Isogaya M, Yamagiwa Y, Fujita S, Sugimoto Y, Nagao T, Kurose H
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
Mol Pharmacol. 1998 Oct;54(4):616-22.
Transmembrane domains (TMDs) I, II, and VII of the beta2-adrenergic receptor (beta2AR) were replaced, individually or in combination, with the corresponding regions of the beta1AR, and vice versa. The beta2-selective binding of salmeterol was not affected by the exchange of TMD I between the beta1- and beta2ARs. The affinity of salmeterol was slightly decreased (32-fold) by replacement of TMD II of the beta2AR with the homologous region of the beta1AR; the affinity was strongly decreased (1870-fold) for the beta2AR with TMD VII of the beta1AR. The affinity of salmeterol was partially restored by the introduction of TMD VII, but not TMD II, of the beta2AR into the beta1AR. By analyzing alanine-substituted mutants, we found that Tyr308 in TMD VII was mainly responsible for the high affinity binding of salmeterol. Two salmeterol derivatives with the ether oxygen at different positions in the side chain showed 33- and 64-fold decreased affinities for the wild-type beta2AR, and a derivative with no ether oxygen showed 147-fold decreased affinity for the wild-type beta2AR. These results indicate that Tyr308 in TMD VII is the major amino acid conferring the beta2-selective binding of salmeterol to the beta2AR and that the position of the ether oxygen in the side chain is also important for beta2-selective binding. A three-dimensional model of the salmeterol-beta2AR complex shows that the phenyl group of Tyr308 interacts with methylene groups near the protonated amine of salmeterol and the ether oxygen interacts with Tyr316.
将β2肾上腺素能受体（β2AR）的跨膜结构域（TMD）I、II和VII分别或组合替换为β1AR的相应区域，反之亦然。β1AR和β2AR之间TMD I的交换不影响沙美特罗对β2的选择性结合。用β1AR的同源区域替换β2AR的TMD II后，沙美特罗的亲和力略有下降（32倍）；用β1AR的TMD VII替换β2AR后，其亲和力大幅下降（1870倍）。将β2AR的TMD VII而非TMD II引入β1AR后，沙美特罗的亲和力部分恢复。通过分析丙氨酸取代突变体，我们发现TMD VII中的Tyr308是沙美特罗高亲和力结合的主要原因。两种侧链醚氧位置不同的沙美特罗衍生物对野生型β2AR的亲和力分别下降了33倍和64倍，一种无醚氧的衍生物对野生型β2AR的亲和力下降了147倍。这些结果表明，TMD VII中的Tyr308是赋予沙美特罗对β2ARβ2选择性结合的主要氨基酸，并且侧链中醚氧的位置对β2选择性结合也很重要。沙美特罗-β2AR复合物的三维模型显示，Tyr308的苯基与沙美特罗质子化胺附近的亚甲基相互作用，醚氧与Tyr316相互作用。
