Abstract

(−)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(−)-RO363] is a highly selective β₁-adrenergic receptor (β₁AR) agonist. To study the binding site of β₁-selective agonist, chimeric β₁/β₂ARs and Ala-substituted β₁ARs were constructed. Several key residues of β₁AR [Leu¹¹⁰ and Thr¹¹⁷ in transmembrane domain (TMD) 2], and Phe³⁵⁹ in TMD 7] were found to be responsible for β₁-selective binding of (−)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (−)-RO363. The model indicated that TMD 2 and TMD 7 of β₁AR form a binding pocket; the methoxyphenyl group ofN-substituent of (−)-RO363 seems to locate within the cavity surrounded by Leu¹¹⁰, Thr¹¹⁷, and Phe³⁵⁹. The amino acids Leu¹¹⁰ and Phe³⁵⁹ interact with the phenyl ring of (−)-RO363, whereas Thr¹¹⁷ forms hydrogen bond with the methoxy group of (−)-RO363. To examine the interaction of these residues with β₁AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-β₁AR mutant. The degree of decrease in the affinity of CA-β₁AR for (−)-RO363 was essentially the same as that of wild-type β₁AR when mutated at Leu¹¹⁰ and Thr¹¹⁷. However, the affinity was decreased in Ala-substituted mutant of Phe³⁵⁹ compared with that of wild-type β₁AR. These results indicated that Leu¹¹⁰ and Thr¹¹⁷ are necessary for the initial binding of (−)-RO363 with β₁-selectivity, and interaction of Phe³⁵⁹ with the N-substituent of (−)-RO363 in an active state is stronger than in the resting state.