Endocrine Abstracts

The mineralocorticoid receptor (MR) has a modular domain structure containing a C-terminal ligand-binding domain (LBD) that consists of 11 α-helices (designated H1 to H12) organized in an antiparallel helical sandwich. The LBD undergoes a conformational change upon aldosterone binding such that H12 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. In the human MR, as in other nuclear receptors, replacement of a highly conserved glutamic acid with alanine in helix 12, eliminates interactions with LxxLL motif-containing co-activators (SRC-1, PGC1α and tesmin) but has little impact on ligand-binding. To determine the functional significance of the MR AF-2, in vivo, we have used CRISPR/Cas9 gene-editing technology to introduce the equivalent AF-2 mutation, E958A, into exon 12 of the mouse MR gene in a C57Bl/6 genetic background. These mice, bred to homozygosity (MRE958A), are viable without the fatal sodium wasting, seen for MR null and MR DBD mutant mice. This argues that intact AF-2 function in mice is not obligatory for MR-mediated sodium transport. Initial evaluation however shows a significant weight difference between both male and female wildtype and MRE958A mice. Metabolic cage analysis showed increased food and water intake in the MRE958A mice potentially as a potential compensation for mild salt and fluid loss. Both plasma and urinary aldosterone levels were markedly elevated and renal renin mRNA levels increased in MRE958A mice, that was further exacerbated on a low sodium diet. Given previous evidence for the importance of the MR LBD/AF2-coregulator interaction in mediating ligand-dependent transactivation, our finding of only a subtle physiological phenotype in the MRE958A mice is unexpected. This suggests that novel non-AF2 mediated mechanisms may play a role in MR-mediated transactivation.