Signaling dynamics distinguish high and low priority neutrophil attractant receptors

Abstract

Human neutrophils respond to multiple chemical attractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils distinguish between attractants to modulate their responses. They prioritize pathogen and injury derived signals over long-range inflammatory signals secreted by host cells. Different attractants can also drive qualitatively different modes of migration. As receptors recognizing both classes of attractant couple to Gi G-proteins, it remains unclear how downstream signaling pathways distinguish and prioritize between the inputs. Here, we use live-cell imaging to demonstrate that the responses differ in their signaling dynamics: low priority attractants cause transient responses, while responses to high priority attractants are sustained. We observe this difference in both primary neutrophils and differentiated HL-60 cells, for signaling outputs of calcium, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. We find that the rapid attenuation of Cdc42 activation in response to LTB4 depends on the threonine 308 and serine 310 phosphorylation sites in the C-terminal tail of its receptor LTB4R, in a manner independent of endocytosis. Mutation of these residues to alanine impairs attractant prioritization, although it does not affect attractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes receptors and contributes to chemoattractant prioritization.