Introduction:
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.
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Methods:
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.
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Preliminary Results:
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.
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