摘要
G蛋白偶联受体(GPCRs)在制药领域中占有极其重要的地位，市售西药中有1／2的作用靶点是GPCR，在中药中以GPCRs为靶点进行药物筛选和分子机理研究尚未见报道。在遗传背景清楚的宿主中表达GPCR并纯化其蛋白不仅可以用作受体亚型、嵌合受体、突变受体等药理和生化分析的研究中，而且还可以分析6PCR与G蛋白的相互作用以及由此所引起的信号传导，寻找可以作为药物的先导物、孤儿GPCR的配体等。
甲醇酵母表达系统是发展迅速、应用广泛的一种真核表达系统与大肠杆菌等原核表达系统相比，它能对表达的蛋白进行翻译后的修饰和加工；与杆状病毒、哺乳动物细胞表达系统相比，它又具有培养成本低、产量高、便于产物的分离和纯化等优点。甲醇酵母中以可调控的AOX1强启动子控制下表达异源基因，其产物较酿酒酵母表达的更接近真核生物甚至人类。因此利用甲醇酵母表达G蛋白偶联受体，不但可以进行快速、高通量的配体和药物先导物的筛选；而且可以提供足量的GPCR蛋白进行蛋白结构、受体亚型、嵌合受体、突变受体的药理和生化分析。因此，以甲醇酵母为宿主的表达体系具有其他系统不可比拟的优点。
内皮细胞分化因子(edg-1)和缓激肽受体(B_2R)同属于G蛋白偶联的受体超家族，都与内皮型一氧化氮合成酶(eNOS)等多种信号途径偶联。其中edg-1与心血管疾病、免疫系统疾病密切相关：而B_2R与心血管疾病及各种炎症密切相关。
edg-1和B_2R都尚未在酵母细胞中表达。为了开发一种操作简便，成本低廉的以edg-1和B_2R为靶点的药物筛选系统，以期获得抗心血管疾病的药物或其他用途的药物。我们用RT-PCR方法从人脐静脉内皮细胞中克隆了edg-1受体和B_R基因，首次利用甲醇酵母Pichia pastoris来表达edg-1和B_2R受体，并对表达受体的细胞定位和功能进行了研究。
在B_2R基因的C-端接上绿色荧光蛋白基因(EGFP)作报告基因，构建了EGFP融合的B_2R-EGFP基因，将B_2R-EGFP融合基因克隆到甲醇酵母整合型表达载体pPIC9k上。利用pPIC9k的α信号肽序列促进膜受体的跨膜分布，电击转化P．pastoris GS115菌株，抗生素G418浓度梯度筛选高抗性转化子，重组菌株用甲醇诱导表达。流式细胞仪分析表明B_2R-EGFP在GS115中获得了很强的表达；Western印迹杂交结果证实了表达产物为B_2R-EGFP融合受体蛋白；在激光共聚焦显微镜下重组菌株观察到很强的绿色荧光；虽然表达的受体蛋白也有内膜分布，但免疫荧光分析证实表达的受体蛋白大量定位到了细胞膜上。
免疫荧光分析还证明了表达的B_2R受体蛋白具有同配体结合的功能。
同样构建pPIC9k-edg-1-EGFP表达载体，电转化P．pastoris GS115菌株，但不筛选高G418抗性转化子，甲醇诱导表达。诱导的转化子流式细胞仪检测表明edg-1-EGFP在GS115中获得了很强的表达；Western印迹杂交结果证实了表达产物为edg-1-EGFP融合受体蛋白；在激光共聚焦显微镜下重组菌株发出很强的绿色荧光；免疫荧光分析证实表达的重组受体大量定位于酵母细胞膜上。
鞘氨醇-1-磷酸是edg-1受体的高亲和性配体，克隆并原核表达了鞘氨醇激酶基因(SphK)，利用纯化的鞘氨醇激酶制备γ-~(32)P标记的鞘氨醇-1-磷酸(S-1-~(32)P)，放射性标记的配体结合分析表明重组edg-1受体具有配体结合功能。
鞘氨醇-1-磷酸明显降低重组酵母GS115：：pPIC9K-edg-1-EGFP的生长速度，表明重组edg-1受体和酵母细胞内信号途径偶联，受体具有生物学功能。我们的研究为进一步利用B_2R和edg-1受体建立药物筛选系统打下了基础。
G protein-coupled receptors(GPCRs) are the largest class of targets for modern drugs by virtue of their roles in the regulation of cellular functions. Heterologous production of GPCRs in a defined host background has been established as an important and valuable tool for the pharmacological and biochemical analysis of defined receptor subtypes, as well as chimeric or mutant receptors. Furthermore, the analysis of G protein interaction and subsequent signal transduction, the detection of 'lead' compounds in the search for new drugs and of ligands for orphan GPCRs by high-throughput screens, as well as the purification of receptor protein suitable for biochemical, biophysical and/or structural studies are important goals. Here, due to their ability of high level production, easy manipulation, and low costs, yeast-based expression systems have proven very attractive. Unlike Escherichia coli, yeasts have the potential to perform eukaryotic post-translational modifications, such as N-glycosylation, which may affect receptor function. The methylotropic yeast strain, Pichia pastoris, has a strong inducible promoter and it is less prone to hyperglycosylation than Saccharomyces cerevisiae. Therefore, recombinant P. pastoris has been developed as an excellent host for the expression of foreign GPCRs.
The human endothelial differentiation gene l(edg-l) and bradykinin B2 receptor (B_2R) belong to the large family of G-protein-coupled receptors. In order to develop a convenient and rapid assay for testing compounds which might be effective as edg-1 and B_2R receptors agonists or antagonists, the gene of edg-1 and B_2R receptors were expressed in the methylotrophic yeast Pichia pastoris.
The expression plasmids were constructed in which the edg-1 or B_2R gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene, the green fluorescent protein(GFP) gene were introduced at the C-terminal of the edg-1 or B_2R gene to permit easy detection.
Fluorescence activated cell sorting (FACS) analysis and Western blot
analysis revealed that the edg-1 and B_2R recombinant receptor proteins were distinctly expressed. The results were further confirmed by Confocal microscopy which produced bright green fluorescence.
The localization of the recombinant edg-1 or B_2R receptor proteins were proved by immunofluorescence microscopy which indicated a distinct expression of the edg-1 or B_2R in the plasma membrane of the transformed yeast cells.
The binding of bradykinin ligand to the B_2R recombinant receptor proteins were also proved by immunofluorescence microscopy which exhibited a high affinities of bradykinin to the GS115-B2-EGFP cell membrane.
In radioligand binding analysis, the edg-1 recombinant receptor proteins also showed high affinities of S— 1 -~(32)P.
Exogenous SPP treatment, inhibited GS115-edg-1-EGFP cells proliferation.
In conclusion, we provide compelling evidence that the edg-1 and B_2R receptor were functionally expressed in P. pastoris and localization on the plasma membrane for the first time. These results strongly suggest that the GS115-B2-EGFP or the GS115-edg-1-EGFP cell line is a useful tool to study the effect of potential pharmacological agents that may modulate the function of edg-1 or B_2R receptor.
引文
陈巨莲，Ge zhi WENG，倪汉祥．2001．G蛋白及其偶联信号传导途径的研究进展．生物工程学报．17(2)：113-117
蒋维，李菊香．2002．孤儿G蛋白偶联受体研究进展．生理科学进展．33(2)：115-120
刘永学，余少平．2003．孤儿G蛋白偶联受体及其作为新药靶点的重要意义．中国药理学通报．19(6)：601-4
谢建平，霍克克，王洪海．2000．G蛋白偶联受体在酵母中的表达及应用．高技术通讯．8：97-101
许治良，高 虹，欧阳克清．2003．G蛋白偶联受体的功能测定和高通量药物筛选 中国药理学通报．19(12)：1330～6
尹燕斌 罗静初，姜颖．2003．G蛋白偶联受体及其生物信息学研究．科学通报．48(4)：307-312
翟中和，王喜中，丁明孝主编：细胞生物学．北京：高等教育出版社，2000
Abdulaev NG, Popp MP, SmithWC. 1997. Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 10: 61-69.
Ames R, Fornwald J, Nuthulaganti P, 2004. BacMam Recombinant Baculoviruses in G Protein-Coupled Receptor Drug Discovery. Receptors Channels. 10(3-4): 99-107
Angermayr M, Strobel G, Muller G, et al. 2000. Stable plasma membrane expression of the soluble domain of the human insulin receptor in yeast. FEBS Lett. 481(1): 8-12
Aparicio SA, Powell J. 2004. Genetic approaches to unraveling G protein-coupled receptor biology. Curt Opin. Drug Discov Devel. 7(5): 658-664.
Arkinstall S, Edgerton M, Maundrell K. 1995. Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 375: 183-187.
Bajaj A, Celic A, Ding FX, et al. 2004. A Fluorescent alpha-Factor Analogue Exhibits Multiple Steps on Binding to Its G Protein Coupled Receptor in Yeast. Biochemistry. 43(42): 13564-13578
Bardwell L. 2004. A walk-through of the yeast mating pheromone response pathway. Peptides. 25(9): 1465-1476
Becker F, Block-Alper L, Nakamura G, et al., 1999. Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast. J Cell Biol. 146(2): 273-284
Berlin B, Freissmuth M, Jockers R. 1994. Cellular signaling by an agonist-activated receptor/Gsc( fusion protein. Proc Nat Acad Sci. 91: 8827-8831.
Bhattacharya M, Babwah AV, Ferguson SS. 2004. Small GTP-binding protein-coupled receptors. Biochem Soc Trans. 32(Pt 6): 1040-1044
Buckholz RG. 1993. Yeast systems for the expression of heterologous gene products. Curt Opin Biotechnol. 4: 538-542.
Burstein ES, Spalding TA, Brauner-Osborne H, et al. 1995. Constitutive activation of muscarinic receptors by the G-protein Gq. FEBS Lett. 363: 261-263.
Carver LA, Jackiw V, Bradfield CA. 1994. The 90-kDa heat shock protein is essential for Ah receptor signaling in a yeast expression system. J Biol Chem. 269(48): 30109-30112
Chidiac P. 1998. Rethinking receptor-G protein-effector interactions. Biochem Pharmacol. 55: 549-556.
Clapham DE, Neer EJ. 1993. New roles for G protein β γ dimmers intransmembrane signaling. Nature. 365: 403～406
David J. Roberts, Magali Waelbroeck. 2004. G protein activation by G protein coupled receptors: ternarycomplex formation or catalyzed reaction? Biochemical Pharmacology. 68: 799-806
Eckart MR, Bussineau CM. 1996. Quality and authenticity of heterologous proteins synthesized in yeast. Curt Opin Biotechnol. 7: 525-530.
Erickson JR, Wu J J, Goddard G. 1998. Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidicacid. J. Bio. Chem. 273: 1506-1510
Evers A, Klebe G. 2004. Successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model. J Med Chem. 47(22): 5381-5392.
Feng W, Cai J, Pierce WM, Song ZH. 2002. Expression of CB2 cannabinoid receptor in Pichia pastoris. Protein Expr Purif. 26(3): 496-505
Ficca AG, Testa L, Tocchini-Valentini GP. 1995. The human β_2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties. FEBS Lett. 377: 140-144.
Ford CE, Skiba NP, Bae H, et al. 1998. Molecular basis for interactions of G protein β γ subunits with effectors. Science. 280: 1271-1274.
Gerstmayer B, Pessara U, Wels W. 1997. Construction and expression in the yeast Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA-4. FEBS Lett. 407(1): 63-68
Gether U. KobilkaBK. 1998. Gprotein-coupledreceptors: Ⅱ. Mechanismof agonist activation. J Biol Chem. 273: 17929-17982
Gether, U. 2000. Uncovering Molecular Mechanisms Involved in Activation of G Protein-Coupled Receptors. Endocr Rev 21: 90-113
Gether, U., Kobilka, B. K. 1998. G Protein-coupled Receptors. Ⅱ. MECHANISM OF AGONIST ACTIVATION. J. Biol. Chem. 273: 17979-17982
Ghanouni, P., Steenhuis, J. J., Farrens, D. L., et al. 2001. Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor. Proc. Natl. Acad. Sci. 98: 5997-6002
Grisshammer R, Tare CG. 1995. Overexpression of integral membrane proteins for structural studies. Quart Rev Biophys. 28: 315-422.
Hallak, H., Seller, A. E. M., Green, J. S., et al.2000. Association of Heterotrimeric Gi with the Insulin-like Growth Factor-I Receptor. RELEASE OF Gbeta gamma SUBUNITS UPON RECEPTOR ACTIVATION. J. Biol. Chem. 275: 2255-2258
Hamm HE. 1998. The many faces of G protein signaling. J Biol Chem. 273: 669-672.
Hamm HE. 2001. How activated receptors couple to G proteins. Proc Natl Acad Sci. 98: 4819-4821.
Helmut R, H Markus W. 1998. Production of G-protein-coupled receptors in yeast. Current Opinion in Biotechnology, 9: 510-517
Hildebrandt V, Fendler K, Heberle J. 1993. Bacteriorhodopsin expressed in Schizosaccharomyces pombe pumps protons through the plasma membrane. Proc Natl Acad Sci. 90: 3578-3582.
Howard A D. Mcallister G. Feighner S D. et al. 2001. Orphan G-protein-coupled receptors and natural ligand discovery. Trends Pharmacol Sci.22: 132-140
Huang C, Hepler JR, Chen LT, et al. 1997. Organization of G proteins and adenyl cyclase at the plasma membrane. Mol Biol Cell. 8: 2365- 2378.
Huang HJ, Liao CF, Yang BC, Kuo TT. 1992. Functional expression of rat M5 muscarinic acetylcholine receptor in yeast. Biochem Biophys Res Commun. 182(3): 1180-1186
Ishizawa, Y., Pidikiti, R., hiebman, P. A. 2002. G Protein-Coupled Receptors as Direct Targets of Inhaled Anesthetics. Mol Pharmacol 61: 945-952
Jensen, A. D., Guarnieri, F., Rasmussen, et al. 2001. Agonist-induced Conformational Changes at the Cytoplasmic Side of Transmembrane Segment 6 in the beta 2 Adrenergic Receptor Mapped by Site-selective Fluorescent Labeling. J. Biol. Chem. 276: 9279-9290
Kalipatnapu S, Chattopadhyay A. 2004. A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin(1A) receptor. FEBS hett. 576(3): 455-60.
Kenakin TP. 2001. Quantitation in receptor pharmacology. Receptors Channels. 7: 371-85.
Krasel C, Vilardaga JP, Bunemann M, hohse MJ. 2004. Kinetics of G-protein-coupled receptor signalling and desensitization. Biochem Soc Trans. Dec 1; 32(6): 1029-1031.
Kukkonen JP, Nasman J, Akerman KE. 2001. Modelling of promiscuous receptor-Gi/Gs-protein coupling and effector response. Trends Pharmacol Sci. 22: 616-622.
Kurjan J. 1993. The pheromone response pathway in Saccharomyces cerevisiae. Annu Rev Genet. 27: 147-179.
Lawson Z, Wheatley M. 2004. The third extracellular loop of G-protein-coupled receptors: more than just a linker between two important transmembrane helices. Biochem Soc Trans. 32(Pt 6): 1048-50.
Leff P, Scaramellini C, Law C, 1997. A three-state receptor model of agonist action. Trends Pharmacol Sci. 18: 355-362.
Lefkowitz RJ, Cotecchia S, Samama P, et al. 1993. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci. 14: 303-7.
Levin DE, Errede B. 1995. The proliferation of MAP kinase signaling pathways in yeast. Curr ODin Cell Biol. 7: 197-202.
Ling, K., Wang, P., Zhao, J., et al. 1999. Five-transmembrane domains appear sufficient for a G protein-coupled receptor: Functional five-transmembrane domain chemokine receptors. Proc. Natl. Acad. Sci. 96: 7922-7927
Lu C, Yang YF, Ohashi H, Walfish PG. 1990. In vivo expression of rat liver c-erbA beta thyroid hormone receptor in yeast (Saccharomyces cerevisiae). Biochem Biophys Res Commun. 171(1): 138-42
Marinissen M J, Gutkind J S. 2001. G protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci. 22: 368-376
Mclntire WE, Myung CS, MacCleery G, et al. 2002. Reconstitution of G protein-coupled receptors with recombinant G protein α and β γ subunits. Meth Enzymol. 343: 372-93.
Menon, S. T., Han, M., Sakmar, T. P. 2001. Rhodopsin: Structural Basis of Molecular Physiology. Physiol. Rev 81: 1659-1688
Miller CA. 1997. Expression of the human aryl hydrocarbon receptor complex in yeast. Activation of transcription by indole compounds. J Biol Chem. 272(52): 32824-32829
Morris, A. J., Malbon, C. C. 1999. Physiological Regulation of G Protein-Linked Signaling. Physiol. Rev 79: 1373-1430
Parrish, W., Eilers, M., Ying, W., et al. 2002. The Cytoplasmic End of Transmembrane Domain 3 Regulates the Activity of the Saccharomyces cerevisiae G-Protein-Coupled {alpha}-Factor Receptor. Genetics 160: 429-443
Pausch MH, Lai M, Tseng E, et al. 2004. Functional expression of human and mouse P2YI2 receptors in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 324(1): 171-177
Pausch MH. 1997. G-protein-coupled receptors in Saccharomyces cerevisiae: high-throughput screening assays for drug discovery. Trends Biotechnol. 15: 487-494
Ports J, Lee EW, Li L, Kitlinska J. 2004. Neuropeptide Y: multiple receptors and multiple roles in cardiovascular diseases. Curr Opin Investig Drugs. 5(9): 957-62.
Price LA, Strnad J, Pausch MH, et al. 1996. Pharmacological characterization of the rat A2a adenosine receptor functionally coupled to the yeast pheromone response pathway. Mol Pharmacol. 50: 829-837.
Psaridi-Linardaki L, Mamalaki A, Remoundos M, et al., 2002. Expression of soluble ligand- and antibody-binding extracellular domain of human muscle acetylcholine receptor alpha subunit in yeast Pichia pastoris. Role of glycosylation in alpha-bungarotoxin binding. J Biol Chem. 277(30): 26980-26986
Rana, B. K, Shiina, T., Insel, P. A 2001. GENETIC VARIATIONS AND POLYMORPHISMS OF G PROTEIN-COUPLED RECEPTORS: Functional and Therapeutic Implications. Annu. Rev. Pharmacol. Toxicol. 41: 593-624
Riond J, Leplatois P, Laurent P, et al., 1991. Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast. Eur J Pharmacol. 208(4): 307-312
Rob Leurs, Maria Sol Rodriguez Pena. 2000. Constitutive activity of G protein coupled receptors and drug action. Pharmaceutica Acta Helvetiae 74: 327-331
Romanos M. 1995. Advances in the use of Pichia pastoris for high-level gene expression. Curr Opin Biotechnol. 6: 527-533.
Sander P, GrLinewald S. 1994. Expression of the human D2s dopamine receptor in the yeasts Saccharomycescerevisiae and Schizosaccharomyces pombe: a comarative study. FEBS Lett. 344: 41-46.
Sander P, Grunewald S, Maul G, et al., 1994. Constitutive expression of the human D2S-dopamine receptor in the unicellular yeast Saccharomyces cerevisiae. Biochim Biophys Acta. 1193(2): 255-262
Scharstuhl, Alwin; Glansbeek, Harrie; Vitters, EllyL. et al.2003. Large scale protein production of the extracellular domain of the transforming growth factor-β type Ⅱ receptor using the Pichia pastoris expression system. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences. 786(1-2): 271-277
Shea LD, Neubig RR, Linderman JJ. 2000. Timing is everything the role of kinetics in G protein activation. Life Sci. 68: 647-658.
Sizmann D, Kuusinen H. 1996. Production of adrenergic receptors in yeast. Recept Channels. 4: 197-203.
Stadel JM, Wilson S, Bergsma D J. 1997. Orphan G protein-coupled receptors: a neglected opportunity for pioneer drug discovery. Trends Pharmacol Sci. 18: 430-437
Sudbery PE. 1996. The expression of recombinant proteins in yeasts. Curr Opin Biotechnol. 7: 517-524.
Sunahara RK, Tesmer JJ, Gilman AG, Sprang SR. 1997. Crystal structure of the adenyl cyclase activator Gs_0. Science. 278: 1943-1947.
Susan R. G, Brian F. O, Samuel P. L. 2002. G-protein-coupled receptor oligomerization and its potential foe drug discovery. Nature. 1: 808-820
Susana R., Prahlad T. R, Ravi Iyengar. 2OO2. G Protein Pathways. Science. 296(5573): 1636
Tae H J. Mathis G. Inhae J. 1998. G protein-coupled receptors: Ⅰ. Diversity of receptor-ligand interactions. J Biol Chem. 273: 17299-17302
Talmont F, Sidobre S, Demange P, et al., 1996. Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris. FEBS Lett. 394(3): 268-272
Tanoue A, Koshimizu T, Tsujimoto G, et al. 2004. Heterogeneity of G protein-coupled receptor generated by post-translational mechanisms and its clinical meanings. Nippon Yakurigaku Zasshi. 124(4): 235-243.
Tate CG, Grisshammer R. 1996. Heterologous expression of G-protein-coupled receptors. Trends Biotechnol. 14: 426-430.
Tokita, K., Hocart, S. J., Katsuno, T., et al.2001. Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is Critical for the High Selectivity of the Peptoid Antagonist PD168368. J. Biol. Chem. 276: 495-504
Tucek S, Michal P, Vlachova V. 2002. Modelling the consequences of receptor-G-protein promiscuity. Trends Pharmacol Sci. 23: 171-176.
UlrikGether, Sansan Lin, and BrianK. et al. 1995. F1uorescent Labeling of Purified β_2-Adrenergic Receptor. J. Biol. Chem. 270: 28268-28275.
Vollmer P, Peters M, Ehlers M, et al., 1996. Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor. J Immunol Methods. 199(1): 47-54
Weiss HM, Haase W, Michel H, et al., 1995. Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization. FEBS Lett. 377(3): 451-456
Wong SK. 2003. G protein selectivity is regulated by multiple intracellular regions of GPCRs. Neurosignals. 12: 1-12
Wright AP, McEwan IJ, Dahlman-Wright K, et al., 1991. High level expression of the major transactivation domain of the human glucocorticoid receptor in yeast cells inhibits endogenous gene expression and cell growth. Mol Endocrinol. 5(10): 1366-1372
Xie XQ, Zhao J, Zheng H. 2004. Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233). Protein Expr Purif. 38(1): 61-68.
Yamamoto M. 1996. The molecular control mechanisms of meiosis in fission yeast. Trends Biochem Sci. 21: 18-22.
Yang Q, Lanier SM. 1999. Influence of G protein type on agonist efficacy. Mol Pharmacol. 56: 651-656.
Yellen G, Migeon JC. 1990. Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences. Gene. 86(2): 145-52
Zuck, P., Lao, Z., Skwish, S., et al. 1999. Ligand-receptor binding measured by laser-scanning imaging. Proc. Natl. Acad. Sci. 96: 11122-11127
Ana M. Genaro, Graciela M. Stranieri, Enri Borda, 2000. Involvement of the endogenous nitric oxide signaling system in bradykinin receptor activation in rat submandibular salivary gland. Archives of Oral Biology. 45, 723-729
Andree Blaukata, Anne Pizardb, Rabary M. Rajerisonb, et al. 1999. Activation of mitogen-activated protein kinase by the bradykinin B2 receptor is independent of receptor phosphorylation and phosphorylation-triggered internalization. FEBS Letters 451, 337-341
Austin, C. E., Faussner, A., Robinson, H. E., et al., 1997. Stable expression of the human kinin B2 receptor in Chinese hamster ovary cells. J. Biol. Chem. 272,(1) 11420-11425.
Bastian, S., Loillier, B., Paquet, J. L., et al., 1997. Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization. Br. J. Pharmacol. 122, 393-399.
Bhoola, K. D., Figueroa, C. D., Worthy, K., 1992. Bioregulation of kinins: kallikreins, kininogens and kininases. Pharmacol. Rev. 44, 1-80.
Blaukat A, Micke P, Kalatskaya I, et a1.,2003. Downregulation of bradykinin B2 receptor in human fibroblasts during prolonged agonist exposure. Am J Physiol Heart Circ Physiol. 284(6): H1909-16.
Camarda V, Rizzi A, Calo G, Wirth K, Regoli D. 2002. Pharmacological characterisation of novel kinin B2 receptor ligands. Can J Physiol Pharmacol. 80(4): 281-6.
Carini F, Guelfi M, Lecci A, et al., 2002. Cardiovascular effects of peptide kinin B2 receptor antagonists in rats. Can J Physiol Pharmacol. 80(4): 310-22.
Chen BC, Yu CC, Lei HC, et al., 2004. Bradykinin B2 receptor mediates NF-kappaB activation and cyclooxygenase-2 expression via the Ras/Raf-1/ERK pathway in human airway epithelial cells. J Immunol. 173(8): 5219-28.
Clemens, J. A., 1994. The human kallikrein gene family: a diversity of expression and function. Mol. Cell. Endocrinol. 99, 1-6.
Dixon, B. S., Sharma, R. V., Dennis, M. J., 1996. Thebradykinin B2 receptor is a delayed early response gene for platelet-derived growth factor in arterial smooth muscle cells. J. Biol. Chem. 271, 13324-13332.
DomenicoRegoli, Suzanne Nsa Allogho, AnnaRizzi, etal, 1998. Bradykinin receptors and their antagonists. European Journal of Pharmacology. 348, 1-10
Dray, A., 1997. Kinins and their receptors in hyperalgesia. Can. J. Physiol. Pharmacol. 75, 704-712.
Dray, h., Perkins, M., 1993. Bradykinin and inflammatory pain. Trends Neurosci. 16, 99-104.
Orouin, J. N., Gaudreau, P., St-Pierre, S.A., Regoli, D., 1979. Biological activities of kinins modified at the N- or the C-terminal end. Can. J. Physiol. Pharmacol. 57, 1018-1023.
Duner T, Conlon JM, Kukkonen JP, et al., 2002. Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor. Biochem J. 364: 817-24.
Eggerickx, D., Raspe, E., Bertrand, D., et al., 1992. Molecular cloning, fuctional expression and pharmacological characterization of a human bradykinin B2 receptor gene. Biochem. Biophys. Res. Commun. 187, 1306-1313.
Feletou, M., Martin, C.A.E., Molimard, M., etal.,1995. In vitro effects of HOE 140 in human bronchial and vascular tissue. Eur. J. Pharmacol. 274, 57-64.
Geppetti, P., 1993. Sensory neuropeptide release by bradykinin: mechanisms and pathological implications. Regul. Pep. 47, 1-23.
Gessi, S., Rizzi, A., Calo, G., et al., 1997. Human vascular kinin receptors of the B2 type characterized by binding. Br. J. Pharmacol. 122, 1450-1454.
Gobeil, F., Filteau, C., Pheng, L. H., et al, 1996d. In vitro and in vivo characterization of bradykinin B2 receptors in the rabbit and the guinea pig. Can. J. Physiol. Pharmacol. 74, 137-144.
Gobeil, F., Pheng, L.H., Badini, I., et al., 1996a. Receptors for kinins in the human isolated umbilical vein. Br. J. Pharmacol. 118, 289-294.
Greco S, Muscella A, Elia MG, et a1.,2004. Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells. J Cell Physiol. 201(1): 84-96.
Hall, J. M., 1992. Bradykinin receptors: pharmacological properties and biological roles. Pharmacol. Ther. 56, 131-190.
Heitsch H. 2000. Bradykinin B2 receptor as a potential therapeutic target. Drug News Perspect. May; 13(4): 213-25.
Heitsch H. 2003. The therapeutic potential of bradykinin B2 receptor agonists in the treatment of cardiovascular disease. Expert Opin Investig Drugs. May; 12(5): 759-70.
Hess, J. F., Borkowski, J. A., Young, G. S., et al., 1992. Cloning and pharmacological characterization of a human bradykinin BK-2 receptor. Biochem. Biophys. Res. Commun. 184, 260-268.
Ikeda Y, Hayashi I, Kamoshita E, et al., 2004. Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth. Cancer Res. 64(15): 5178-85.
Judith M. Hall. 1997. Bradykinin Receptors. Gen. Pharmac. 28(1), 1-6
Kang DS, Leeb-Lundberg LM. 2002. Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G(q/11)-mediated[correction of G(9/11)-mediated]phospholipase Cbeta activity. Mol Pharmacol. 62(2): 281-8
Kang DS, Ryberg K, Morgelin M, et al., 2004. Spontaneous formation of a proteolytic B1 and B2 bradykinin receptor complex with enhanced signaling capacity. J Biol Chem. May 21; 279(21): 22102-7.
Levesque, L., Harvey, N., Rioux, F., et al., 1995. Development of a binding assay for the B1 receptors for kinins. Immunopharmacology 29, 141-147.
Lohse, M. J., 1993. Molecular mechanisms of membrane receptor desensitization. Biochim. Biophys. Acta 1179, 171-188.
Lopes, P., Couture, R., 1992. Cardiovascular responses elicited by intrathecal kinins in the conscious rat. Eur. J. Pharmacol. 210, 137-147.
Marceau, F., Levesque, L., Drapeau, G., et al., 1994. Effects of peptide and non peptide antagonists of bradykinin B2 receptors on the venoconstrictor action of bradykinin. J. Pharmacol. Exp.Ther. 269, 1136-1143.
Menke, J. G., Borkowski, J. A., Bierlo, K. K., et al., 1994. Expression cloning of a human B1 bradykinin receptor. J. Biol. Chem. 269, 21583-21586.
Michineau S, Muller L, Pizard A, et al., 2004. N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling. Biol Chem. 385(1):49-57.
Mombouli, J. V., Vanhoutte, P.M., 1995. Kinins and endothelial control of vascular smooth muscle. Annu. Rev. Pharmacol. Toxicol. 35, 679-705.
Nsa Allogho, S., Gobeil, F., Pheng, L.H., et al., 1995. Kinin Bl and B2 receptors in the mouse. Can. J. Physiol. Pharmacol. 73,1759-1764.
Phagoo, S.B., Poole, S., Leeb-Lundberg, L M. F., 1999. Autoregulation of bradykinin receptors: agonists in the presence of Interleukin-1b shift the repertoire of receptor subtypes from B2 to Bl in human lung fibroblasts. Mol. Pharmacol. 56, 325-333.
Qadri F, Hauser W, Johren O, Dominiak P. 2002. Kinin Bl and B2 receptor mRNA expression in the hypothalamus of spontaneously hypertensive rats. Can J Physiol Pharmacol. 80(4):258-63.
Qadri F, Schwartz EC, Hauser W, et al., 2003. Kinin B2 receptor localization and expression in the hypothalamo-pituitary-adrenal axis of spontaneously hypertensive rats. Int Immunopharmacol. 3(3):285-92.
Regoli, D., Gobeil, F., Nguyen, Q.T., et al., 1994a. Bradykinin receptor types and B2 subtypes. Life Sci. 55, 735 - 749.
Regoli, D., Jukic, D., Gobeil, F., Rhaleb, N. E., 1993. Receptors for bradykinin and related kinins: a critical analysis. Can. J. Physiol. Pharmacol. 71, 556 - 567.
Regoli, D., Rhaleb, N. E., Dion, S., Drapeau, G., 1990b. New selective bradykinin receptor antagonists and bradykinin B2 receptor character- ization. Trends Pharmacol. Sci. 11, 156-161.
Regoli, D., Rhaleb, N. E., Drapeau, G., Dion, S., 1990a. Kinin receptor subtypes. J. Cardiovasc. Pharmacol. 15, 530 - 538, Suppl. 6.
Rizzi, A., Gobeil, F., Calo, G., et al., 1997. FR173657, a new potent and selective nonpeptide kinin B2 receptor antagonist: an in vitro study. Hypertension 29, 951-956.
Rodell, T. C., 1996. The kallikreinrkinin system andkininantagonists in trauma. Immunopharmacology 33, 279-283.
Sabine Btickmann, Inge Paegelow, 1995. Bradykinin receptors and signal transduction pathways in peritoneal guinea pig macrophages. European Journal of Pharmacology. 291, 159-165
Salvino, J. M., Seoane, P. R., Douty, B.D., et al., 1993. Design of potent non-peptide competitive antagonists of the human bradykinin B2 receptor. J. Med. Chem. 36, 2583-2584.
Sawutz, D. J., Salvino, J. M., Oolle, R. E., et al., 1994. The nonpeptide WIN 64338 is a bradykinin B2 receptor antagonist. Proc. Natl. Acad. Sci. 91, 4693-4697.
Schini, V. B., Boulanger, C., Regoli, D., et al., 1990. Bradykinin stimulates the production of cyclic GMP via activation of B2 kinin receptors in cultured porcine aortic endothelial cells. J. Pharmacol. Exp. Ther. 252, 581-585.
Schmidlin, F., Scherrer, D., Daeffler, et al., 1998. Interleukin-1b induces bradykinin B2 receptor gene expression through a prostanoid cyclic AMP-dependent pathway in human bronchial smooth muscle cells. Mol. Pharmacol. 53, 1009-1015.
Seegers HC, Avery PS, McWilliams DF, et al., 2004. Combined effect of bradykinin B2 and neurokinin-I receptor activation on endothelial cell proliferation in acute synovitis. FASEB J. 18(6): 762-4.
Smith, J. A. M., Webb, C., Holford, J., et al., 1995. Signal transduction pathways for BI and B2 bradykinin receptors in bovine pulmonary artery endothelial cells. Mol. Pharmacol. 47, 525-534.
Sobey CG. 2003. Bradykinin B2 receptor antagonism: a new direction for acute stroke therapy?. Br J Pharmacol. 139(8): 1369-71.
Souza DG, Pinho V, Pesquero JL, et al.,2003. Role of the bradykinin B2 receptor for the local and systemic inflammatory response that follows severe reperfusion injury. Br J Pharmacol. 139(1): 129-39.
Stephen B. Phagoo, Mohammed Yaqoob, Esteban Herrera-Martinez, et al. 2000. Regulation of bradykinin receptor gene expression in human lung fibroblasts. European Journal of Pharmacology. 397, 237-246
Steranka, L. R., Manning, D. C., Dehaas, C. J., et al., 1988. Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic action. Proc. Natl. Acad. Sci. 85, 3245-3249.
Sui-Po Zhang, Hoau-Yan Wang, Timothy W. Lovenberg, et al. 2001. Functional studies of bradykinin receptors in Chinese hamster ovary cells stably expressing the human B2 bradykinin receptor. International Immunopharmacology, 1, 955-965
Sylvia Mueller, Claus Liebmann, Siegmund Reissmann, 2002. intramolecular signal transduction by the bradykinin B2 receptor. International Immunopharmacology. 2, 1763-1770
Trabold F, Pons S, Hagege AA, 2002. Cardiovascular phenotypes of kinin B2 receptor- and tissue kallikrein-deficient mice. Hypertension. 40(1): 90-5.
Vavrek, R., Stewart, J. M., 1985. Competitive antagonists of bradykinin. Peptides 6, 161- 164.
Yang CM, Chien CS, Ma YH, et al., 2003. Bradykinin B2 receptor-mediated proliferation via activation of the Ras/Raf/MEK/MAPK pathway in rat vascular smooth muscle cells. J Biomed Sci. 10(2): 208-18
Yuh-Jiin Ivy Jong, Linda R. Dalemar, Beverlyn Wilhelm, et al. 1996. Human lung fibroblasts express multiple means for enhanced activity of bradykinin receptor pathways. Immunopharmacology. 33, 9-15
Marceau F. Regoli D. 2004. Bradykinin receptor ligands: therapeutic perspectives. Nat Rev Drug Discov. 3(10): 845-52.
Alirio J. Melendez a, Estelle Carlos-Dias a, Mark Gosink b, et al. 2000. Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution. Gene 251, 19-26
An, S., Bleu, r., &Zheng, Y. 1999. Transductionofintracellularcalcium signals through G protein-mediated activation of phospholipase C by recombinant sphingosine 1-phosphate receptors. Mol Pharmacol 55, 787-794.
An, S., Zheng, Y., & Bleu, T. 2000. Sphingosine l-phosphate-induced cell proliferation, survival and related events mediated by G protein-coupled receptors EDG3 and EDGS. J Biol Chem 275, 288-296.
Ancellin, N., & Hla, T. 1999. Differential pharmacological properties and signal transduction of the sphingosine 1-phosphate receptors EDGI, EDG3, and EDG5. J Biol Chem 274, 18997-19002.
Catherine H. Liu, Shobha Thangada, et al. 1999. Ligand-induced Trafficking of the Sphingosine-1-phosphate Receptor EDG-1. Mol Biol Cell. 10 (4): 1179-1190
Cuvillier, O., Pirianov, G., Kleuser, B., et al. 1996. Suppression of ceramide-mediated programmed cell death by sphingosine 1-phosphate. Nature 381, 800-803.
Cuvillier, 0., Rosenthal, D. S., Smulson, et a1.1998. Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramidemediated apoptosis in Jurkat T lymphocytes. J Biol Chem 273, 2910-2916.
Dagmar Meyer zu Heringdorf , Holger Lass, Igor Kuchar, et al. 2001. Stimulation of intracellular sphingosine-1-phosphate production by G-protein-coupled sphingosine-1-phosphate receptors. European Journal of Pharmacology 414, 145-154
Dagmar Meyer zu Heringdorf a, Myriam E. M. Vincenta, Matthias Lipinski. 2003. Inhibition of Ca~(2+) signalling by the sphingosine 1-phosphate receptor S1P. Cellular Signalling 15, 677-687
Dickson RC, Nagiec EE, Skrzypek M, et al. 1997. Sphingolipids are potential heat stress signals in Saccharomyces. J Biol Chem. 272: 30196.
Edsall LC, Pirianov GG, Spiegel S. 1997. Involvement of sphingosine 1-phosphate in nerve growth factormediated neuronal survival and differentiation. J Neurosci. 17: 6952.
Gottlieb D, Heideman W, Saba JD. 1999. The DPL1 gene is involved in mediating the response to nutrient deprivation in Saccharomyces cerevisiae. Mol Cell Biol Res Commun. 1: 66.
Graler, M. H., Bernhardt, G., & Lipp, M. 1998. A lymphoid tissue-specific receptor, EDG6, with potential immune modulatory functions mediated by extracellular lysolipids. Genomics 53, 164-169.
Herve Le Stunff a, Courtney Peterson b, Hong Liu a, et al. 2002. Sphingosine-1-phosphate and lipid phosphohydrolases. Biochimica et Biophysica Acta. 1582, 8-7
Hla T, Lee, MJ, Ancellin N, et al. 1999. Sphingosine-l-phosphate: extracellular mediator or intracellular second messenger? Biochem Pharmacol. 8: 201.
Hong, G., Baudhuin, L. M., & Xu, Y. 1999. Sphingosine 1-phosphate modulates growth and adhesion of ovarian cancer cells. FEBS Lett. 460, 513-18.
Igarashi JS, Michel T. 2000. Activation of eNOS by sphingosine 1-phosphate mediated by the EDG-1 receptor and the inhibitory role of caveolin in sphingolipid signaling. Circulation. 102 (18): 528 Suppl.
Igarashi, Y., & Yatomi, Y. 1998. Sphingosine 1-phosphate is a blood constituent released from activated platelets, possible playing a variety of physiological and pathophysiological. roles. Acta Biochim Pol. 45, 299-09.
Im, D. -S., Heise, C. E., Ancellin, N., et al. Characterisation of a novel sphingosine 1- phosphate receptor, ED6-8. J Biol Chem. 275, 14281-4286.
Kohama, T., Olivera, A., Edsall, L., et al. 1998. Molecular cloning and functional characterisation of murine sphingosine kinase. J Biol Chem. 273, 23722-23728.
Lee, M. -J., Evans, M., & Ella, T. 1996a. The inducible G protein-coupled receptor edg-1 signals via the Gi/mitogen-activated protein kinase pathway. J Biol Chem 271, 11272-11279.
Lee, M. -J., Thangada, S., Claffey, K. P., et al. 1999. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine 1-phosphate. Cell 99, 301-312.
Lee, M. -J., Thangada, S., Liu, C. H., et al. 1998. Lysophosphatidic acid stimulates the G-protein-coupled receptor EDG-1 as a low affinity agonist. J Biol Chem 273, 22105-22112.
Lee, M. -J., Van Brocklyn, J. R., Thangada, et al. 1996b. Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science 279, 1552-1555.
Lee, O. -H., Kim, Y. -M., Lee, Y. M., et al. 1999. Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signalling mechanism in human umbilical endothelial cells. Biochem Biophys Res Commun 264, 743-750.
Lina M. Obeida, b, c, Yasuo Okamotoc, Cungui Mao. 2OO2. Yeast sphingolipids: metabolism and biology. Biochimica et Biophysica Acta 1585, 163-171
Lynch, K. R., & Im, D. -S. 1999. Life on the edg. Trends Pharmacol Sci 20, 473-475.
Maria Laura Allende, Richard L. Proia. 2002. Sphingosine-1-phosphate receptors and the development of the vascular system. Biochimica et Biophysica Acta 1582, 222-227.
Mathias, S., Pena, L. A., & Kolesnick, R. N. 1998. Signal transduction of stress via ceramide. Biochem J. 335, 465-480.
Michael J. Kluk, Timothy Hla. 2002. Signaling of sphingosine-1-phosphate via the SIP/EDG-family of G-protein-coupled receptors. Biochimica et Biophysica Acta. 1582, 72-80.
Moolenaar, W. H. 1999. Bioactive lysophospholipids and their G proteincoupled receptors. Exp Cell Res. 253, 230-238.
Okamoto, H., Takuwa, N., Gonda, K., et al. 1998. EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a 6(i/o) to multiple signaling pathways, including phospholipase C activation, Ca~(2+) mobilization, ras-mitogen-activated protein kinase activation and adenylate cyclase inhibition. J Biol Chem 273, 27104-27110.
Olivera, A., Kohama, T., Edsall, L., et al. 1999b. Sphingosine kinase expression increases intracellular sphingosine 1-phosphate and promotes cell growth and survival. J Cell Biol 147, 545-557.
Olivera, A., Kohama, T., Tu, Z., et al. 1998. Purification and characterization of rat kidney sphingosine kinase. 3 Biol Chem 273, 12576-12583.
Orlati, S., Porcelli, A. M., Hrelia, S., et al.2000. Sphingosine 1-phosphate activates phospholipase D in human airway epithelial cells via a G-protein-coupled receptor. Arch Biochem Biophys. 375, 69-77.
Perry, D. K., &Hannun, Y. A. 1999. The role of ceramide in cell signaling. Biochim Biophys Acta 1436, 233-243.
Postma, F. R., Jalink, K., Hengeveld, r., et a1.1996. Sphingosine 1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor. EMBO J 15, 2388-2392.
Rakhit, S., Conway, A. -M., rate, R., et al. 1999. Sphingosine 1-phosphate stimulation of the p42/p44 mitogenactivated protein kinase pathway in airway smooth muscle: role of endothelial differentiation gene-1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem J. 338, 643-649.
Rius, R. A., Edsall, L. C., &Spiegel, S. 1997. Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors. FEBS Lett. 417, 173-176.
Spiegel, S. and Milstien, S. 2000. Sphingosine-l-phosphate: signaling inside and out. FEBS Lett. 476, 55-67
Spiegel, S. and Milstien, S. 2000. Functions of a new family of sphingosine-1-phosphate receptors. Biochim. Biophys. Acta 1484, 107-116
Susan PYNE, Nigel J. PYNE. 2000. Sphingosine 1-phosphate signalling in mammalian cells. Biochem. J. 349, 385-402.
Susan Pyne, Nigel J. Pyne. 2002. Sphingosine 1-phosphate signalling and termination at lipid phosphate receptors. Biochimica et Biophysica Acta 1582, 121- 131.
Susan Pyne, Nigel Pyne. 2000. Sphingosine 1-phosphate signalling via the endothelial differentiation gene family of G-protein-coupled receptors. Pharmacology & Therapeutics 88, 115-131.
Van Brocklyn, J. R., Lee, M. -J., Menzeleev, R., et al. 1998. Dual actions of sphingosine 1- phosphate: extracellular through the Gi-coupled receptor EDG1 and intracellular to regulate proliferation and survival. J Cell Biol. 142, 229-240.
Wang, F., Nobes, C. D., Hall, A., & Spiegel, S. 1997. Sphingosine 1-
phosphate stimulates Rho-mediated tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 fibroblasts. Biochem J 324, 481-488.
Windh, R. T., Lee, M. -J., Hla, T., et a1.1999. Differential coupling of the sphingosine 1-phosphate receptors ED61, EDG3 and H218/EDG5 to the Gi, Gq, and G12 families of heterotrimeric g proteins. J Biol Chem 274, 27351-27358.
Wu, J., Spiegel, S., & Sturgill, T. W. 1995. Sphingosine 1-phosphate rapidly activates the mitogen-activated protein kinase pathway by a O-protein-dependent mechanism. J Biol Chem 270, 11484-11488.
Xia, P., Vadas, M. A., Rye, K. -A., et al. 1999a. High-density lipoproteins (HDL) interrupts the sphingosine kinase signalling pathway. A possible mechanism for protection against atherosclerosis by HDL. J Biol Chem. 274, 33143-33147.
Yatomi, Y., Ruan, F. Q., Hakomori, et al. 1995. Sphingosine 1-phosphate: a platelet-activating sphingolipid released from agonist stimulated human platelets. Blood. 86, 193-202.
Zhang, H., Desai, N. N., Olivera, A., et al. 1991. Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol. 114, 155-167.
Zondag, G. C., Postma, F. R., Van Etten, A., et a1.1998. Sphingosine 1-phosphate signaling through the G-proteincoupled receptor Edg-1. Biochem J. aao, 608-609.