Androgen receptor (AR) is a nuclear hormone receptor whose activity is a critical molecular determinant in the development of prostate cancer in men. In the absence of androgens, AR is localized to the cytoplasm bound by chaperones as an inactive protein complex. Upon binding androgens, AR translocates into the nucleus where it modulates the expression of downstream target genes. Proteins that bind AR are critical regulators of AR activity and thought to play an important role in inappropriate AR activation during prostate tumorigenesis. Thus, we have employed a large-scale immunoprecipitation strategy coupled to isotopic protein labeling via the incorporation of isotope coded affinity tags (ICATTM)to identify AR binding proteins using quantitative mass spectrometry.

Heterologous 293 human embryonic kidney (293HEK) cell lines expressing n-terminal and c-terminal tandem affinity tag (TAP) AR were used to identify AR containing protein complexes located in the cytosolic,membranous, and nuclear protein fractions through affinity purification followed by isotopic labeling and tandem mass spectrometry (MS/MS). A DNA affinity capture purification scheme was also utilized to identify AR/DNA protein complexes that bound to androgen response elements (ARE)located in the androgen-responsive rat probasin promoter.

Subsets of known and unknown AR proteins were detected based upon enriched ICAT ratios during AR purification. For example, heat shock 90 and 70 chaperones, known regulators of AR function were detected by our analysis. In depth bioinformatic analyses revealed AR co-purified with proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link to these pathways. Therefore,using traditional affinity purification strategies coupled to isotopic labeling and tandem MS/MS provided an effective platform for identifying protein networks that bind to AR in mammalian cells, which provides a molecular framework for probing AR function in normal and cancerous prostate epithelial cells.