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 B₄ (LTB₄) 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 LTB₄-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 LTB₄ 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.