G protein–coupled receptors (GPCRs) are major drug targets. Developing a method to measure the activities of GPCRs is essential for pharmacology and drug screening. However, it is difficult to measure the effects of a drug by monitoring the receptor on the cell surface; thus, changes in the concentrations of downstream signaling molecules, which depend on the signaling pathway selectivity of the receptor, are often used as an index of receptor activity. We show that single-molecule imaging analysis provides an alternative method for assessing the effects of ligands on GPCRs. Using total internal reflection fluorescence microscopy (TIRFM), we monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions. Our single-molecule tracking analysis demonstrated that increases and decreases in the average diffusion coefficient of mGluR3 quantitatively reflected the ligand-dependent inactivation and activation of receptors, respectively. Through experiments with inhibitors and dual-color single-molecule imaging analysis, we found that the diffusion of receptor molecules was altered by common physiological events associated with GPCRs, including G protein binding, and receptor accumulation in clathrin-coated pits. We also confirmed that agonist also decreased the average diffusion coefficient for class A and B GPCRs, demonstrating that this parameter is a good index for estimating ligand effects on many GPCRs regardless of their phylogenetic groups, the chemical properties of the ligands, or G protein–coupling selectivity.

G蛋白偶联受体（GPCR）是主要的药物靶标。开发一种测量GPCR活性的方法对于药理学和药物筛选至关重要。然而，通过监测细胞表面的受体来衡量药物的作用是困难的。因此，取决于受体的信号传导途径选择性的下游信号传导分子浓度的变化通常被用作受体活性的指标。我们表明单分子成像分析提供了一种替代的方法来评估配体对GPCRs的影响。使用全内反射荧光显微镜（TIRFM），我们在各种配体条件下监测了C型CPCR代谢型谷氨酸受体3（mGluR3）的扩散动力学。我们的单分子跟踪分析表明，mGluR3的平均扩散系数的增加和减少分别定量反映了配体依赖性的失活和受体的活化。通过使用抑制剂的实验和双色单分子成像分析，我们发现受体分子的扩散被与GPCR相关的常见生理事件（包括G蛋白结合和受体在网格蛋白包被的小孔中的积累）改变了。我们还证实了激动剂还降低了A类和B类GPCR的平均扩散系数，表明该参数是估算配体对许多GPCR的影响的良好指标，而不论它们的系统进化基团，配体的化学性质或G蛋白如何-耦合选择性。