Northup John
Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD, USA.
Methods Mol Biol. 2004;261:93-112. doi: 10.1385/1-59259-762-9:093.
G-protein-coupled receptors (GPCRs) initiate a variety of cellular responses to diverse array of extracellular stimuli. Surface plasmon resonance detection offers a powerful approach to the study of protein-protein interactions in real time. In this chapter we outline procedures for the immobilization of the prototype GPCR structure, rhodopsin or the G-protein alpha and betagamma subunits, for analysis of the molecular interactions initiating G-protein signaling. The attachment of rhodopsin via its extracellular carbohydrate residues provides a convenient, and universally applicable, procedure for GPCR immobilization in a form that retains full biochemical activity and ability to interact with intracellular signaling components. SPR detection then allows for the analysis of the kinetic and equilibrium binding properties of the immobilized receptor with G-protein subunits and potentially other interacting molecules.
G蛋白偶联受体（GPCRs）可引发多种细胞反应，以应对各种各样的细胞外刺激。表面等离子体共振检测提供了一种实时研究蛋白质-蛋白质相互作用的强大方法。在本章中，我们概述了固定原型GPCR结构、视紫红质或G蛋白α和βγ亚基的程序，用于分析启动G蛋白信号传导的分子相互作用。通过视紫红质的细胞外碳水化合物残基进行附着，为以保留完全生化活性和与细胞内信号成分相互作用能力的形式固定GPCR提供了一种方便且普遍适用的程序。然后，SPR检测允许分析固定化受体与G蛋白亚基以及潜在其他相互作用分子的动力学和平衡结合特性。
