Gether U, Lin S, Kobilka B K
Howard Hughes Medical Institute, Stanford University Medical School, California 94305, USA.
J Biol Chem. 1995 Nov 24;270(47):28268-75. doi: 10.1074/jbc.270.47.28268.
The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.
本研究的目的是开发一种方法，以直接监测G蛋白偶联受体在药物结合时的结构变化。纯化的人β2肾上腺素能受体用对半胱氨酸有反应的荧光探针N,N'-二甲基-N-(碘乙酰基)-N'-(7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基)乙二胺（IANBD）进行共价标记。IANBD的荧光对其所处环境的极性高度敏感。我们发现，完全激动剂异丙肾上腺素可引起IANBD标记的β2受体荧光的立体选择性和剂量依赖性降低。荧光变化可与异丙肾上腺素浓度绘制为简单的双曲线结合等温线，表明与受体中的单一结合位点相互作用。几种肾上腺素能拮抗剂逆转该反应的能力证实，该结合位点与β2受体中已充分描述的结合位点相同。将对异丙肾上腺素的反应与一系列具有不同生物学效能的肾上腺素能激动剂进行比较，发现生物学效能与荧光变化之间存在线性相关性。这表明激动剂介导的IANBD标记的β2受体荧光降低是由于与受体激活和G蛋白偶联所涉及的相同构象变化。与激动剂相反，负性拮抗剂导致基线荧光有小幅但显著的增加。尽管该反应幅度较小，但支持拮抗剂自身可能改变受体结构的观点。总之，我们的数据为G蛋白偶联受体中发生的配体特异性构象变化提供了首个直接证据。此外，数据证明了荧光光谱作为进一步阐明G蛋白偶联受体药物作用分子机制工具的潜力。
