IDENTIFICATION OF CRITICAL RESIDUES FOR HETERODIMERIZATION WITHIN THELIGAND-BINDING DOMAIN OF RETINOID-X-RECEPTOR

Nuclear receptors regulate transcription by binding to specific DNA re sponse elements as homodimers or heterodimers with the retinoid X rece ptors (RXRs), The identity box (I-box), a 40-amino acid region within the ligand-binding domains of RXRs and other nuclear receptors, was re cently shown to determine identity in the heterodimeric interactions. Here, we dissected this region in the yeast two-hybrid system by analy zing a series of chimeric receptors between human RXR alpha and rat he patocyte nuclear factor 4 (HNF4), a distinct member of the nuclear rec eptor superfamily that prefers homodimerization, We found that the C-t erminal Il-amino acid region of the RXR I-box was sufficient to direct chimeric receptors based on the HNF4 ligand-binding domain to heterod imerize with retinoic acid receptors or thyroid hormone receptors. Fur thermore, we identified the hRXR alpha amino acids A416 and R421 of th e Il-amino acid subregion as most critical determinants of heterodimer ic interactions; i.e. mutant HNF4s incorporating only the hRXR alpha A 416 or R421 heterodimerized with retinoic acid receptor.