Visualization of ligand-induced Gi-protein activation in chemotaxing cells

FASEB J. 2017 Mar;31(3):910-919. doi: 10.1096/fj.201601102R. Epub 2016 Nov 23.

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 responses via 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-Gi, by linking the Gi-protein α subunit to the regulator of G-protein signaling domain. The R10-Gi probe 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-Gi probe exhibited an increased FRET signal in accord with the LTB4-dependent activation of Gi Furthermore, 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 LTB4 These 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.

Keywords: FRET; GPCR; HL-60; RGS; heterotrimeric G protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chemotaxis*
  • Fluorescence Resonance Energy Transfer / methods*
  • GTP-Binding Protein alpha Subunits / genetics
  • GTP-Binding Protein alpha Subunits / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HL-60 Cells
  • Humans
  • Leukotriene B4 / metabolism
  • Microscopy, Fluorescence / methods
  • Protein Binding
  • Protein Transport
  • RGS Proteins / genetics
  • RGS Proteins / metabolism
  • Receptors, Leukotriene B4 / metabolism

Substances

  • GTP-Binding Protein alpha Subunits
  • LTB4R protein, human
  • RGS Proteins
  • Receptors, Leukotriene B4
  • Green Fluorescent Proteins
  • Leukotriene B4