Involvement of phospholipase D2 in lysophosphatidate-induced transactivation of platelet-derived growth factor receptor-β in human bronchial epithelial cells

Lysophosphatidate (LPA) mediates multiple cellular responses via heterotrimeric G protein coupled LPA-1, LPA-2, and LPA-3 receptors. Many G protein-coupled receptors stimulate ERK following tyrosine phosphorylation of growth factor receptors; however, the mechanism(s) of transactivation of receptor tyrosine kinases are not well defined. Here, we provide evidence for the involvement of phospholipase D (PLD) in LPA-mediated transactivation of platelet-derived growth factor receptorbeta (PDGF-Rbeta). In primary cultures of human bronchial epithelial cells (HBEpCs), LPA stimulated tyrosine phosphorylation of PDGF-Rbeta and threonine/tyrosine phosphorylation of ERK1/2. The LPA-mediated activation of ERK and tyrosine phosphorylation of PDGF-Rbeta was attenuated by tyrphostin AG 1296, an inhibitor of PDGF-R kinase, suggesting transactivation of PDGF-R by LPA. Furthermore, LPA-, but not PDGF beta-chain homodimer-induced tyrosine phosphorylation of PDGF-Rbeta was partially blocked by pertussis toxin, indicating coupling of LPA-R(s) to G(i). Exposure of HBEpCs to LPA activated PLD. Butan-1-ol, which acts as an acceptor of phosphatidate generated by the PLD pathway, blocked LPA- mediated transactivation of PDGF-Rbeta. This effect was not seen with butan-3-ol, suggesting PLD involvement. The role of PLD1 and PLD2 in the PDGF-Rbeta transactivation by LPA was investigated by infection of cells with adenoviral constructs of wild type and catalytically inactive mutants of PLD. LPA activated both PLD1 and PLD2 in HBEpCs; however, infection of cells with cDNA for wild type PLD2, but not PLD1, increased the tyrosine phosphorylation of PDGF-Rbeta in response to LPA. Also, the LPA- mediated tyrosine phosphorylation of PDGF-Rbeta was attenuated by the catalytically inactive mutant mPLD2-K758R. Infection of HBEpCs with adenoviral constructs of wild type hPLD1, mPLD2, and the inactive mutants of hPLD1 and mPLD2 resulted in association of PLD2 wild type and inactive mutant proteins with the PDGF-Rbeta compared with PLD1. These results show for the first time that transactivation of PDGF-Rbeta by LPA in HBEpCs is regulated by PLD2.