title: Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription.
abstract: Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the "AR-interactome." Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Jordy J
authors.initials: JJ
authors.lastname: Hsiao
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Melinda M
authors.initials: MM
authors.lastname: Smits
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Brandon H
authors.initials: BH
authors.lastname: Ng
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Jinhee
authors.initials: J
authors.lastname: Lee
authors.affiliation: From the Department of Molecular Physiology and Biophysics, Carver College of Medicine, Iowa City, Iowa 52242 michael-e-wright@uiowa.edu.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Wright
mesh_terms: D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D020755:Coat Protein Complex I / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D020755:Coat Protein Complex I; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011944:Receptors, Androgen; D014157:Transcription Factors; C077335:TMF1 protein, human
keywords: androgen; androgen receptor; mass spectrometry (MS); protein trafficking (Golgi); transcription; transcription regulation