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Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor‐mediated increase in 1,2‐diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin‐stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous ‘alcohol’ DG (acceptor), yielding bis(1,2‐diacylglycero)‐3‐sn‐phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long‐term phorbol ester treatment blocks bradykinin‐induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2–10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor‐induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone‐ and growth factor‐like activities. Exogenous LPA stimulates GTP‐dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA‐responsive cell types. A 32P‐labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine‐LPA, labels a membrane protein of apparent molecular mass of 38–40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38–40 kDa protein is competitively inhibited by unlabeled 1‐oleoyl‐LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38–40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.