Immunocytochemistry of phospholipase D1 and D2 in cultured cells

Activation of Gq protein-coupled receptors triggers the phospholipase C (PLC) pathway, which yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA), which serves as another second messenger. Along with PLC-DGK pathway, PA is produced directly by the action of phospholipase D (PLD), which hydrolyzes the major membrane phospholipid: phosphatidylcholine (PC). PA is converted to DG by phosphatidic acid phosphatase, suggesting that PLD, together with DGK, is a key enzyme regulating DG and PA. PLD has been implicated in a broad range of cellular processes. However, cellular expression and subcellular localization of PLD remain elusive because of a lack of specific antibodies against PLDs. For this study, we raised specific antibodies against major mammalian PLD isoforms: PLD1 and PLD2. Immunocytochemical analysis using specific antibodies showed clearly that native PLD1 and PLD2 localize to distinct subcellular regions as dot-like structures in cultured cells. PLD1 predominantly localizes to the plasma membrane, whereas PLD2 mostly localizes within the cytoplasm. These findings suggest that PLD1 and PLD2 have different roles in the phosphoinositide signaling pathway in distinct subcellular regions.