Leukotriene D4 Triggers an Association between Gβγ Subunits and Phospholipase C-γ1 in Intestinal Epithelial Cells*
- From the Division of Experimental Pathology, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden
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.
Footnotes
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↵* This work was supported by grants from the Swedish Medical Research Council, the Inga and John Hains Foundation, the Åke Wiberg Foundation, the Crafoord Foundation, and foundations at MalmöUniversity Hospital (to A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Recipient of fellowships from the Wenner-Gren Foundation and the Swedish Institute.
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↵§ To whom correspondence should be addressed: Division of Experimental Pathology, Department of Laboratory Medicine, MalmöUniversity Hospital, Entrance 78, Floor 3,SE-205 02 Malmö, Sweden. Tel.: 46-40-337223; Fax: 46-40-337353; E-mail: anita.sjolander@ exppat.mas.lu.se.
- Abbreviations:
- LT
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leukotriene
- PLC
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phospholipase C
- Int 407
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intestine 407 cells
- PH
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pleckstrin homology
- SH2
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Src homology 2
- GST
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glutathioneS-transferase
- PAGE
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polyacrylamide gel electrophoresis
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- Received November 22, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
