Swaminath Gayathri, Deupi Xavier, Lee Tae Weon, Zhu Wen, Thian Foon Sun, Kobilka Tong Sun, Kobilka Brian
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 157 Beckman Circle, Stanford, CA 94305, USA.
J Biol Chem. 2005 Jun 10;280(23):22165-71. doi: 10.1074/jbc.M502352200. Epub 2005 Apr 7.
The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.
β₂肾上腺素能受体（β₂AR）是典型的A类G蛋白偶联受体（GPCR），也是研究GPCR激活机制的优秀模型系统。β₂AR激动剂结合位点已得到充分表征，并且有大量结构相关但功能各异的配体。在本研究中，我们使用儿茶酚（1,2 - 苯二酚，儿茶酚胺激动剂的结构成分）作为分子探针，以识别儿茶酚胺激动剂（如异丙肾上腺素）和结构相关的非儿茶酚部分激动剂沙丁胺醇激活β₂AR的机制差异。通过生物物理和药理学方法，我们表明沙丁胺醇的芳香环与β₂AR上的结合位点不同于儿茶酚胺的芳香环。这种差异在受体激活中很重要，因为据推测儿茶酚胺的芳香环通过旋转异构体切换机制与第六跨膜段中保守的芳香族残基簇相互作用，在触发受体激活中发挥作用。我们的实验表明，沙丁胺醇的芳香环既不能直接也不能间接激活这一机制。此外，部分激动剂的非儿茶酚环与第五跨膜螺旋中的丝氨酸残基的相互作用并不理想，而这些丝氨酸残基已被证明在儿茶酚胺激活过程中起重要作用。这些结果证明了结构相似的激动剂和部分激动剂在结合和激活方面存在意想不到的差异。此外，它们提供了证据表明GPCR的激活是一个多步骤过程，可以使用激动剂片段将其分解为各个组成部分。
