OR12-03 Mineralocorticoid and Glucocorticoid Receptors Adopt Distinct Quaternary Structures and Can Form Heteromultimers That Affect Chromatin-Binding Profiles

The mineralocorticoid and glucocorticoid receptors (MR and GR) are evolutionarily related nuclear receptors with high sequence conservation and a shared hormone response element (HRE). Both receptors are activated by glucocorticoids, but MR can be selectively activated by aldosterone. Using the imaging technique Number & Brightness (N&B) it has recently been proposed that liganded GR dimers form tetramers upon binding to HREs in live cells. We now show that agonist-bound MR adopts a tetrameric organization in the nucleoplasm and forms complexes with an average of 7 receptor units upon binding an HRE. Interestingly, MR antagonists eplerenone and spironolactone induced intermediate oligomerization arrangements, strongly suggesting that higher order oligomerization is essential for receptor activity. Site-directed mutagenesis and deletion analysis suggest that the N-terminus of MR is a main determinant of higher order oligomerization. Both with corticosterone and aldosterone, GR can incorporate into MR complexes partially displacing MR monomers. Genome-wide chromatin binding studies suggest that the presence of GR in the same cells profoundly change MR interaction with distinct sets of enhancers in a ligand-dependent way, contributing to receptor-specific signaling. Certain genes respond to only one receptor while others respond to both receptors. The interaction of these two closely related receptors has important implications for the mechanisms for glucocorticoid signaling and transcription factors in general.

Abstract P100: Interaction of the Mineralocorticoid Receptor with RACK1

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.