Arginine of retinoic acid receptor β which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARα, RARβ, and RARγ) and retinoid X receptors (RXRα, RXRβ, and RXRγ). Consistent with the X-ray crystal structures of RARα and RARγ, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (αArg276, βArg269, and γArg278) for coordination with the carboxyl group of RA. However, mutation of Arg269 to Ala in RARβ causes only a 3- to 6-fold increase in the Kd for RA and EC50 in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARα or RARγ causes a 110-fold and a 45-fold increase in EC50 value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of βR269A to a mutant which mimics either RARα ligand selectivity (βA225S/R269A) or RARγ ligand selectivity (βI263M/R269A/V338A). Our results demonstrate that in RARβ mutants that acquire either RARα or RARγ ligand specificity the Arg269 position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARβ. Furthermore, three mutant receptors (βA225S/R269A, βA225S/F279, and αF286A) were found to have a greater than wild-type affinity for the RARα-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARβ and the X-ray crystal structures of the LBD of both RARα and RARγ are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg269 in RARβ compared to its homologous Arg in RARα and RARγ.