Molecular cloning of cDNAs encoding α1, α2, and β subunits of rat brain platelet-activating factor acetylhydrolase1The nucleotide sequence data reported in this paper have been submitted to the GenBank database under the accession number AF016047 for α1, AF016048 for α2 and AF016049 for β, respectively.1

AbstractBrain intracellular platelet-activating factor acetylhydrolase (PAF-AH(Ib)) is a tertiary G-protein-complex-like heterotrimeric enzyme which is composed of α1, α2, and β subunits and is implicated in stages of brain development such as the formation of the brain cortex. We have isolated and sequenced cDNA clones encoding these three subunits of rat brain PAF-AH(Ib). The amino acid sequences of brain PAF-AH has shown an extremely high homology among mammalian species. The tissue distribution of the three subunits was examined by Northern blot analysis. Although the mRNAs were detected in various organs, the ratio of the level of mRNA expression for the three subunits differed among rat tissues, raising the possibility that isoform(s) other than the heterotrimeric isoform exist in certain tissues

Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA), mediates the induction of nerve-injured neuropathic pain

Recently, we reported that lysophosphatidic acid (LPA) induces long-lasting mechanical allodynia and thermal hyperalgesia as well as demyelination and upregulation of pain-related proteins through one of its cognate receptors, LPA1. In addition, mice lacking the LPA1 receptor gene (lpa1-/- mice) lost these nerve injury-induced neuropathic pain behaviors and phenomena. However, since lpa1-/- mice did not exhibit any effects on the basal nociceptive threshold, it is possible that nerve injury-induced neuropathic pain and its machineries are initiated by LPA via defined biosynthetic pathways that involve multiple enzymes. Here, we attempted to clarify the involvement of a single synthetic enzyme of LPA known as autotaxin (ATX) in nerve injury-induced neuropathic pain. Wild-type mice with partial sciatic nerve injury showed robust mechanical allodynia starting from day 3 after the nerve injury and persisting for at least 14 days, along with thermal hyperalgesia. On the other hand, heterozygous mutant mice for the autotaxin gene (atx+/-), which have 50% ATX protein and 50% lysophospholipase D activity compared with wild-type mice, showed approximately 50% recovery of nerve injury-induced neuropathic pain. In addition, hypersensitization of myelinated Aβ˜- or Aδ-fiber function following nerve injury was observed in electrical stimuli-induced paw withdrawal tests using a Neurometer®. The hyperalgesia was completely abolished in lpa1-/- mice, and reduced by 50% in atx+/- mice. Taken together, these findings suggest that LPA biosynthesis through ATX is the source of LPA for LPA1 receptor-mediated neuropathic pain. Therefore, targeted inhibition of ATX-mediated LPA biosynthesis as well as LPA1 receptor and its downstream pathways may represent a novel way to prevent nerve injury-induced neuropathic pain

A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

Free Fatty Acid Receptor 2 is a GPCR activated by short chain fatty acids produced in high levels in the lower gut by microbial fermentation of non-digestible carbohydrates. A major challenge in studying this receptor is that the mouse ortholog does not have significant affinity for antagonists that are able to block the human receptor. Docking of exemplar antagonists from two chemical series to homology models of both human and mouse Free Fatty Acid Receptor 2 suggested that a single lysine - arginine variation at the extracellular face of the receptor might provide the basis for antagonist selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face of the receptor thus plays key roles in both agonist and antagonist function

Identifcation of P2Y receptors involved in oleamide-suppressing infammatory responses in murine microglia and human dendritic cells

Abstract Microglia, a type of immune cell in the central nervous system, are involved in inflammation leading to neurodegenerative diseases. We previously identified oleamide from fermented dairy products as a neuroprotective compound suppressing microglial inflammation. Oleamide is an endocannabinoid and displays anti-inflammatory activity via the cannabinoid-2 (CB2) receptor; however, the mechanism underlying this anti-inflammatory activity has not been fully elucidated. Here, we found that the suppressive effect of oleamide on microglial tumor necrosis factor-α (TNF-α) production was canceled by inhibitors of G-protein-coupled receptor (GPCR) downstream signaling but not by a CB2 antagonist, suggesting that GPCRs other than CB2 are involved in the anti-inflammatory effects of oleamide. An extensive screen for GPCRs using a transforming growth factor-α shedding assay system identified P2Y1, P2Y4, P2Y6, P2Y10, and P2Y11 as candidates for the oleamide target. P2Y1 and P2Y10 agonists suppressed microglial TNF-α production, while a pan P2 receptor antagonist canceled the suppressive effect. Furthermore, we observed a relationship between the P2Y1 agonistic activities and the suppressive activities of oleamide and its analogs. Taken together, our results suggest that, in addition to CB2, P2Y type receptors are the potential targets of oleamide, and P2Y1 plays a role in the suppression of microglial inflammatory responses by oleamide. (200/200 words

Endogenous agonist–bound S1PR3 structure reveals determinants of G protein–subtype bias

脂質受容体の新たな活性化機構を解明 --脂質がまっすぐ伸びて活性化--. 京都大学プレスリリース. 2021-06-10.Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the “quartet core”) exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein–coupled receptors and will help the design of biased ligands for optimized therapeutics

Genetic and Epigenetic Alterations of Lysophosphatidic Acid Receptor Genes in Rodent Tumors by Experimental Models

Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA1), LPA2, LPA3, LPA4, LPA5 and LPA6. These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models

Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production

Autotaxin (ATX) is a tumor cell motility–stimulating factor, originally isolated from melanoma cell supernatants. ATX had been proposed to mediate its effects through 5′-nucleotide pyrophosphatase and phosphodiesterase activities. However, the ATX substrate mediating the increase in cellular motility remains to be identified. Here, we demonstrated that lysophospholipase D (lysoPLD) purified from fetal bovine serum, which catalyzes the production of the bioactive phospholipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC), is identical to ATX. The Km value of ATX for LPC was 25-fold lower than that for the synthetic nucleoside substrate, p-nitrophenyl-tri-monophosphate. LPA mediates multiple biological functions including cytoskeletal reorganization, chemotaxis, and cell growth through activation of specific G protein–coupled receptors. Recombinant ATX, particularly in the presence of LPC, dramatically increased chemotaxis and proliferation of multiple different cell lines. Moreover, we demonstrate that several cancer cell lines release significant amounts of LPC, a substrate for ATX, into the culture medium. The demonstration that ATX and lysoPLD are identical suggests that autocrine or paracrine production of LPA contributes to tumor cell motility, survival, and proliferation. It also provides potential novel targets for therapy of pathophysiological states including cancer

Lysophosphatidic acid-3 receptor-mediated feed-forward production of lysophosphatidic acid: an initiator of nerve injury-induced neuropathic pain

<p>Abstract</p> <p>Background</p> <p>We previously reported that intrathecal injection of lysophosphatidylcholine (LPC) induced neuropathic pain through activation of the lysophosphatidic acid (LPA)-1 receptor, possibly via conversion to LPA by autotaxin (ATX).</p> <p>Results</p> <p>We examined <it>in vivo </it>LPA-induced LPA production using a biological titration assay with B103 cells expressing LPA₁receptors. Intrathecal administration of LPC caused time-related production of LPA in the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve or sciatic nerve. LPC-induced LPA production was markedly diminished in ATX heterozygotes, and was abolished in mice that were deficient in LPA₃, but not LPA₁or LPA₂receptors. Similar time-related and LPA₃receptor-mediated production of LPA was observed following intrathecal administration of LPA. In an <it>in vitro </it>study using spinal cord slices, LPA-induced LPA production was also mediated by ATX and the LPA₃receptor. Intrathecal administration of LPA, in contrast, induced neuropathic pain, which was abolished in mice deficient in LPA₁or LPA₃receptors.</p> <p>Conclusion</p> <p>These findings suggest that feed-forward LPA production is involved in LPA-induced neuropathic pain.</p