Identification of critical residues for heterodimerization within the ligand-binding domain of retinoid X receptor

Nuclear receptors regulate transcription by binding to specific DNA response elements as homodimers or heterodimers with the retinoid X receptors (RXRs). The identity box (I-box), a 40-amino acid region within the ligand-binding domains of RXRs and other nuclear receptors, was recently shown to determine identity in the heterodimeric interactions. Here, we dissected this region in the yeast two-hybrid system by analyzing a series of chimeric receptors between human RXRalpha and rat hepatocyte nuclear factor 4 (HNF4), a distinct member of the nuclear receptor superfamily that prefers homodimerization. We found that the C-terminal 11-amino acid region of the RXR I-box was sufficient to direct chimeric receptors based on the HNF4 ligand-binding domain to heterodimerize with retinoic acid receptors or thyroid hormone receptors. Furthermore, we identified the hRXRalpha amino acids A416 and R421 of the 11-amino acid subregion as most critical determinants of heterodimeric interactions; i.e. mutant HNF4s incorporating only the hRXRalpha A416 or R421 heterodimerized with retinoic acid receptor.