Leukotriene D4 Triggers an Association between Gβγ Subunits and Phospholipase C-γ1 in Intestinal Epithelial Cells

Abstract The proinflammatory mediator leukotriene D4 (LTD4) binds to the seven-transmembrane receptor CYSLT1. Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD4 induced tyrosine phosphorylation and translocation of phospholipase (PLC)-γ1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD4induced a rapid interaction between Gβγ subunits and PLC-γ1; results obtained with GST fusion proteins of PLC-γ1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-γ1. Moreover, LTD4 induced an increased association of c-Src with PLC-γ1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD4-induced tyrosine phosphorylation of PLC-γ1 and the association of PLC-γ1 with Gβγ subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gβ, anti-PLC-γ1, or anti-c-Src antibodies and by pretreatment with PP1. LTD4-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gβγ subunits can, directly or indirectly, serve as membrane-bound partners for PLC-γ1 and c-Src and that each of these proteins is essential for LTD4-induced downstream PLC-γ1 signaling.