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

Abstract

G protein-coupled receptors (GPCRs) are major drug targets and have high potential for drug discovery. The development of a method for measuring 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, and changes in the concentrations of downstream signaling molecules, which are specific to each receptor, are used as an index of the receptor activity. Here, we show that singlemolecule imaging analysis provides an alternative method for assessing GPCR activity.We monitored the dynamics of the diffusion of metabotropic glutamate receptor 3 (mGluR3), a class C GPCR, under various ligand conditions by using total internal reflection fluorescence microscopy (TIRFM). The single-molecule tracking analysis demonstrate that changes in the average diffusion coefficient of mGluR3 quantitatively reflect the ligand-dependent activity. Then, we reveal that the diffusion of receptor molecules is altered by the common physiological events associated with GPCRs, including G protein binding or accumulation in clathrin-coated pits, by inhibitor assay and dual-color single-molecule imaging analysis. We also confirm the general applicability of the method to class A and B GPCRs, demonstrating that the diffusion coefficient is a good index for estimating the activities of many GPCRs regardless of the phylogenetic groups, chemical properties of the ligands, and G protein-coupling selectivity.

Optical sensors of heterotrimeric G protein signaling

“…For example, SMI (using CLIPtag labeled [94] G i1 and G s subunits) has allowed uncovering G protein signaling from cellular "hotspots" delineated by cytoskeleton filaments [95]. A similar construct (G i1 labeled with a SNAPtag) was used to demonstrate precoupling of the G i1 protein to the metabotropic glutamate receptor 3 caused by the receptor's basal activity [96]. G s and G 2 subunits labeled with a HaloTag have been used to show [97] that after  2 -adrenoceptor stimulation, G s protein subunits become less mobile and, in contrast to -arrestin 2, do not accumulate in clathrin-coated pits.…”