Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
J Mol Biol. 2011 Jun 17;409(4):513-28. doi: 10.1016/j.jmb.2011.03.059. Epub 2011 Apr 6.
We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor--agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.
我们描述了一种快速探测细胞表面配体结合蛋白突变的方法，这些突变会影响结合配体的环境。该方法使用荧光激活细胞分选筛选随机突变的受体，以寻找改变环境敏感荧光配体荧光发射光谱的取代。当应用于酵母α因子受体 Ste2p（一种 G 蛋白偶联受体）时，该程序鉴定出 22 个取代可使荧光激动剂的发射红移，包括与配体结合和其他部位相关的取代。在筛选改变荧光α因子拮抗剂发射的突变时，确定了一组单独的取代，发生在不太可能直接与配体接触的部位。相反，这些突变改变受体构象以增加配体结合亲和力，并对正常受体的拮抗剂做出信号响应。这些结果表明，受体-配体相互作用至少涉及两个部位，其中只有一个部位是受体激活构象的特异性部位。
