title: Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.
abstract: Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.
authors.affiliation: Department of Toxicology, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
authors.forename: B F
authors.initials: BF
authors.lastname: Tate
authors.forename: G
authors.initials: G
authors.lastname: Allenby
authors.forename: R
authors.initials: R
authors.lastname: Janocha
authors.forename: S
authors.initials: S
authors.lastname: Kazmer
authors.forename: J
authors.initials: J
authors.lastname: Speck
authors.forename: L J
authors.initials: LJ
authors.lastname: Sturzenbecker
authors.forename: P
authors.initials: P
authors.lastname: Abarzúa
authors.forename: A A
authors.initials: AA
authors.lastname: Levin
authors.forename: J F
authors.initials: JF
authors.lastname: Grippo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C000607071:RARA protein, human; C000607072:Rara protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin