Lysophosphatidylcholines activate G2A inducing Gαi-1-/Gαq/11- Ca2+ flux, Gβγ-Hck activation and clathrin/β-arrestin-1/GRK6 recruitment in PMNs

Abstract

Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca2+ via release of Gα and Gβγ subunits, kinase activation, and the recruitment of clathrin, β-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5–180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in Gαi-1 and Gαq/11 release leading to increases in cytosolic Ca2+, which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the Gβγ subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr411). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of Gαi-1, Gαq/11 and Gβγ, resulting in cytosolic Ca2+ flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.