Research ArticleGPCR SIGNALING

Single-molecule diffusion-based estimation of ligand effects on G protein–coupled receptors

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Sci. Signal.  18 Sep 2018:
Vol. 11, Issue 548, eaao1917
DOI: 10.1126/scisignal.aao1917

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Tracking receptor dynamics

Methods to determine the effect of a drug on a given G protein–coupled receptor (GPCR) often rely on monitoring intracellular molecules affected by the signaling pathway of that receptor. However, how can the effects of drugs be determined for GPCRs whose signaling pathways are unclear? Using single-molecule imaging of the diffusion of different classes of known GPCRs in the plasma membrane, Yanagawa et al. showed that the dynamics of GPCR movement could be classified into four groups depending on whether the interacting drug activated or inhibited the receptor. The coupling of GPCRs to G proteins and receptor endocytosis also resulted in defined diffusion characteristics, suggesting that analyzing the diffusion coefficient of a GPCR provides an estimate of the effect that a candidate drug has.


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.

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