An Inverse Agonist GSK5182 Increases Protein Stability of the Orphan Nuclear Receptor ERRγ via Inhibition of Ubiquitination

Abstract

The orphan nuclear receptor, estrogen-related receptor gamma (ERR gamma) is a constitutively active transcription factor involved in mitochondrial metabolism and energy homeostasis. GSK5182, a specific inverse agonist of ERR gamma that inhibits transcriptional activity, induces a conformational change in ERR gamma, resulting in a loss of coactivator binding. However, the molecular mechanism underlying the stabilization of the ERR gamma protein by its inverse agonist remains largely unknown. In this study, we found that GSK5182 inhibited ubiquitination of ERR gamma, thereby stabilizing the ERR gamma protein, using cell-based assays and confocal image analysis. Y326 of ERR gamma was essential for stabilization by GSK5182, as ligand-induced stabilization of ERR gamma was not observed with the ERR gamma-Y326A mutant. GSK5182 suppressed ubiquitination of ERR gamma by the E3 ligase Parkin and subsequent degradation. The inhibitory activity of GSK5182 was strong even when the ERR gamma protein level was elevated, as ERR gamma bound to GSK5182 recruited a corepressor, small heterodimer partner-interacting leucine zipper (SMILE), through the activation function 2 (AF-2) domain, without alteration of the nuclear localization or DNA-binding ability of ERR gamma. In addition, the AF-2 domain of ERR gamma was critical for the regulation of protein stability. Mutants in the AF-2 domain were present at higher levels than the wild type in the absence of GSK5182. Furthermore, the ERR gamma-L449A/L451A mutant was no longer susceptible to GSK5182. Thus, the AF-2 domain of ERR gamma is responsible for the regulation of transcriptional activity and protein stability by GSK5182. These findings suggest that GSK5182 regulates ERR gamma by a unique molecular mechanism, increasing the inactive form of ERR gamma via inhibition of ubiquitination.