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

β1-肾上腺素受体高亲和力结合和激活的结构分析和分子建模

• 批准号: 11672210
• 负责人: KUROSE Hitoshi
• 金额: $ 2.24万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672210/
• 关键词: β1-adrenergic receptor; transmembrane region; selective agonist; (-)-RO-363; constitutively active receptor; structure of ligand; アラニン置換型受容体; β_1アドレナリン受容体; 三次元モデル

(-)-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.

（-）- ro363是β_1选择性激动剂之一，其对β_1的亲和力分别比β_2和β_3高100倍和3000倍。因此，(-)- ro363可以作为检测β_1选择性激动剂对β_1受体激活的结合位点和作用的工具。我们制备了β_1/β_2ARs和α -取代β_1ARs嵌合，发现了几个与β_1选择性结合(-)- ro363（第二跨膜结构域（TMD 2）的Leu^<110>和Thr^<117>， TMD 7的Phe^<359>）的关键氨基酸。为了分析这些氨基酸在激活步骤中的作用，我们制造了几个具有组成活性(CA-) β_1- ar的突变体，其中负责β_1选择性结合的关键氨基酸被Ala取代。在Leu^<110>和Thr^<117>突变时，CA-β_1AR对(-)- ro363的亲和力下降程度与野生型β_1AR基本相同。然而，与野生型β_1AR相比，Phe^<359>的ala取代突变体的亲和力降低。因此，我们得出结论，(-)- ro363结合诱导β_1AR发生构像变化可能需要Phe^<359>, (-)- ro363高亲和力的初始结合可能需要Leu^<110>和Thr^<117>，并且Phe^<359>与(-)- ro363的n取代基相互作用。基于这些结果，我们建立了(-)- ro363结合域的三维模型。模型表明，β_1AR的TMD 2和TMD 7形成一个结合袋；（-）- ro363 n -取代基的甲氧基苯基似乎位于由Leu^<110>、Thr^<117>和Phe^<359>包围的空腔内，Leu^<110>和Phe^<359>与(-)- ro363的苯基环相互作用，而Thr^<117>与(-)- ro363的甲氧基形成氢键。这些结果也可以用于更好的药物设计。

项目成果

Sato, T.: "The contribution of third serine residue of β_2-adrenoceptor to interaction with and activation by isoproterenol"British Journal of Pharmacology. 128. 272-274 (1999)

Sato，T.：“β_2-肾上腺素受体的第三丝氨酸残基对异丙肾上腺素相互作用和激活的贡献”英国药理学杂志 128. 272-274 (1999)。

Shiina,T.: "Interaction with β-arrestin determines the difference in internalization behavior between β_1-and β_2-adrenergic receptors."J.Biol.Chem.. 275. 29082-29090 (2000)

Shiina, T.：“与 β-arrestin 的相互作用决定了 β_1-和 β_2-肾上腺素受体之间内化行为的差异。J.Biol.Chem.. 275. 29082-29090 (2000)

Shiina,T.: "Interaction with β-arrestin determines the difference in internalization behavior between β_1- and β_2-adrenergic receptors."J.Biol.Chem.. 275. 29082-29090 (2000)

Shiina, T.：“与 β-arrestin 的相互作用决定了 β_1- 和 β_2-肾上腺素受体之间内化行为的差异。J.Biol.Chem.. 275. 29082-29090 (2000)

Nishida,M.: "G_<iα> and G_<oα> are target proteins of reactive oxygen species."Nature. 408. 492-495 (2000)

Nishida, M.：“G_<iα> 和 G_<oα> 是活性氧的靶蛋白。”《自然》408. 492-495 (2000)

Shiina, T.: "Interaction with β-arrestin determines the difference in internalization behavior between β_1-and β_2-adrenergic receptors."J.Biol.Chem.. 275. 29082-29090 (2000)

Shiina, T.：“与 β-arrestin 的相互作用决定了 β_1-和 β_2-肾上腺素受体之间内化行为的差异。J.Biol.Chem.. 275. 29082-29090 (2000)