Watching activation and signalling in individual GPCRs

G蛋白偶联受体（GPCR）是药物开发中最重要的药物靶点家族之一。它们是7-跨膜结构域（7 TMD）膜受体，控制大多数神经功能，并且包括哺乳动物蛋白质的最大家族（~1%;数量> 1，000）。因此，它们是药物设计和功能研究的重点，尤其是因为约75%的药物只解决了5%的已知GPCR。我们现在（截至2007年10月）有一个配体结合A类GPCR的结构，β 2-肾上腺素能受体，以补充视紫红质的3D结构。在β 2-ADR结构中，鉴定了推定的胆固醇结合位点和二聚化界面。GPCR的二聚化可能是其信号激活机制的基础，并且脂质相互作用对配体结合和/或信号传导过程至关重要，这两者都是有重大争议的领域，并且仅部分解决而未完全解决。3D结构是这类研究的一个很好的开端，但动力学、膜参与和动力学介导在功能描述中都是必不可少的，甚至在3D结构之前。特别是，有关配体化学计量，界面相互作用和脂质参与信号传导的问题都是理解GPCR激活和信号传导的重要问题。对于所选择的GPCR（神经降压素受体1），已经克服了获得用于此类研究的纯化的、重构的和功能上胜任的GPCR的障碍。我们现在发表的研究（哈丁，生物物理学。J.，2008）证明了特定组合物的脂质双层中的二聚化。在这里，我们将利用这一投资，并使用单分子方法来剖析GPCR功能的细节，首次使用新的体外方法对GPCR进行研究。

G-protein coupled receptors (GPCRs) represent one of the most important families of drug targets in pharmaceutical development. They are 7-transmembrane domain (7TMD) membrane receptors that control most neurological functions and comprise the largest family (~1%; >1,000 in number) of mammalian proteins. They are therefore at the focus of intense efforts in drug design and functional studies, not least because ~75% of drugs address only 5% of known GPCRs. We now (as of October 2007) have a structure of a ligand-binding Class A GPCR, the beta2-adrenergic receptor, to complement the 3D structures of rhodopsin. In the beta2-ADR structures, putative cholesterol binding sites and dimerisation interfaces were identified. Dimerisation of GPCRs may be fundamental to their signal activation mechanism, and lipid interactions crucial to ligand binding and/or the signalling process, both of which are areas of significant controversy and have only been partially addressed and not fully resolved. 3D structures are a very good start in such studies, but dynamics, the membrane involvement and kinetic mesurements are all essential in functional descriptions, even ahead of 3D structures. In particular, questions about ligand stoichiometry, interface interactions and lipid involvement in signalling are all important issues for understanding GPCR activation and signalling. The hurdle of obtaining a purified, reconstituted and functionally competent GPCR for such studies has been overcome for the chosen GPCR (neurotensin receptor 1). Studies now published by us (Harding, Biophys. J., 2008) demonstrate dimerisation in lipid bilayer of a specific composition. Here we will capitalize on this investment and use single molecule methods to dissect details about GPCR function using novel in vitro approaches for the first time on a GPCR.