title: Use of phage display to identify novel mineralocorticoid receptor-interacting proteins.
abstract: The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter.
authors.affiliation: MIMR-PHI Medical Research Institute (J.Y., P.J.F., J.M., C.D.C., M.J.Y.), Department of Medicine (J.Y., P.J.F., M.J.Y.), Monash University, Clayton, Victoria 3168, Australia; Department of Endocrinology, Metabolism, Rheumatology, and Nephrology (H.S.), Oita University, Yufu 879-5593, Japan; and Department of Pharmacology and Cancer Biology (D.P.M.), Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Fuller
authors.forename: James
authors.initials: J
authors.lastname: Morgan
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Shibata
authors.forename: Donald P
authors.initials: DP
authors.lastname: McDonnell
authors.forename: Colin D
authors.initials: CD
authors.lastname: Clyne
authors.forename: Morag J
authors.initials: MJ
authors.lastname: Young
mesh_terms: D034961:Antigens, Nuclear / Q000378:metabolism; D017123:Bacteriophage T7 / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D057809:HEK293 Cells; D006609:High Mobility Group Proteins / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D008024:Ligands; D008856:Microscopy, Fluorescence; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D019151:Peptide Library; D011944:Receptors, Androgen / Q000378:metabolism; D018161:Receptors, Mineralocorticoid / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D035602:Transcriptional Elongation Factors / Q000378:metabolism
chemical_list: D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C094814:EEF1A1 protein, human; D006609:High Mobility Group Proteins; D008024:Ligands; D020648:Peptide Elongation Factor 1; D019151:Peptide Library; D011944:Receptors, Androgen; D018161:Receptors, Mineralocorticoid; C073537:SSRP1 protein, human; D035602:Transcriptional Elongation Factors; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen