The androgen receptor (AR) is a member of the nuclear receptor superfamily, a large group of ligand-activated transcription factors crucial to the development of prostate cancer and functions as part of a multiprotein complex which binds to the promoter regions of androgen responsive genes via specific DNA sequences called androgen response elements (AREs). Identification and characterization of the constituents of the AR transcription complex is essential to the development of strategies for controlling prostate cancer. Although a number of proteins have been shown to interact with the AR from studies using the yeast two-hybrid system and chromatin immunoprecipitation (CHIP), the AR transcription complex has yet to be purified and directly examined. This proposal will explore the technical feasibility of capturing the AR transcription complex using immobilized promoter DNA segments and applying a novel mass spectrometry (MS)-based proteomic method to examine these captured AR complexes. Two strategies will be used to identify the proteins in the complex. The first will compare the complexes captured on ARE-containing DNA template from LNCaP nuclear extract before and after stimulation with androgen, which causes translocation of AR from cytoplasm to nucleus to facilitate DNA binding. A second strategy will compare AR complexes bound to the three ARE-containing DNA templates with the same templates containing point mutations in the AREs that abrogate AR binding. Secondary aims will characterize the AR transcription complex in the presence of the androgen antagonist bicalutamide which has been reported to recruit transcriptional corepressors and to characterize the involvement of the proteosome in AR mediated transcription using the proteosome inhibitor MG-132. Once implemented, this method has the potential to reveal differences in transcription complex compositions under various physiological conditions such as escape from androgen ablation in cancer therapy. It also can be adapted to study the entire nuclear receptor family (by changing the hormone-specific DNA binding sites used for capture) to identify changes in the complexes that correlate with health, development and disease.