title: Mutagenesis of the ligand binding domain of the human retinoic acid receptor alpha identifies critical residues for 9-cis-retinoic acid binding.
abstract: We have recently identified a small region (amino acids 405-419) within the ligand binding domain of a truncated human retinoic acid receptor alpha (delta 419) that is required for binding of 9-cis-retinoic acid (RA), but not all-trans-retinoic acid (t-RA). To probe the structural determinants of this high affinity 9-cis-RA binding site, a series of delta 419 mutants were prepared whereby an individual alanine residue was substituted for each amino acid within this region. These modified receptors were expressed in mammalian COS-1 cells and assayed for their ability to bind 9-cis-RA as well as t-RA. Only two of the mutants, M406A (mutation of methionine 406 to alanine), and I410A (mutation of isoleucine 410 to alanine) exhibit no detectable binding of 9-cis-RA when analyzed using saturation binding kinetics. Substitution of methionine 406 with the amino acids leucine, isoleucine, and valine yields mutant receptors that exhibit decreased binding for 9-cis-RA as the length or hydrophobicity of the R group decreases. Further substitution of methionine 406 with the small polar amino acid, threonine, results in a loss of detectable 9-cis-RA binding. Since amino acids 405-419 on a human RAR alpha (hRAR alpha) are predicted to form a short amphipathic alpha-helix, modeling of this structure into a helical wheel indicates that these two amino acids, methionine 406 and isoleucine 410, are actually positioned proximal to each other. Data presented here suggest that high affinity 9-cis-RA binding to a hRAR alpha depends on an interaction with the two amino acids methionine 406 and isoleucine 410.
authors.affiliation: Department of Metabolic Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110, USA.
authors.forename: B F
authors.initials: BF
authors.lastname: Tate
authors.forename: J F
authors.initials: JF
authors.lastname: Grippo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D000072482:Retinoic Acid Receptor alpha; D013237:Stereoisomerism; D013379:Substrate Specificity; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism*
chemical_list: C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D000072482:Retinoic Acid Receptor alpha; D014212:Tretinoin