Using Proteomics and Immunofluorescence to Investigate Potential Mechanisms Responsible for SARM Tissue-Selectivity

The androgen receptor (AR) is a member of the superfamily of nuclear receptors (NRs) that regulate gene expression in a ligand-dependent manner. Selective androgen receptor modulators (SARMs) are ligands that activate AR in a tissue-selective manner to produce unique physiological effects. To investigate the mechanism responsible for SARM tissue-selectivity, the conformation of AR while bound to the SARM-014, dihydrotestosterone (DHT), or bicalutamide was investigated using the NHS-biotin labeling technique. Briefly, ligand-bound AR was affinity purified from Sf9 cells and incubated with NHS-biotin to biotinylate surface accessible lysine residues. It was anticipated that ligand-specific biotinylation patterns would be detected using tandem MS. Unfortunately, no biotinylation was observed for any of the ligand-bound AR samples. The possibility that AR was blocked from biotinylation because of associated chaperone and cofactor proteins was subsequently investigated using native gels and immunoprecipitation (IP) in combination with LC/ESI-ITMS and tandem MS. The native gel results showed multiple ligand-specific bands, which suggests ligand-bound AR exists in multiple ligand-specific protein complexes. Consequently, when the largest protein band (> 680 kDa) for both the DHT and SARM treated samples was analyzed using tandem MS, ligand-specific cofactors were identified. Specifically, the nuclear receptor corepressor protein (N-CoR), which plays a role in regulating SERM tissueselective activity, was identified in only the SARM treated sample. The potential role of N-CoR in moderating SARM tissue-selective activity was subsequently explored using N-CoR IP experiments in LNCaP and Sf9 cells after ligand treatment. The IP results confirmed a higher degree of AR/NCoR association for SARM versus DHT treated cells. Lastly, slower cytoplasmic-nuclear transport and nuclear foci formation rates were observed for SARMversus DHT-bound AR using Western blots and immunofluorescence, respectively. Overall, by using a combination of proteomics and immunofluorescence, support for the presence of functionally distinct AR-SARM containing protein complexes, in terms of AR cellular transport and transactivation, has been generated. Most importantly, these functionally distinct protein complexes may account for SARM tissue-selectivity.