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Autotaxin (ATX) is a secreted phosphodiesterase that produces the lipid mediator lysophosphatidic acid (LPA). LPA acts through specific guanine-nucleotide-binding protein (G protein)-coupled receptors to stimulate migration, proliferation, survival and other functions in many cell types. ATX is important in vivo for processes as diverse as vasculogenesis, lymphocyte trafficking and tumour progression. However, the inner workings of ATX have long been elusive, in terms of both its substrate specificity and how localized LPA signalling is achieved. Structural studies have shown how ATX recognizes its substrates and may interact with the cell surface to promote specificity in LPA signalling.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells1,2. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3–6), due to activation of a Na+/H+ exchanger in the plasma membrane4, and this alkalinization is necessary for the initiation of DNA synthesis7. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger8–10. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli11,12. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12–15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.

The mechanisms by which growth factors stimulate metabolism and cell proliferation are largely unknown. Recent evidence suggests that mitogens rapidly activate a Na+/H+ exchange mechanism in the plasma membrane of their target cells, implicating cytoplasmic pH (pH1) as a potential 'messenger'. Indeed, growth stimulation of quiescent fibroblasts leads to intracellular alkalinization at approximately 1 h after mitogen addition, as measured by weak-acid distribution methods. We have used an internalized fluorescent pH1 indicator to examine the pH1-regulating mechanisms in diploid human fibroblasts and to obtain the first continuous pH1 recordings of the response to growth factors. We report here that (1) pH1 in human fibroblasts is controlled by a membrane-bound Na+/H+ exchanger, which rapidly restores pH1 after an acute cytoplasmic acidification, and (2) epidermal growth factor (EGF) and serum factors induce a rapid and persistent elevation of pH1 by modifying the pH1 sensitivity of the Na+H+ exchanger. We conclude that in addition to having a basic role in pH1 regulation, Na+/H+ exchange may function as a transmembrane signal transducer in the action of growth factors.