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    Visualization of ligand-induced G-protein activation in chemotaxing cells.

    FASEB J 2017 03 23;31(3):910-919. Epub 2016 Nov 23.
    Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and
    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 responses the 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-G, by linking the G-protein α subunit to the regulator of G-protein signaling domain. The R10-G probe was coupled with a chemoattractant leukotriene B (LTB) receptor 1 (BLT1) that induced the receptor to display a high-affinity ligand binding activity ( = 0.91 nM) in HEK293 cells. The R10-G probe exhibited an increased FRET signal in accord with the LTB-dependent activation of G Furthermore, neutrophil-like differentiated human leukemia cell line 60 that expressed the intrinsic BLT1 displayed temporal G-protein activation in an area localized to the leading edge during chemotaxis in a shallow gradient of LTB These findings afford an opportunity to clarify the mechanisms underlying the subcellular regulation of G-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 G-protein activation in chemotaxing cells.
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