Mutagenesis of the Ligand Binding Domain of the Human Retinoic Acid Receptor Identifies Critical Residues for 9-cis-Retinoic Acid Binding

• Published in: The Journal of biological chemistry 1995-09, Vol.270 (35), p.20258
• Main Authors: Bonnie F. Tate, Joseph F. Grippo
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.35.20258

We have recently identified a small region (amino acids 405-419) within the ligand binding domain of a truncated human retinoic acid receptor α (Δ419) 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 Δ419 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 RARα (hRARα) are predicted to form a short amphipathic α-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 hRARα depends on an interaction with the two amino acids methionine 406 and isoleucine 410.