Vol. 301, Issue 1, 51-58, April 2002

₁-Selective Agonist ()-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [()-RO363] Differentially Interacts with Key Amino Acids Responsible for ₁-Selective Binding in Resting and Active States

Yoshiyuki Sugimoto¹ , Reiko Fujisawa, Ryuji Tanimura, Anne Laure Lattion, Susanna Cotecchia, Gozoh Tsujimoto, Taku Nagao and Hitoshi Kurose

Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (Y.S., R.F., T.N., H.K.); Toray Industries, Inc., Basic Research Laboratories, Kanagawa, Japan (R.T.); Department of Molecular, Cell Pharmacology, National Children's Medical Research Center, Tokyo, Japan (G.T.); and Institute de Pharmacologie et de Toxicologie, Lausanne, Switzerland (A.L.L., S.C.)

()-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [()-RO363] is a highly selective ₁-adrenergic receptor (₁AR) agonist. To study the binding site of ₁-selective agonist, chimeric ₁/₂ARs and Ala-substituted ₁ARs were constructed. Several key residues of ₁AR [Leu¹¹⁰ and Thr¹¹⁷ in transmembrane domain (TMD) 2], and Phe³⁵⁹ in TMD 7] were found to be responsible for ₁-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 ₁AR form a binding pocket; the methoxyphenyl group of N-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 ₁AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-₁AR mutant. The degree of decrease in the affinity of CA-₁AR for ()-RO363 was essentially the same as that of wild-type ₁AR when mutated at Leu¹¹⁰ and Thr¹¹⁷. However, the affinity was decreased in Ala-substituted mutant of Phe³⁵⁹ compared with that of wild-type ₁AR. These results indicated that Leu¹¹⁰ and Thr¹¹⁷ are necessary for the initial binding of ()-RO363 with ₁-selectivity, and interaction of Phe³⁵⁹ with the N-substituent of ()-RO363 in an active state is stronger than in the resting state.