The mineralocorticoid receptor (MR) is a nuclear receptor that plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to cardiac fibrosis and failure. The molecular mechanisms for tissue-specific MR activation are poorly understood. Coregulators have been implicated as they are critical for nuclear receptor-mediated gene expression and are diverse in their structure, tissue distribution and function. Unfortunately, there is only a limited repertoire of MR coregulators described. To address this issue, we used phage display to screen for novel MR coregulators.
Our initial studies using M13 phage display identified a consensus motif amongst MR-interacting peptides, MPxLxxLL [1]. Gem-associated protein 4 (Gemin4) contains this motif. In assays performed in two different cell lines, Gemin4 repressed agonist-induced MR transactivation in a cell-specific manner. Furthermore, overexpression of Gemin4 significantly decreased, while knockdown of Gemin4 increased, the mRNA expression of specific endogenous MR target genes. A physical interaction between Gemin4 and MR is suggested by their nuclear co-localisation upon agonist treatment.
We then used T7 phage display to screen tissue-specific cDNA libraries and identified 30 MR binding peptides. Three were chosen for further characterization based on their nuclear localization, interaction with other MR-interacting proteins, and/or known status as a nuclear receptor coregulator. Eukaryotic elongation factor 1A1 (EEF1A1), structure-specific recognition protein 1 (SSRP1), and x-ray repair cross-complementing protein 6 (XRCC6) modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner, and co-localized 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 Gemin4, EEF1A1, SSRP1 and XRCC6 act as novel MR coregulators whose activity is dependent on ligand, cellular context and target gene promoter.