Kobilka B, Gether U, Seifert R, Lin S, Ghanouni P
Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University Medical Center, CA 94305-5345, USA.
J Recept Signal Transduct Res. 1999 Jan-Jul;19(1-4):293-300. doi: 10.3109/10799899909036652.
Drugs acting at G protein coupled receptors can be classified in biological assays as either agonists, partial agonists, neutral antagonists, or as inverse agonists. Very little is known about the actual molecular events and structural changes that occur in the receptor following ligand binding and during transmission of a signal across the membrane. Therefore, the structural basis for the biological classification of drug action remains unknown. To date, the conformational state of G protein coupled receptors has been inferred from the activity of the effector enzyme modulated by the G protein. We have used two different approaches to monitor conformational changes in beta 2 adrenergic receptor. Fluorescence spectroscopy can be used to directly monitor structural changes in purified beta 2 adrenergic receptor in real-time. The emission from many fluorescent molecules is strongly dependent on the polarity of the environment in which they are located. Thus, fluorescent probes covalently bound to proteins can be used as sensitive indicators of conformational changes and protein-protein interactions. In addition, we examined functional differences between agonists and partial agonists using fusion proteins between wild-type beta 2 receptor or a constitutively active beta 2 receptor mutant and Gs alpha. These receptor-G protein fusion proteins guarantee highly efficient coupling with a defined stoichiometry. The results of these experiments will be discussed in the context of current models of G protein coupled receptor activation.
作用于G蛋白偶联受体的药物在生物学检测中可分为激动剂、部分激动剂、中性拮抗剂或反向激动剂。关于配体结合后以及信号跨膜传递过程中受体实际发生的分子事件和结构变化，我们了解得很少。因此，药物作用生物学分类的结构基础仍然未知。迄今为止，G蛋白偶联受体的构象状态是根据由G蛋白调节的效应酶的活性推断出来的。我们采用了两种不同的方法来监测β2肾上腺素能受体的构象变化。荧光光谱可用于实时直接监测纯化的β2肾上腺素能受体的结构变化。许多荧光分子的发射强烈依赖于它们所处环境的极性。因此，与蛋白质共价结合的荧光探针可作为构象变化和蛋白质 - 蛋白质相互作用的敏感指标。此外，我们使用野生型β2受体或组成型活性β2受体突变体与Gsα之间的融合蛋白研究了激动剂和部分激动剂之间的功能差异。这些受体 - G蛋白融合蛋白保证了以确定的化学计量比进行高效偶联。这些实验的结果将在G蛋白偶联受体激活的当前模型背景下进行讨论。
