title: Real-time endocytosis imaging as a rapid assay of ligand-GPCR binding in single cells.
abstract: 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.
authors.affiliation: State Key Laboratory of Biomembrane and Membrane Biotechnology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research, Institute of Molecular Medicine and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
authors.forename: Liang-Hong
authors.initials: LH
authors.lastname: Zheng
authors.forename: Chang-He
authors.initials: CH
authors.lastname: Wang
authors.forename: Shu-Jiang
authors.initials: SJ
authors.lastname: Shang
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Zhang
authors.forename: Ye-Shi
authors.initials: YS
authors.lastname: Wang
authors.forename: Qi-Hui
authors.initials: QH
authors.lastname: Wu
authors.forename: Mei-Qin
authors.initials: MQ
authors.lastname: Hu
authors.forename: Zu-Yin
authors.initials: ZY
authors.lastname: Chai
authors.forename: Xi
authors.initials: X
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.forename: Lie-Cheng
authors.initials: LC
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Xiong
authors.forename: Zhuan
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000818:Animals; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000378:metabolism; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011941:Receptors, Adrenergic / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018080:Receptors, GABA-B / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism; D011985:Receptors, Serotonin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: C086686:FM2 10; D005456:Fluorescent Dyes; D008024:Ligands; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011941:Receptors, Adrenergic; D011950:Receptors, Cholinergic; D043562:Receptors, G-Protein-Coupled; D018080:Receptors, GABA-B; D017475:Receptors, Nerve Growth Factor; D058472:Receptors, Purinergic P2Y1; D011985:Receptors, Serotonin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: DRG neuron; FM imaging; GPCR; confocal microscopy; endocytosis