H11-H12 LOOP RETINOIC ACID RECEPTOR MUTANTS EXHIBIT DISTINCT TRANSACTIVATING AND TRANS-REPRESSING ACTIVITIES IN THE PRESENCE OF NATURAL OR SYNTHETIC RETINOIDS

Retinoids, such as the naturally occurring all-trans-retinoic acid (at RA) and synthetic ligand CD367 modulate ligand-dependent transcription through retinoic acid receptors (RARs), Retinoid binding to RAR is be lieved to trigger structural transitions in the ligand-binding domain (LBD), leading to helix H1 and helix H12 repositioning and coactivator recruitment and corepressor release. Heir, we carried out a detailed mutagenesis analysis of the H11-H12 loop (designated the L box) to stu dy its contribution to hRAR alpha activation process. Point mutations that reduced transactivation by atRA also reduced atRA-induced transre pression of AP1 transcription, correlating ligand-induced activation a nd repression. However, a correlation was not observed with these muta tions when tested with another ligand CD367, a synthetic agonist with binding properties identical to those of atRA. Transcription was stron gly inhibited in the presence of CD367 for some mutants, thus leading to an inverse agonist activity of this ligand. None of these mutations significantly altered binding affinity for either ligand, indicating that altered transcription was not caused by altered ligand binding by these mutations, Although simple correlations with transcriptional ac tivities were not found, these mutations were also characterized by al tered ligand-induced structural transitions, which were distinct for t he atRA-hRAR alpha or CD367-hRAR alpha complexes, These results indica te that amino acids in the L box are involved in specifying trans-repr essive and transactivating properties of the hRAR alpha, and support t he notion that different agonists induce distinct conformations in the LED of the receptor.