OR26-2 mTOR Interacts with the Mineralocorticoid Receptor to Regulate its Activation by Ligand

Abstract

Abstract Background Phosphorylation of the mineralocorticoid receptor (MR) at serine 843 inhibits its activity. mTOR phosphorylation of ULK1 prevents this MR phosphorylation. Purpose To study the relationship between mTOR and MR and its role in the modulation of aldosterone signaling. Methods Mouse cortical collecting duct M1 cells stably expressing the rat wild type MR or mutated MR (mu/S843A) and a Gaussia luciferase gene reporter under a hormone response promoter tyrosine aminotransferase (TAT) were incubated with mTOR activator and inhibitors in the presence and absence of aldosterone or corticosterone. Co-immunoprecipitation was performed to study protein interactions with the MR, and a phospho-serine antibody was used to demonstrate MR and ULK1 phosphorylation. Effects of mTOR knockdown in M1 cells by lentiviral transduction with CRISPR/gRNA for Raptor and Rictor were also studied. Results mTOR and the adaptor proteins of mTOR complex 1 and 2, Raptor and Rictor, respectively, co-precipitated with the MR of M1-rMR cells. Expression levels of these proteins were not changed by aldosterone. mTOR inhibitors significantly reduced ligand-induced MR reporter gene transactivation, however the mTOR activator MHY1485 had no effect. CRISPR/gRNA gene knockdown of Raptor or Rictor also inhibited MR activation stimulated by ligands, supporting a role of mTOR in maintaining the active MR. The mTOR inhibitor AZD8055 attenuated ligand-mediated MR gene transcription assessed by the reporter gene product and measurement of products of the endogenous MR target genes ACSBG1, LCN2 and Psca. AZD8055 also decreased phospho-ULK1 levels in a dose-dependent manner. We thus assumed that mTOR decreases ULK1 activity and prevents ULK1 phosphorylation of the MR at Serine (S843). Indeed, phosphorylation of the MR was increased in M1-rMR cells treated with AZD8055. However, AZD8055 still inhibited ligand-induced MR activity in cells carrying the mutated MR in which the Serine at 843 was replaced with an Alanine. These results suggest that mTOR has an additional role in MR activity unrelated to the specific phosphorylation at S843 by ULK1. This merits further investigation. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.