title: Visualization of ligand-induced Gi-protein activation in chemotaxing cells.
abstract: Cell migration to chemoattractants is critically important in both normal physiology and the pathogenesis of many diseases. In GPCR-mediated chemotaxis, GPCRs transduce the gradient of an extracellular chemotactic ligand into intracellular responsesviathe activation of heterotrimeric G proteins. However, ligand-induced G-protein activation has not been directly imaged as yet in mammalian chemotaxing cells. We developed a Förster resonance energy transfer (FRET) probe, R10-Gi, by linking the Gi-protein α subunit to the regulator of G-protein signaling domain. The R10-Giprobe was coupled with a chemoattractant leukotriene B4(LTB4) receptor 1 (BLT1) that induced the receptor to display a high-affinity ligand binding activity (Kd= 0.91 nM) in HEK293 cells. The R10-Giprobe exhibited an increased FRET signal in accord with the LTB4-dependent activation of GiFurthermore, neutrophil-like differentiated human leukemia cell line 60 that expressed the intrinsic BLT1 displayed temporal Gi-protein activation in an area localized to the leading edge during chemotaxis in a shallow gradient of LTB4These findings afford an opportunity to clarify the mechanisms underlying the subcellular regulation of Gi-protein activity, as well as GPCR-mediated ligand sensing, during chemotaxis in mammalian cells.-Masuda, K., Kitakami, J., Kozasa, T., Kodama, T., Ihara, S., Hamakubo, T. Visualization of ligand-induced Gi-protein activation in chemotaxing cells.
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Kitakami
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kozasa
authors.affiliation: Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.affiliation: Laboratory of Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
authors.forename: Sigeo
authors.initials: S
authors.lastname: Ihara
authors.affiliation: Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and hamakubo@qbm.rcast.u-tokyo.ac.jp.
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
mesh_terms: D002633:Chemotaxis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D007975:Leukotriene B4 / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D021381:Protein Transport; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism; D018102:Receptors, Leukotriene B4 / Q000378:metabolism
chemical_list: D044385:GTP-Binding Protein alpha Subunits; C513240:LTB4R protein, human; D020710:RGS Proteins; D018102:Receptors, Leukotriene B4; D049452:Green Fluorescent Proteins; D007975:Leukotriene B4
keywords: FRET; GPCR; HL-60; RGS; heterotrimeric G protein