IDENTIFICATION OF A KEY AMINO-ACID OF THE BETA(2)-ADRENERGIC RECEPTORFOR HIGH-AFFINITY BINDING OF SALMETEROL

Transmembrane domains (TMDs) I, II, and VII of the beta(2)-adrenergic receptor (beta(2)AR) were replaced, individually or in combination, wi th the corresponding regions of the beta(1)AR, and vice versa. The bet a(2)-selective binding of salmeterol was not affected by the exchange of TMD I between the beta(1)- and beta(2)ARs. The affinity of salmeter ol was slightly decreased (32-foId) by replacement of TMD II of the be ta(2)AR with the homologous region of the beta(1)AR; the affinity was strongly decreased (1870-fold) for the beta(2)AR with TMD VII of the b eta(1)AR. The affinity of salmeterol was partially restored by the int roduction of TMD VII, but not TMD II, of the beta(2)AR into the beta(1 )AR. By analyzing alanine-substituted mutants, we found that Tyr308 in TMD VII was mainly responsible for the high affinity binding of salme terol. Two salmeterol derivatives with the ether oxygen at different p ositions in the side chain showed 33- and 64-fold decreased affinities for the wild-type beta(2)AR, and a derivative with no ether oxygen sh owed 147-fold decreased affinity for the wild-type beta(2)AR. These re sults indicate that Tyr308 in TMD VII is the major amino acid conferri ng the beta(2)-selective binding of salmeterol to the beta(2)AR and th at the position of the ether oxygen in the side chain is also importan t for beta(2)-selective binding. A three-dimensional model of the salm eterol-beta(2)AR complex shows that the phenyl group of Tyr308 interac ts with methylene groups near the protonated amine of salmeterol and t he ether oxygen interacts with Tyr316.