Structural analysis and molecular modeling of high affinity binding and activation of β1-adrenergic receptor

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(-)-RO363 is one of the β_1-selective agonists, and the affinity of (-)-RO363 for β_1AR is about 100-fold and 3000-fold higher than for β_2AR and β_3AR, respectively. Therefore, (-)-RO363 can be used as a tool to examine the binding site of β_1AR and a role in receptor activation by β_1-selective agonists. We made chimeric β_1/β_2ARs and Ala-substituted β_1ARs, and found several key amino acids responsible for β_1-selective binding of (-)-RO363 (Leu^<110> and Thr^<117> in the second transmembrane domain (TMD 2), and Phe^<359> in TMD 7). To analyze a role of these amino acids in the activation step, we made several mutants of a constitutively active (CA-) β_1AR with Ala substitution of the key amino acids responsible for β_1-selective binding. The degree of decrease in the affinity of CA-β_1AR for (-)-RO363 was essentially the same as that of wild type β_1AR when mutated at Leu^<110> and Thr^<117>. However, the affinity was decreased in Ala-substituted mutant of Phe^<359> compared to that of wild type β_1AR.Therefore, we concluded that Phe^<359> may be required for a conformational change of β_1AR induced by binding of (-)-RO363, and Leu^<110> and Thr^<117> may be necessary for the initial binding of (-)-RO363 with high affinity and Phe^<359> interacts with the N-substituent of (-)-RO363. 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 β_1AR form a binding pocket ; the methoxy phenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu^<110>, Thr^<117>, and Phe^<359>, and Leu^<110> and Phe^<359> interact with the phenyl ring of (-)-RO363, whereas Thr^<117> forms hydrogen bond with the methoxy group of (-)-RO363. These results can be also used for better drug design.