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NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.

Autotaxin (ATX) or ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) is a secretory glycoprotein and functions as the key enzyme for lysophosphatidic acid generation. The mechanism of ATX protein trafficking is largely unknown. Here, we demonstrated that p23, a member of the p24 protein family, was the protein-sorting receptor required for endoplasmic reticulum (ER) export of ATX. A di-phenylalanine (Phe-838/Phe-839) motif in the human ATX C-terminal region was identified as a transport signal essential for the ATX-p23 interaction. Knockdown of individual Sec24 isoforms by siRNA revealed that ER export of ATX was impaired only if Sec24C was down-regulated. These results suggest that ATX is selectively exported from the ER through a p23, Sec24C-dependent pathway. In addition, it was found that AKT signaling played a role in ATX secretion regulation to facilitate ATX ER export by enhancing the nuclear factor of activated T cell-mediated p23 expression. Furthermore, the di-hydrophobic amino acid motifs (FY) also existed in the C-terminal regions of human ENPP1 and ENPP3. Such a p23, Sec24C-dependent selective ER export mechanism is conserved among these ENPP family members.

Zinc-requiring ectoenzymes, including both secreted and membrane-bound enzymes, are considered to capture zinc in their active site for their activation in the early secretory pathway. This idea has been confirmed by our studies conducted using tissue-nonspecific alkaline phosphatase (TNAP), which is elaborately activated by means of a two-step mechanism by zinc transporter 5 (ZNT5)-ZNT6 heterodimers and ZNT7 homodimers, through protein stabilization followed by enzyme activation with zinc in the early secretory pathway. However, the molecular basis of the activation process in other zinc-requiring ectoenzymes remains largely unknown. In this study, we investigated this activation process by using three cancer-promoting zinc-requiring ectoenzymes, autotaxin (ATX), matrix metalloproteinase 9 (MMP9), and carbonic anhydrase IX (CAIX), and the chicken DT40 cell mutants that we generated; we specifically focused on clarifying whether the same or a similar activation mechanism operates in these ectoenzymes. ATX activation required ZNT5-ZNT6 heterodimers and ZNT7 homodimers in a manner similar to TNAP activation, although the protein stability of ATX was differently regulated from that of TNAP. MMP9 required ZNT5-ZNT6 heterodimers and ZNT7 homodimers for its activation as well as secretion; MMP9 was not secreted into the spent medium unless both zinc-transport complexes were present. Finally, CAIX activation by zinc was mediated not only by ZNT5-ZNT6 heterodimers and ZNT7 homodimers but also by ZNT4 homodimers; thus, these three zinc-transport complexes redundantly contribute to CAIX activation. Our results provide pivotal insights into the activation processes of zinc-requiring ectoenzymes, and furthermore, they offer novel insights for potential cancer therapy applications given the cancer-promoting potencies of ATX, MMP9, and CAIX.

Contains fulltext : 117828.pdf (Publisher’s version ) (Open Access) Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), playing a key role in diverse physiological and pathological processes. ATX exists in distinct splice variants, but isoform-specific functions remain elusive. Here we characterize the ATXalpha isoform, which differs from the canonical form (ATXbeta) in having a 52-residue polybasic insertion of unknown function in the catalytic domain. We find that the ATXalpha insertion is susceptible to cleavage by extracellular furin-like endoproteases, but cleaved ATXalpha remains structurally and functionally intact due to strong interactions within the catalytic domain. Through ELISA and surface plasmon resonance assays, we show that ATXalpha binds specifically to heparin with high affinity (K(d) ~10(-8) M), whereas ATXbeta does not; furthermore, heparin moderately enhanced the lysophospholipase D activity of ATXalpha. We further show that ATXalpha, but not ATXbeta, binds abundantly to SKOV3 carcinoma cells. ATXalpha binding was abolished after treating the cells with heparinase III, but not after chondroitinase treatment. Thus, the ATXalpha insertion constitutes a cleavable heparin-binding domain that mediates interaction with heparan sulfate proteoglycans, thereby targeting LPA production to the plasma membrane.

Autotaxin (ATX) is a secreted lysophospholipase D that generates the bioactive lipid mediator lysophosphatidic acid (LPA). We and others have reported that ATX binds to integrins, but the function of ATX-integrin interactions is unknown. The recently reported crystal structure of ATX suggests a role for the solvent-exposed surface of the N-terminal tandem somatomedin B-like domains in binding to platelet integrin αIIbβ(3). The opposite face of the somatomedin B-like domain interacts with the catalytic phosphodiesterase (PDE) domain to form a hydrophobic channel through which lysophospholipid substrates enter and leave the active site. Based on this structure, we hypothesize that integrin-bound ATX can access cell surface substrates and deliver LPA to cell surface receptors. To test this hypothesis, we investigated the integrin selectivity and signaling pathways that promote ATX binding to platelets. We report that both platelet β1 and β3 integrins interact in an activation-dependent manner with ATX via the SMB2 domain. ATX increases thrombin-stimulated LPA production by washed platelets ~10-fold. When incubated under conditions to promote integrin activation, ATX generates LPA from CHO cells primed with bee venom phospholipase A(2), and ATX-mediated LPA production is enhanced more than 2-fold by CHO cell overexpression of integrin β(3). The effects of ATX on platelet and cell-associated LPA production, but not hydrolysis of small molecule or detergent-solubilized substrates, are attenuated by point mutations in the SMB2 that impair integrin binding. Integrin binding therefore localizes ATX activity to the cell surface, providing a mechanism to generate LPA in the vicinity of its receptors.

Autotaxin (ATX) is a secreted lysophospholipase D that hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), initiating signaling cascades leading to cancer metastasis, wound healing, and angiogenesis. Knowledge of the pathway and kinetics of LPA synthesis by ATX is critical for developing quantitative physiological models of LPA signaling. We measured the individual rate constants and pathway of the LPA synthase cycle of ATX using the fluorescent lipid substrates FS-3 and 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-LPC. FS-3 binds rapidly (k(1) ≥500 μm(-1) s(-1)) and is hydrolyzed slowly (k(2) = 0.024 s(-1)). Release of the first hydrolysis product is random and rapid (≥1 s(-1)), whereas release of the second is slow and rate-limiting (0.005-0.007 s(-1)). Substrate binding and hydrolysis are slow and rate-limiting with LPC. Product release is sequential with choline preceding LPA. The catalytic pathway and kinetics depend strongly on the substrate, suggesting that ATX kinetics could vary for the various in vivo substrates. Slow catalysis with LPC reveals the potential for LPA signaling to spread to cells distal to the site of LPC substrate binding by ATX. An ATX mutant in which catalytic threonine at position 210 is replaced with alanine binds substrate weakly, favoring a role for Thr-210 in binding as well as catalysis. FTY720P, the bioactive form of a drug currently used to treat multiple sclerosis, inhibits ATX in an uncompetitive manner and slows the hydrolysis reaction, suggesting that ATX inhibition plays a significant role in lymphocyte immobilization in FTY720P-based therapeutics.

Autotaxin or NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2) is a secreted lysophospholipase-D that promotes metastasis and tumor growth by its ability to generate lysophosphatidic acid. Considerable evidence suggests that inhibitors of NPP2 can be used as a novel therapy for the treatment of cancer. Although most attention is currently directed toward the development of inhibitors of the catalytic site, we have explored whether NPP2 can also be targeted through its non-catalytic nuclease-like domain. We demonstrate here that the catalytic and nuclease-like domains are covalently linked by an essential disulfide bridge between Cys413 and Cys805. Within the nuclease-like domain, residues 829–850 are involved in the secretion of NPP2, and Lys852 is required for the expression of catalytic activity. These data show that the nuclease-like domain is crucial for catalysis by NPP2 and is a possible target to generate inhibitors.

Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8 \Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.

Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.

Autotaxin/NPP2, a secreted lysophospholipase-D, promotes cell proliferation, survival, and motility by generating the signaling molecule lysophosphatidic acid. Here we show that ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is N-glycosylated on Asn-53, Asn-410, and Asn-524. Mutagenesis and deglycosylation experiments revealed that only the glycosylation of Asn-524 is essential for the expression of the catalytic and motility-stimulating activities of NPP2. The N-glycan on Asn-524 was identified as Man8/9GlcNAc2, which is rarely present on mature eukaryotic glycoproteins. Additional studies show that this Asn-524-linked glycan is not accessible to α-1,2-mannosidase, suggesting that its non-reducing termini are buried inside the folded protein. Consistent with a structural role for the Asn-524-linked glycan, only the mutation of Asn-524 augmented the sensitivity of NPP2 to proteolysis and increased its mobility during Blue Native PAGE. Asn-524 is phylogenetically conserved and maps to the catalytic domain of NPP2, but a structural model of this domain suggests that Asn-524 is remote from the catalytic site. Our study defines an essential role for the Asn-524-linked glycan chain of NPP2.

Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid (LPA), was previously identified in human serum. Although cPA possesses distinct physiological activities not elicited by LPA, its biochemical origins have scarcely been studied. In the present study, we assayed cPA formation from lysophosphatidylcholine in fetal bovine serum and found significant activity of transphosphatidylation that generated cPA. The cPA-producing enzyme was purified from fetal bovine serum using five chromatographic steps yielding a 100-kDa protein with cPA biosynthetic activity. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of its tryptic peptides revealed that the enzyme shared identical fragments with human autotaxin, a serum lysophospholipase D that produces LPA. Western blot analysis demonstrated that the 100-kDa protein was specifically recognized by an anti-human autotaxin antibody. Moreover, recombinant rat autotaxin was found to generate cPA in addition to LPA. No significant cPA- or LPA-producing activity was detected in autotaxin-depleted serum from bovine or human prepared by immunoprecipitation with an anti-autotaxin monoclonal antibody. These results indicate that the generation of cPA and LPA in serum is mainly attributed to autotaxin.

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2, NPP2) is an autocrine motility factor initially characterized from A2058 melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities that accounts for many biological properties of serum. LPA is thought to be produced during serum formation based on the fact that the LPA level is much higher in serum than in plasma. In this study, to better understand the pathways of LPA synthesis in serum, we evaluated the roles of platelets, plasma, and phospholipases by measuring LPA using a novel enzyme-linked fluorometric assay. First, examination of platelet-depleted rats showed that half of the LPA in serum is produced via a platelet-dependent pathway. However, the amount of LPA released from isolated platelets after they are activated by thrombin or calcium ionophore accounted for only a small part of serum LPA. Most of the platelet-derived LPA was produced in a two-step process: lysophospholipids such as lysophosphatidylcholine (LPC), lysophosphatidylethanolamine, and lysophosphatidylserine, were released from activated rat platelets by the actions of two phospholipases, group IIA secretory phospholipase A(2) (sPLA(2)-IIA) and phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)), which were abundantly expressed in the cells. Then these lysophospholipids were converted to LPA by the action of plasma lysophospholipase D (lysoPLD). Second, accumulation of LPA in incubated plasma was strongly accelerated by the addition of recombinant lysoPLD with a concomitant decrease in LPC accumulation, indicating that the enzyme produces LPA by hydrolyzing LPC produced during the incubation. In addition, incubation of plasma isolated from human subjects who were deficient in lecithin-cholesterol acyltransferase (LCAT) did not result in increases of either LPC or LPA. The present study demonstrates multiple pathways for LPA production in serum and the involvement of several phospholipases, including PS-PLA(1), sPLA(2)-IIA, LCAT, and lysoPLD.

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A(1) (PLA(1)) that produces 2-acyl-LPA. The PLA(1) was identified in the GenBank(TM) data base as a close homologue of phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA(3), a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA(1) and suggest that it has a role in LPA production.

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [32P]orthophosphate, and the production of [32P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA1 and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A1 and A2 and then to LPA by plasma lysophospholipase D.

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.

A family of extracellular type I phosphodiesterases has recently been isolated by cDNA cloning, but a physiological function linked to the phosphodiesterase active site has remained unknown. We now present evidence that the phosphodiesterase catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating activity of autotaxin (ATX), one member of the exophosphodiesterase family. Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously purified recombinant ATX, based on the teratocarcinoma sequence, retains these same activities. A single amino acid in the phosphodiesterase catalytic site, Thr210, is found to be necessary for motility stimulation, phosphodiesterase activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and Asp210-ATX, lack motility stimulation and lack both enzymatic activities; Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with sustained phosphodiesterase activity but exhibits no detectable phosphorylation. This mutation eliminates the phosphorylation reaction and indicates that the dephosphorylated state is an active motility-stimulating form of the ATX molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to motility stimulation, these data reveal a novel role for this family of exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades as a regulatory mechanism for cellular motility.

A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein, reveals that this protein is an ecto/exo-enzyme with significant homology to the plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein, previously isolated from a human melanoma cell line (A2058), which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were employed to screen an A2058 cDNA expression library made in lambda gt11. The partial cDNA sequence which was obtained was then extended by utilizing reverse transcriptase on total cellular RNA followed by polymerase chain reaction amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA message size was approximately 3.3 kilobases. The deduced amino acid sequence of autotaxin matched 30 previously sequenced peptides and comprised a protein of 915 amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I phosphodiesterase expressed on the surface of activated B cells and plasma cells. ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel motility-regulating function for this class of ecto/exo-enzymes.

Autotaxin (ATX) is a potent human motility-stimulating protein that has been identified in the conditioned medium from A2058 melanoma cells. This protein has been purified to homogeneity utilizing a strategy involving five column steps. Homogeneity of ATX was verified by two-dimensional gel electrophoresis. The molecular size of ATX is 125 kDa, and it has an isoelectric point of 7.7 +/- 0.2. Purified ATX was digested with cyanogen bromide and trypsin, and the resulting ATX peptides were purified by reverse-phase high performance liquid chromatography. Eleven peptides were subjected to amino acid sequence analysis, and 114 residues were identified. The partial amino acid sequences and the amino acid composition obtained for ATX show that it does not exhibit any significant homology to known growth factors or previously described motility factors. At picomolar concentrations, ATX stimulates both random and directed migration of human A2058 melanoma cells. Pretreatment of the melanoma cells with pertussis toxin abolishes the response to purified ATX, indicating that ATX stimulates motility through a receptor acting via a pertussis toxin-sensitive G protein.