MON-270 An E958A Knock-in Mutation in the AF-2 Domain of the Mouse Mineralocorticoid Receptor Ligand Binding Domain Causes Activation of the Renin-Angiotensin-Aldosterone System

Abstract Disclosure: T.J. Cole: None. J. Ng: None. V. Wang: None. M.J. Young: None. J. Yang: None. P.J. Fuller: None. The mineralocorticoid receptor (MR) is an intracellular nuclear receptor that mediates physiological actions of the adrenal steroid ligands, aldosterone and cortisol. The MR contains a C-terminal ligand-binding domain (LBD) that consists of 11 α-helices organized in an antiparallel helical sandwich. The LBD undergoes a conformational change upon aldosterone binding such that helix 12 forms a stable interaction with helices 3, 4 and 5 to create an AF-2 domain, a hydrophobic cleft on the surface of the LBD, which serves as a docking platform for transcriptional coactivators. To determine the relative contribution to MR-mediated transactivation of the MR AF-2 domain in vivo, we have used CRISPR/Cas9 gene-editing technology to introduce a previously described, AF-2 mutation, E958A, into helix 12 of the MR LBD. These mice, bred to homozygosity (MRE958A), are viable without the fatal sodium wasting phenotype, seen for MR-null and MR-DBD C603S mutant mice, which argues that AF-2 function in mice is not obligatory for MR-mediated sodium transport. Initial phenotyping shows a significant weight difference between both male and female wildtype and MRE958A mice. Metabolic cage analyses showed increased food and water intake in the MRE958A mice potentially as compensation for mild salt and fluid loss. Both plasma and urinary aldosterone levels, and plasma renin were markedly elevated. Increased renal renin mRNA levels in MRE958A mice, were further exacerbated on a low sodium diet. The adrenal gland had increased zona glomerulosa immunostaining for aldosterone synthase with evidence of zona glomerulosa hyperplasia. Given previous evidence for the importance of the MR LBD/AF2-coregulator interaction in mediating ligand-dependent transactivation, these findings of a subtle physiological phenotype in the MRE958A mice are unexpected and suggest that novel non-AF2 mediated mechanisms may play a central role in MR-mediated transactivation. Presentation: Monday, July 14, 2025