Heterodimer formation with retinoic acid receptor RXRα modulates coactivator recruitment by peroxisome proliferator-activated receptor PPARγ

  • Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET–based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.
Author:Whitney KiluORCiDGND, Daniel MerkORCiDGND, Dieter SteinhilberORCiDGND, Ewgenij ProschakORCiDGND, Jan Peter HeeringORCiDGND
Parent Title (English):The journal of biological chemistry
Publisher:ASBMB Publications
Place of publication:Bethesda, Md.
Document Type:Article
Date of Publication (online):2021/05/31
Date of first Publication:2021/05/31
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2023/02/13
Tag:PPARγ; RXRα; allostery; cofactor recruitment; drug target; heterodimer; homodimer; homogeneous time-resolved FRET (HTRF)
Issue:1, art. 100814
Article Number:100814
Page Number:16
First Page:1
Last Page:16
This work was supported by the Landes-Offensive zur Entwicklung Wissenschaftlich oekonomischer Exzellenz (LOEWE) of the State of Hessen, and Research Center for Translational Medicine and Pharmacology TMP.
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Biochemie, Chemie und Pharmazie / Pharmazie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung 4.0