Visualization of ligand‐induced G i ‐protein activation in chemotaxing cells

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‐G i , by linking the G i ‐protein α subunit to the regulator of G‐protein signaling domain. The R10‐G i probe was coupled with a chemoattractant leukotriene B 4 (LTB 4 ) receptor 1 (BLT1) that induced the receptor to display a high‐affinity ligand binding activity ( K d = 0.91 nM) in HEK293 cells. The R10‐G i probe exhibited an increased FRET signal in accord with the LTB 4 ‐dependent activation of G i . Furthermore, neutrophil‐like differentiated human leukemia cell line 60 that expressed the intrinsic BLT1 displayed temporal G i ‐protein activation in an area localized to the leading edge during chemotaxis in a shallow gradient of LTB 4 . These findings afford an opportunity to clarify the mechanisms underlying the subcellular regulation of G i ‐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 i ‐protein activation in chemotaxing cells FASEB J. 31, 910–919 (2017). www.fasebj.org