Identification of residues required for ligand binding to the β-adrenergic receptor

The functional significance of conserved polar amino acids within the putative transmembrane region of the β-adrenergic receptor (βAR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding βAR and expression of the mutant genes in COS-7 cells. Although a substitution of aspartate at position 113 with an asparagine residue did not affect expression or processing of the protein, the resulting mutant βAR did not show detectable binding toward the antagonist [125I]-iodocyanopindolol. Replacement of the aspartate and asparagine residues at positions 79 and 318, respectively, had no effect on the affinity of the receptor toward antagonists but reduced the affinity of the receptor toward agonist by 1 order of magnitude. Furthermore, the authors observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the βAR, presumably reflecting a role for this residue in the folding of the receptor. Together with previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the βAR