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

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Abstract
The functional significance of conserved polar amino acids within the putative transmembrane region of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster gene encoding beta AR 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 beta AR did not show detectable binding toward the antagonist 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 agonists by 1 order of magnitude. Furthermore, we observed that substitution of the proline at position 323 with a serine residue resulted in improper or incomplete processing of the beta AR, presumably reflecting a role for this residue in the folding of the receptor. Together with our previous results from deletion mutagenesis studies, these observations indicate that the ligand binding site involves the transmembrane region of the beta AR.