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Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G_i, G_q, and G₁₂ families of heterotrimeric G proteins

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第39期274卷 27351-27358页

作者： Windh, RT Lee, MJ Hla, T An, SZ Barr, AJ Manning, DR Univ Penn Sch Med Dept Pharmacol Philadelphia PA 19104 USA Univ Connecticut Sch Med Dept Physiol Farmington CT 06030 USA Univ Calif San Francisco Dept Med San Francisco CA 94143 USA

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [S-35]GTP gamma S binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates Gi in response not only to S1P but also sphingosylphosphorylcholine;no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

关键词：细胞系 DNA结合蛋白质类/代谢 GTP结合蛋白质类/代谢鸟苷5'-O-(3-硫代三磷酸)/代谢 I-κB蛋白质类即早蛋白质类/代谢动力学溶血磷脂素类大分子物质受体细胞表面/代谢受体 G-蛋白偶联受体溶血磷脂重组蛋白质类/代谢鞘氨醇/类似物和衍生物鞘氨醇/代谢斜纹夜蛾属硫放射性同位素转染毒力因子鲍特菌属/药理学动物人类

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Sphingosine 1-phosphate as a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells

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BLOOD 2000年第10期96卷 3431-3438页

作者： Yatomi, Y Ohmori, T Ge, RL Kazama, F Okamoto, H Sano, T Satoh, K Kume, S Tigyi, G Igarashi, Y Ozaki, Y Yamanashi Med Univ Dept Lab Med Yamanashi 4093898 Japan Hokkaido Univ Grad Sch Pharmaceut Sci Dept Biomembrane & Biofunct Chem Sapporo Hokkaido Japan Univ Tennessee Ctr Hlth Sci Dept Physiol Memphis TN 38163 USA

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [P-32]Sph-1-P, but not [P-32]LPA, was observed In [P-32]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca++ mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have-been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects. (C) 2000 by The American Society of Hematology.

关键词：生物转运血小板/代谢钙信号/药物作用细胞运动/药物作用色谱法薄层细胞骨架/药物作用内皮血管/细胞学内皮血管/代谢胎血/细胞学胎血/代谢溶血磷脂素类/分离和提纯溶血磷脂素类/代谢磷酸盐类/诊断应用磷酸盐类/代谢磷放射性同位素/诊断应用 RNA 信使/代谢受体细胞表面/遗传学受体 G-蛋白偶联受体溶血磷脂酸受体溶血磷脂血清白蛋白/药理学鞘氨醇/类似物和衍生物鞘氨醇/代谢人类

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Purification of free sphingoid bases by solid-phase extraction on weak cation exchanger cartridges

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ANALYTICAL BIOCHEMISTRY 2000年第2期279卷 245-248页

作者： Bodennec, J Famy, C Brichon, G Zwingelstein, G Portoukalian, J Fac Med Lyon Sud Lab Tumor Glycobiol F-69921 Oullins France Univ Lyon 1 Inst Michel Pacha F-83500 La Seyne Sur Mer France

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Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第50期275卷 39379-39384页

作者： Parrill, AL Wang, DA Bautista, DL Van Brocklyn, JR Lorincz, Z Fischer, DJ Baker, DL Liliom, K Spiegel, S Tigyi, G Univ Tennessee Ctr Hlth Sci Dept Physiol Memphis TN 38163 USA Univ Memphis Dept Chem Memphis TN 38152 USA Univ Memphis Computat Res Mat Inst Memphis TN 38152 USA Ohio State Univ Dept Pathol Columbus OH 43210 USA Georgetown Univ Dept Biochem Washington DC 20007 USA

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine I-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine l-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [S-35]GTP gammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

关键词：氨基酸序列精氨酸/化学结合部位印迹法蛋白质细胞系计算机模拟谷氨酸/化学即早蛋白质类/遗传学即早蛋白质类/代谢免疫组织化学离子配体溶血磷脂素类模型分子分子序列数据诱变定点突变蛋白质结合蛋白质结构二级受体细胞表面/代谢受体 G-蛋白偶联受体溶血磷脂序列同源性氨基酸鞘氨醇/类似物和衍生物鞘氨醇/遗传学鞘氨醇/代谢转染肿瘤细胞培养的动物人类大鼠

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Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期267卷 438-444页

作者： Sabatini, M Rolland, G Léonce, S Thomas, M Lesur, C Pérez, V de Nanteuil, G Bonnet, J Inst Rech Servier Div Rheumatol F-92150 Suresnes France Inst Rech Servier Div Oncol F-92150 Suresnes France Inst Rech Servier Div Med Chem F-92150 Suresnes France

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C-2 stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) RI. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat, Furthermore, in isolated chondrocytes, C-2 stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis. (C) 2000 Academic Press.

关键词：膜联蛋白A5/代谢细胞凋亡/药物作用细胞凋亡/生理学软骨关节/细胞学软骨关节/药物作用软骨关节/生理学细胞培养的神经酰胺类/药理学胶原酶类/遗传学白细胞介素1/药理学动力学基质金属蛋白酶13 基质金属蛋白酶类/拮抗剂和抑制剂基质金属蛋白酶类/遗传学基质金属蛋白酶类/代谢苯丙氨酸/类似物和衍生物苯丙氨酸/药理学蛋白酶抑制药/药理学蛋白聚糖类/代谢逆转录聚合酶链反应鞘磷脂磷酸二酯酶/代谢鞘磷脂磷酸二酯酶/药理学鞘氨醇/类似物和衍生物鞘氨醇/药理学噻吩类/药理学转录遗传/药物作用动物男(雄)性兔

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Sphingosine-1-phosphate and lysophosphatidic acid trigger invasion of primitive hematopoietic cells into stromal cell layers

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BLOOD 2000年第1期96卷 139-144页

作者： Yanai, N Matsui, N Furusawa, T Okubo, T Obinata, M Tohoku Univ Inst Dev Aging & Canc Dept Cell Biol Aoba Ku Sendai Miyagi 9808575 Japan

A new primitive hematopoietic cell line (THS119), exhibiting Lin(-)/Sca-1(+)/c-Kit(+) a surface phenotype, grew and survived underneath stromal cells (TBR59). The ability of the THS119 cells to invade these stromal cell layers was dependent on the inclusion of serum in the culture medium, This was apparently due to a requirement for lipids contained in serum. Their invasion of the stromal cell layers in serum-free cultures could be triggered by addition of sphingosine-1-phosphate (S1P) or lysophosphatidic acid (LPA) and was dependent on both Rho- and Ras-signaling pathways. Between the 2 possible receptors of S1P and LPA, edg-1 and edg-2, expression of edg-2 only was found to be correlated with immaturity and/or invasive activity of the primitive hematopoietic cells. These results suggest the importance of specific lipids and their specific receptors on the invasive activity of primitive hematopoietic cells in the hematopoietic microenvironment. (Blood. 2000;96:139-144) (C) 2000 by The American Society of Hematology.

关键词：骨髓细胞/细胞学细胞系细胞培养的共同培养技术培养基造血干细胞/细胞学造血干细胞/药物作用造血干细胞/生理学脂类/血液溶血磷脂素类/药理学小鼠近交C57BL 信号传导/药物作用信号传导/生理学鞘氨醇/类似物和衍生物鞘氨醇/药理学间质细胞/细胞学间质细胞/生理学动物牛小鼠

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A longevity assurance gene homolog of tomato mediates resistance to Alternaria alternata f. sp lycopersici toxins and fumonisin B₁

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第9期97卷 4961-4966页

作者： Brandwagt, BF Mesbah, LA Takken, FLW Laurent, PL Kneppers, TJA Hille, J Nijkamp, HJJ Free Univ Amsterdam Inst Mol Biol Sci Bioctr Amsterdam Dept Genet NL-1081 HV Amsterdam Netherlands

The phytopathogenic fungus Alternaria alternata f. sp, lycopersici (AAL) produces toxins that are essential for pathogenicity of the fungus on tomato (Lycopersicon esculentum). AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which cause inhibition of sphingolipid biosynthesis in vitro and are toxic for some plant species and mammalian cell lines. Sphingolipids can be determinants in the proliferation or death of cells. We investigated the tomato Alternaria stem canker (Asc) locus, which mediates resistance to SAM-induced apoptosis. Until now, mycotoxin resistance of plants has been associated with detoxification and altered affinity or absence of the toxin targets. Here we show that SAM resistance of tomato is determined by Asc-1, a gene homologous to the yeast longevity assurance gene LAG1 and that susceptibility is associated with a mutant Asc-1. Because both sphingolipid synthesis and LAG1 facilitate endocytosis of glycosylphosphatidylinositol-anchored proteins in yeast, we propose a role for Asc-1 in a salvage mechanism of sphingolipid-depleted plant cells.

关键词：衔接蛋白质类信号转导链格孢属/致病力氨基酸序列细胞凋亡羧酸类/毒性细胞死亡细胞分裂伏马菌素类真菌蛋白质类/化学真菌蛋白质类/遗传学 GTP结合蛋白质类基因植物番茄/药物作用番茄/遗传学番茄/微生物学膜蛋白质类/化学膜蛋白质类/遗传学分子序列数据真菌毒素类/毒性植物疾病/遗传学酿酒酵母蛋白质类序列比对序列同源性氨基酸鞘脂类/生物合成鞘脂类/代谢鞘氨醇/类似物和衍生物鞘氨醇/毒性

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Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G_i protein-linked phospholipase C pathway

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期268卷 47-53页

作者： Lee, OH Lee, DJ Kim, YM Kim, YS Kwon, HJ Kim, KW Kwon, YG Hallym Univ Hallym Acad Sci Inst Environm & Life Sci Chunchon 200702 Kangwon Do South Korea Pusan Natl Univ Dept Mol Biol Pusan 609735 South Korea Sejong Univ Dept Biosci & Technol Seoul 143747 South Korea

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et at, Biochem. Biophys. Res. Commun. 264, 743-750, 1999), To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca2+ signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells. (C) 2000 Academic Press.

关键词：细胞黏附分子/化学细胞黏附分子/代谢细胞培养的趋化作用/药物作用内皮血管/细胞学内皮血管/药物作用内皮血管/代谢酶激活/药物作用黏着斑激酶1 黏着斑蛋白酪氨酸激酶类 GTP结合蛋白质α亚单位 Gi-Go/代谢溶血磷脂素类/药理学磷酰化蛋白酪氨酸激酶类/化学蛋白酪氨酸激酶类/代谢信号传导鞘氨醇/类似物和衍生物鞘氨醇/代谢鞘氨醇/药理学 C型磷脂酶类/代谢酪氨酸/代谢人类

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Antibody against a novel, myriocin (ISP-I)-based immunosuppressant, FTY720

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BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 2000年第4期10卷 337-339页

作者： Fujita, T Matsumoto, N Uchida, S Kohno, T Shimizu, T Hirose, R Yanada, K Kurio, W Watabe, K Setsunan Univ Fac Pharmaceut Sci Hirakata Osaka 5730101 Japan Taito Co Ltd Res Lab Nagata Ku Kobe Hyogo 6530023 Japan

An antibody was prepared by immunizing rabbits with an ovalbumin conjugate of 2-amino-2-(2-(4-(4-mercaptobutyl)phenyl)ethyl)propane- 1,3-diol HCl (AMPD-4), which contains the essential structure of the novel immunosuppressant FTY720. As the antibody reacted to not only AMPD-4, but also FTY720, it should be useful for immunoassay of FTY720 in body fluids, tissues and cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：抗体/免疫学抗体生成抗体特异性交叉反应酶联免疫吸附测定脂肪酸类单不饱和/化学合成脂肪酸类单不饱和/化学脂肪酸类单不饱和/免疫学免疫抑制剂/化学合成免疫抑制剂/化学免疫抑制剂/免疫学丙醇胺类/化学合成丙醇胺类/化学丙醇胺类/免疫学丙二醇类/化学合成丙二醇类/化学丙二醇类/免疫学鞘氨醇/类似物和衍生物动物兔

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Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6

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BLOOD 2000年第8期95卷 2624-2629页

作者： Van Brocklyn, JR Gräler, MH Bernhardt, G Hobson, JP Lipp, M Spiegel, S Georgetown Univ Med Ctr Dept Biochem & Mol Biol Washington DC 20007 USA Max Delbruck Ctr Mol Med Dept Tumor Genet & Immunogenet Berlin Germany

EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-8, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [P-32]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6, Binding of [P-32]SPP was saturable, demonstrating high affinity (K-D = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP, LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results Indicate that EDG-6 is a high affinity receptor for SPP, which couples to a G(i/o) protein, resulting in the activation of growth-related signaling pathways. (Blood, 2000;95:2624-2629) 2000 by The American Society of Hematology.

关键词： CHO细胞仓鼠亚科基因转移技术配体溶血磷脂素类蛋白质结合放射配体测定受体细胞表面/遗传学受体细胞表面/代谢信号传导/药物作用鞘氨醇/类似物和衍生物鞘氨醇/代谢鞘氨醇/药理学动物人类

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