Quantitative image cytometry for analyzing intracellular trafficking of G protein-coupled receptors on a...

G protein-coupled receptors (GPCRs) are important targets in medical and pharmaceutical research fields, because they play key roles in a variety of biological processes. Recently, intracellular trafficking of GPCRs involving endosomal internalization and recycling to the plasma membrane has been studied as a regulation mechanism for GPCR activities. However, the absence of a quantitative single-cell analysis method has hampered conditional GPCR trafficking studies and the possibility of gaining significant insights into the mechanism of regulation of GPCR signaling. Here, we report a facile image cytometry method to analyze the trafficking of GPCRs. In this method, GPCR-expressing cells were arrayed with a photo-responsive cell-immobilizing reagent in a single-cell manner, and the tagged GPCR was visualized by pulse-labeling with a fluorescent dye through sortase-mediated peptide-tag ligation. We quantified the intracellular distribution changes of a pH-dependent GPCR, G2A, by time-course observation under mildly acidic and slightly basic pH conditions. The difference in pH-dependent G2A trafficking between individual cells was automatically detected by an image analysis custom software program, and simultaneously, the average distribution ratios were also determined for understanding the properties of G2A. The present method should be applicable for investigating the dynamic intracellular trafficking of a wide variety of GPCRs under various conditions in a high-throughput manner.