SYSTEMATIC ANALYSIS OF THE AF-2 DOMAIN OF HUMAN RETINOIC ACID RECEPTOR-ALPHA REVEALS AMINO-ACIDS CRITICAL FOR TRANSCRIPTIONAL ACTIVATION AND CONFORMATIONAL INTEGRITY

We previously identified a carboxy-terminal transactivation function t ermed AF-2 within the last 15 amino acids of the ligand binding domain of the human retinoic acid receptor alpha (hRAR alpha). Truncation of this region abolished transcriptional activity, Here we provide a sys tematic analysis using alanine scanning mutagenesis of amino acids fro m Ser(405) to Gly(419) on a truncated hRAR alpha (Delta 419) to identi fy residues within this region that are responsible for transcriptiona l activity, Whereas mutations in positions 405, 408, 411, and 415-419 have little or no effect on the ability of modified receptors to activ ate a DR5 response element, mutations in positions 406, 407, 409, 410, and 412-413 modify either the potency or efficacy of all-trans retino ic acid (tRA) -induced gene transcription, Therefore, receptors with m utations in positions 409, 410, 413, and 414 have low transcriptional activity over a wide range of tRA concentrations, Receptors with mutat ions in positions 406, 407, and 412 exhibit a maximum transcriptional activity similar to wild-type hRAR alpha, but require higher concentra tions of tRA, Replacing residues 405-419 on Delta 419 with the conserv ed AF-2 domain from the vitamin DQ receptor or the estrogen receptor r esults in a receptor with wild-type or low transcriptional activity, r espectively, A full-length hRAR alpha mutant with an alanine substitut ion at position 406 (hRAR alpha M406A) binds tRA, but unlike the trunc ated M406A, which lacks the ''F'' region, it is not transcriptionally active, Protease mapping experiments detect a consistent difference in the conformation of hRAR alpha M406A compared to wild-type hRAR alpha , These data define amino acids from Ser(405) to Gly(419) on Delta 419 that are critical for transcriptional activity and point to the impor tance of the conformational integrity of receptor domains in maintaini ng ligand-induced transcriptional activation.