Abstract P100: Interaction of the Mineralocorticoid Receptor with RACK1

Abstract

The mineralocorticoid receptor (MR) is a multifunctional ligand dependent transcription factor in the steroid receptor superfamily. The MR mediates aldosterone regulation of electrolytes and blood pressure. MR transcriptional co-regulators to influence specific gene transcription. We used a yeast two-hybrid system to find proteins that interact with a full-length MR as bait. Results: Among other proteins, we found a specific interaction of MR with RACK1 (Receptor for Activated C Kinase 1), a scaffolding protein. Overexpression of RACK1 using a tetracycline-inducible lentivirus in mouse cortical collecting duct M1 cells stably expressing a reporter Gaussia luciferase gene under a hormone-response element promoter resulted in enhanced agonist-dependent MR transactivation. Inhibition of RACK1 protein expression by short hairpin RNA led to a significant reduction in MR activation of the reporter gene. RACK1 regulation of MR action is mediated through the PKC-β signaling pathway. MR and RACK1 co-precipitated using a MR antibody in Sprague-Dawley brain tissue and M1 cells and immunofluorescent histochemistry showed MR and RACK1 co-localization in Male Sprague-Dawley brains and M1 cells. Conclusion: The scaffolding protein RACK1 is associated with MR under basal and agonist stimulated conditions and facilitates agonist stimulated MR actions through PKC-β. These findings indicate that RACK1 function as a novel co-activator of MR.