Vol. 54, Issue 4, 616-622, October 1998

Identification of a Key Amino Acid of the ₂-Adrenergic Receptor for High Affinity Binding of Salmeterol

Masafumi Isogaya,¹ Yoko Yamagiwa, Shigeo Fujita, Yoshiyuki Sugimoto, Taku Nagao, and Hitoshi Kurose

Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan (M.I., Y.S., T.N., H.K.), and Molecular Chemistry Research Laboratory, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ibaraki 305-8585, Japan (Y.Y., S.F.)

Transmembrane domains (TMDs) I, II, and VII of the ₂-adrenergic receptor (₂AR) were replaced, individually or in combination, with the corresponding regions of the ₁AR, and vice versa. The ₂-selective binding of salmeterol was not affected by the exchange of TMD I between the ₁- and ₂ARs. The affinity of salmeterol was slightly decreased (32-fold) by replacement of TMD II of the ₂AR with the homologous region of the ₁AR; the affinity was strongly decreased (1870-fold) for the ₂AR with TMD VII of the ₁AR. The affinity of salmeterol was partially restored by the introduction of TMD VII, but not TMD II, of the ₂AR into the ₁AR. 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 ₂AR, and a derivative with no ether oxygen showed 147-fold decreased affinity for the wild-type ₂AR. These results indicate that Tyr308 in TMD VII is the major amino acid conferring the ₂-selective binding of salmeterol to the ₂AR and that the position of the ether oxygen in the side chain is also important for ₂-selective binding. A three-dimensional model of the salmeterol-₂AR 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.