Protein Inhibitor of Activated Signal Transducer and Activator of Transcription 1 Interacts with the N-Terminal Domain of Mineralocorticoid Receptor and Represses Its Transcriptional Activity: Implication of Small Ubiquitin-Related Modifier 1 Modification

Abstract

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators. To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASxbeta, described as SUMO (small ubiquitin-related modifier) E3-ligases. Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated. Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation. The agonist or antagonist nature of the bound ligand also determines PIAS1 corepressive action. We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo. Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties. Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status. Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications.

Structure–function relationships in the mineralocorticoid receptor

“…Both PIAS1 and PIAS xb behave as small ubiquitin-related modifier-3 (SUMO-E3) ligases able to sumoylate MR both in vitro and in vivo (Metivier et al 2000, Mihailidou et al 2004; PIAS1 is a MR-specific corepressor which interacts with the NTD. Interestingly, repression of transcriptional activity of MR mediated by PIAS1 is both dependent and independent of the MR's sumoylation status (Pascual-Le Tallec et al 2003). The SUMO-E2 activating enzyme, Ubc9, interacts with the MR-NTD/ DBD (1-670 amino acids) to potentiate aldosteronedependent MR transactivation (Yokota et al 2007).…”

The Synthetic Androgen Methyltrienolone (R1881) Acts as a Potent Antagonist of the Mineralocorticoid Receptor

“…The N-terminal domain harbors a ligand-independent activation function. This domain is important for interactions with transcriptional coactivators (Kitagawa et al, 2002;Pascual-Le Tallec et al, 2003) and with the ligand binding domain (LBD) (Rogerson and Fuller, 2003). The centrally located DNA binding domain is involved in DNA binding and in receptor homo-and heterodimerization.…”

GEMIN4 functions as a coregulator of the mineralocorticoid receptor

“…It is thought that allosteric changes in nuclear receptor conformation resulting from the interaction of the receptor with distinct DNA response elements may alter its binding affinity for coactivators (Wood et al 2001, Klinge et al 2004. Promoter dependence has been demonstrated for the MR corepressor, protein inhibitor of activated STAT1 (PIAS1), which depended on sumoylation of the MR for its repressive effect at the glucocorticoid response element promoter, but was sumoylation independent at the MMTV promoter (Pascual-Le Tallec et al 2003). Based on the literature, it seems plausible that the response elements for the various MR target genes in HEK293 cells may each have their own unique structures and induce distinct MR conformations such that GEMIN4 binds differentially to each promoter and produces gene-specific transcriptional modulation.…”