Identification of a key amino acid of the β₂-adrenergic receptor for high affinity binding of salmeterol

Transmembrane domains (TMDs) I, II, and VII of the β₂-adrenergic receptor (β₂AR) were replaced, individually or in combination, with the corresponding regions of the β₁AR, and vice versa. The β₂-selective binding of salmeterol was not affected by the exchange of TMD I between the β₁- and β₂ARs. The affinity of salmeterol was slightly decreased (32- fold) by replacement of TMD II of the β₂AR with the homologous region of the β₁AR; the affinity was strongly decreased (1870-fold) for the β₂AR with TMD VII of the β₁AR. The affinity of salmeterol was partially restored by the introduction of TMD VII, but not TMD II, of the β₂AR into the β₁AR. By analyzing alanine-substituted mutants, we found that Tyr308 in TMD VII was mainly responsible for the high affinity binding of salmeterol. Two salmeterol derivatives with the ether oxygen at different positions in the side chain showed 33- and 64-fold decreased affinities for the wild-type β₂AR, and a derivative with no ether oxygen showed 147-fold decreased affinity for the wild-type β₂AR. These results indicate that Tyr308 in TMD VII is the major amino acid conferring the β₂-selective binding of salmeterol to the β₂AR and that the position of the ether oxygen in the side chain is also important for β₂-selective binding. A three-dimensional model of the salmeterol-β₂AR complex shows that the phenyl group of Tyr308 interacts with methylene groups near the protonated amine of salmeterol and the ether oxygen interacts with Tyr316.