Corgiat Brian A, Nordman Jacob C, Kabbani Nadine
Department of Molecular Neuroscience, Krasnow Institute for Advanced Study, George Mason University Fairfax, VA, USA.
Front Pharmacol. 2014 Jan 7;4:171. doi: 10.3389/fphar.2013.00171.
Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor-protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor-protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.
受体功能依赖于与各种细胞内蛋白质的相互作用，这些蛋白质确保受体的定位和信号传导。虽然已经优化了许多方法用于细胞中受体 - 蛋白质相互作用网络（互作组）的分离、纯化和蛋白质组学表征，但捕获受体互作组及其动态特性仍然是一个挑战。特别是，研究与受体以低亲和力结合或能迅速从大分子复合物中解离的互作组成分很困难。在此，我们描述了化学交联（CC）如何有助于受体 - 蛋白质相互作用的分离和蛋白质组学分析。将CC添加到标准亲和纯化和质谱分析方案中，可增强蛋白质组学检测中蛋白质捕获的能力，并能够在各种细胞和受体状态下鉴定特定的结合伴侣。CC在受体互作组研究中的效用在烟碱型乙酰胆碱受体以及其他几种受体类型中得到了突出体现。更好地理解受体及其与蛋白质的相互作用是分子生物学的前沿，为基础医学的一个重要组成部分提供信息，这有助于药物开发、药物作用以及理解疾病的病理生理学。
