proposal will contribute to the understanding of molecular mechanisms of androgen regulated gene transcription.
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