Real-time endocytosis imaging as a rapid assay of ligand-GPCR binding in single cells

Most G protein-coupled receptors (GPCRs) do not generate membrane currents in response to ligand-receptor binding (LRB). Here, we describe a novel technique using endocytosis as a bioassay that can detect activation of a GPCR in a way analogous to patch-clamp recording of an ion channel in a living cell. The confocal imaging technique, termed FM endocytosis imaging (FEI), can record ligand-GPCR binding with high temporal (second) and spatial (micrometer) resolution. LRB leads to internalization of an endocytic vesicle, which can be labeled by a styryl FM dye and visualized as a fluorescent spot. Distinct from the green fluorescence protein-labeling method, FEI can detect LRB endocytosis mediated by essentially any receptors (GPCRs or receptors of tyrosine kinase) in a native cell/cell line. Three modified versions of FEI permit promising applications in functional GPCR studies and drug screening in living cells: 1) LRB can be recorded in "real time" (time scale of seconds); 2) internalized vesicles mediated by different GPCRs can be discriminated by different colors; and 3) a high throughput method can screen ligands of a specific GPCR.