黏附家族G蛋白偶联受体GPR126在血管新生及结肠癌发生发展中的功能和机理研究

详细信息    本馆镜像全文|  推荐本文 |  |   获取CNKI官网全文

英文题名：An Adhesion Family G Protein-coupled Receptor GPR126Regualtes Angiogenesis and Colorectal Cancer Progression 作者：崔恒祥 论文级别：博士 学科专业名称：生物医学 学位年度：2011 导师：刘明耀 ; 李大力 学科代码：0831 学位授予单位：华东师范大学 论文提交日期：2011-10-01

摘要

G蛋白偶联受体(G protein-coupled receptor, GPCR)是细胞膜上具有七次跨膜结构的受体蛋白,也是最大的一类膜蛋白分子。人类GPCR共有800多个成员,约占全部基因组编码基因的3-4%。GPCR与配体作用后,激活G蛋白,生成细胞内信使物质(如cAMP, cGMP, N0, DAG, IP等),调节细胞增殖、分化、迁移、死亡,参与几乎所有生理活动的调控。由于数目众多并且功能广泛,GPCR及其下游信号在多种重大疾病中扮演关键的角色,而且位于细胞膜上容易被药物识别和发生作用,因此GPCR是令人瞩目的药物治疗靶点。
     一直以来,GPCR研究与人类健康、医药产业和经济发展直接相关,是生物医学基础、转化与临床研究以及创新药物研发的重大前沿领域。但在众多的GPCR中,很多成员的功能还不确切,并且有一半以上是配体未知的孤儿受体。黏附家族GPCR是数目第二多的GPCR家族,一般认为在发育中他们参与调节细胞与细胞、细胞与细胞外基质之间的相互作用,但是这些受体的功能大多未知。因此深入研究黏附GPCR的功能,具有非常重要的理论和现实意义。
     作为黏附家族GPCR成员,据文献报导,GPR126在蛋白质一级结构和功能上都非常保守。在斑马鱼和小鼠中,Gpr126促进施旺细胞(Schwann cell)细胞的成熟,影响外周神经系统髓鞘的形成。GPR126完全敲除,将致小鼠在胚胎期E10.5-E12.5期间死亡,并且约50%的胚胎内存在出血现象。另外有报道GPR126可能参与调节炎症,能被LPS在内皮细胞内诱导表达。上述结论提示,GPR126可能在血管发育中调节血管生成或血管新生。本研究从GPR126在内皮细胞的表达开始,深入对该受体在血管新生过程的作用进行了研究。同时发现GPR126在人类结肠癌细胞系和组织芯片的肿瘤组织中高表达,进而对其在结肠癌增殖中所起的作用进行了研究。研究主要分以下两个部分：
     一.GPR126在血管新生中的功能研究
     1.GPR126在血管内皮表达丰富
     利用拟胚体形成模型证实了GPR126在VEGFR2阳性细胞群中的表达显著高于阴性细胞；免疫组织化学法证明GPR126在肢芽、心脏、肾和肺组织的血管中特异表达；蛋白印迹杂交实验证明,HMEC-1和HUVEC细胞系中GPR126表达丰富；在HMEC-1细胞内,GPR126能被VEGF、IGF-1和FGF-2能促新生血管生长因子诱导；RT-PCR方法证明,在分选的斑马鱼内皮细胞中,Gpr126表达丰富；
     2.干扰GPR126在内皮细胞的表达,削弱了内皮细胞的血管新生活性。
     HMEC-1细胞内沉默表达,削弱了内皮细胞在三维基质胶中的出芽形成过程、抑制了内皮细胞的增殖、迁移和在基质胶上的成管能力；
     3.抑制GPR126表达抑制体内生理和病理血管新生。
     Matrigel Plug实验和视网膜造影实验证明,GPR126调节VEGF诱导的基质胶内血管新生和低氧诱导的视网膜血管新生。
     4.在斑马鱼胚胎中沉默Gpr126的表达导致血管新生缺陷。
     利用模式生物斑马鱼为研究材料,靶向Gpr126的反义吗啉代引起胚胎的体节间血管(ISVs)新生过程受到了严重的抑制；人源GPR126mRNA和GPR126的反义吗啉代寡核苷酸显微注射入斑马鱼胚胎,ISVs的血管新生过程部分到恢复；进一步研究证实,靶向Gpr126的反义吗啉代寡核苷酸主要影响节段动脉(segmental arteries)内皮细胞的数目,调节内皮细胞从背主动脉出芽以及顶端细胞(tip cell)的迁移和增殖。
     5.GPR126通过GATA2和STAT5调节VEGFR2的表达。
     在HMEC-1细胞中干扰GPR126的表达,抑制了VEGF诱导的ERK和FAK活性。通过对该信号通路的进一步研究发现,GPR126调节了VEGFR2的表达；通过RT-PCR法对已报导的调节血管新生、特别是调节VEGFR2表达的转录因子进行筛选,干扰GPR126在HMEC-1细胞的表达下调节了GATA2和STAT5的mRNA水平；蛋白质印迹也证明GPR126在内皮细胞的沉默表达也促使GATA2和STAT5蛋白水平的下调。GATA2和STAT5都能直接结合到VEGFR2的转录调节区,调节VEGFR2的表达；PKA激活剂forskolin增加了内皮细胞HMEC-1内CREB活性,并且以剂量依赖的方式诱导增加了STAT5和GATA2的表达；进一步发现STAT5和GATA2的启动调节区存在CREB的直接结合。总结起来,GPR126通过PKA-CREB调节STAT5和GATA2的表达,进而调节了VEGFR2的表达。
     6. STAT5和GATA2可回复因GPR126干扰而致的斑马鱼血管新生缺陷
     GPR126反义吗啉代联合注射STAT5mRNA或者和GATA2mRNA,部分恢复只注射GPR126反义吗啉代导致的ISVs血管新生缺陷；STAT5mRNA和GATA2mRNA同时与Gpr126反义吗啉代联合注射,将很大程度上恢复只注射GPR126反义吗啉代导致的ISVs血管新生缺陷；在HMEC-1细胞中,高表达STAT5或/和GATA2,都能部分恢复由于GPR126表达下调而受到削弱的内皮细胞成管能力,也恢复了由于GPR126表达下调而受到削弱的VEGF诱导的ERK和FAK的激活。
     二.GPR126在结肠癌增殖过程中的作用研究
     1.GPR126在结肠癌细胞中的表达
     RT-PCR和蛋白免疫印迹证明,GPR126在各结肠癌细胞中有比较高的表达。利用免疫组织化学法检测了结肠癌组织芯片中GPR126的表达,结果表明GPR126在恶性结肠癌组织中的表达显著高于同一病人的癌旁和正常组织的表达。
     2.体外实验证明GPR126调节结肠癌细胞的增殖
     GPR126在结肠癌细胞中的干扰表达,抑制了多种结肠癌细胞的增殖活性。
     3.GPR126在体内调节结肠癌细胞的生长
     荷瘤实验证明,干扰GPR126在HT-29, HCT116和Lovo细胞中的表达,抑制了这些细胞在裸鼠中形成的肿瘤的生长。
     4.GPR126可能通过调节mTOR途径调节结肠癌细胞生长
     利用高通量RNA测序方法在转录组水平研究GPR126调节结肠癌细胞系HT-29增殖的分子机理。
G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors (7TM receptors) comprise the largest protein family of transmembrane proteins. There are nearly800different human GPCR genes, about4%of the entire protein-coding genome, being predicted from genome sequence analysis. When ligands binding and activating these receptors, they transducer intracellular signal molecules, such as cAMP, cGMP, NO, DAG and IP, which modulating cell proliferation and differentiation, cell migration and death, thus participating in nearly all physiological processes. Due to broad spectrum of physiological functions and structural advantages, GPCRs are the most successful targets for moden medicine.
     Functional investigation of GPCR, a frontier field for medicinal chemistry, clinical research and drug discovery, has been shown to be important for pharmaceutical industry and human health. However, many members of the GPCRs have not been well characterized, and even more than half of them are still orphans, meaning their endogenous ligands are still unknown. As the second largest family, adhesion GPCRs are commonly considered to be involved in interaction of cell to cell, or cell to ECM, but the precise physiological roles of most of the adhesion GPCRs are to be identified. GPR126, as a member of adhesion GPCRs, is identified to be required for Schwann cell maturation in zebrafish and mouse during peripheral nerve development. GPR126knock-out mice were observed a sharp loss of viability occurs from10.5dpf to12.5dpf, accompanying with intra-embryonic hemorrhage in about50%(24/49) of embryos. And GPR126was initially isolated from HUVEC cultures that had been challenged with lipopolysaccharides or thrombin. All these indicate that GPR126might be implicated in endothelial cell function and in vascular development including processes of vasculogenesis and angiogenesis. In this thesis, we started the investigation of GPR126from its expression in endothelial cells, and subsequently revealed the role of GPR126in vascular angiogenesis and colon cancer progression.
     I. Role of GPR126in angiogenesis
     1. Gpr126is highly enriched in endothelial cells and induced by pro-angiogenic factors in HMEC-1cells
     Using model of embryoid bodies (EB) formation and subsequent differentiation of embryonic stem cells, we identified Gpr126mRNA expression was highly enriched in Flkl-positive cells at day4of EB formation. And immunohistochemistry staining revealed that GPR126positive cells were specifically detected in the endothelial cells of vessels of embryonic limb bud, lung, heart and kidney. Western blot showed that GPR126expresses in HUVEC and HMEC-1cells. Meanwhile, VEGF, IGF-1and FGF-2increased GPR126expression in HMEC-1cells. And also RT-PCR assay revealed GPR126mRNA is highly enriched in Flk+endothelial cells sorted from zebrafish.
     2. Knockdown of GPR126impaired endothelial sprout formation in three-dimensional collagen, and also inhibited endothelial activity of proliferation, migration and tube formation.
     3. Inhibition of GPR126expression impairs physiological and pathological angiogenesis. Matrigel Plug assay and oxygen-induced retinopathy assay demonstrated that GPR126regulated VEGF induced angiogenesis in matrigel and ischemia-induced retinal neovascularization.
     4. Silencing of GPR126causes angiogensis defects during embryogenesis. Knockdown of Gpr126in zebrafish resulted to defects of intersegmental vessel formation, while coinjecting antisense morpholinos targeting Gpr126(Gpr126MO) with human GPR126mRNA in the embryos restored intersegmental vessel formation. Further investigation showed that antisense morpholinos targeting Gpr126dramatically reduced cell numbers of segmental arteries. The phenotypes of zebrafish Gpr126knockdown showed that the formation of dorsal aorta and the subsequent ECs sprouting from dorsal aorta to the horizontal myoseptum were obviously affected, and the subsequent endothelial tip cell migration and proliferation were severely inhibited.
     5. GPR126regulated VEGFR2expression via STAT5and GATA2.In GPR126deficient HMEC-1cells, VEGF induced ERK and FAK activities were downregulated. Further GPR126was found to regulate VEGFR2transcription.
     By RT-PCR and western blot methods, we identified both mRNA and protein of STAT5and GATA2were down-regulated. While both GATA2and STAT5were demonstrated to directly bind to the promoter of VEGFR2and regulate expression of VEGFR2in HMEC-1cells. Forskolin, a PKA agonist, increased CREB activity and upregulated GATA2and STAT5expression in dose dependent way in HMEC-1cells. Furthermore, chip assays revealed that there contain direct binding in promoters of GATA2and STAT5respectively.
     Together, GPR126regulates GATA2and STAT5expression through PKA-CREB pathway, through which further modulates VEGFR2expression in HMEC-1cells.
     6. STAT5and GATA2restored the angiogenic activity of endothelial cells attenuated by silencing of GPR126in vivo and in vitro. Coinjection of GATA2mRNA or STAT5mRNA with Gpr126MO partially restored ISVs growth which was disrupted by Gpr126MO alone. When STAT5and GATA2mRNA together were introduced with Gpr126MO at the same time, the impaired ISV morphology was greatly rescued than individual injection of STAT5mRNA or GATA2mRNA. And forced expression of STAT5and/or GATA2in GPR126deficit HMEC-1cells restored the accumulated length of tubes significantly compared to that of GPR126knockdown group in tube formation assay, and also regained VEGF induced activation of ERK and FAK which were attenuated by GPR126knockdown.
     II. Role of GPR126in Colorectal Cancer Proliferation
     1. GPR126overexpressed in colorectal cancer cell
     RT-PCR and Western blot experiment showed that GPR126mRNA and protein level was high. Taking advantage of immunohistochemistry staining, we confirmed that GPR126expression in the malignant tissues from colon cancer is significantly higher than that in tissues beside the malignant ones from colon cancer.
     2. Knockdown of GPR126in vitro inhibited proliferation of colorectal cancer cells.
     3. Knockdown of GPR126inhibited tumor growth in vivo. Using xenograft nude mice, we demonstrated that silence of GPR126in HT-29, HCT116and Lovo cells inhibited tumor growth.
     4. GPR126might regulate proliferation of colorectal cancer cells via mTOR pathway. RNA sequencing technology was used to investigate the mechanism of GPR126modulating proliferation of colorectal cancer cells. The result showed that knockdown of GRR126in HT-29cells decreased mTOR protein.

引文

1. King, N., C.T. Hittinger, and S.B. Carroll, Evolution of key cell signaling and adhesion protein families predates animal origins. Science,2003.301(5631):p.361-3.
    2. Zhang, Y., M.E. Devries, and J. Skolnick, Structure modeling of all identified G protein-coupled receptors in the human genome. PLoS Comput Biol,2006.2(2):p. e13.
    3. Bjarnadottir, T.K., et al.. Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse. Genomics,2006.88(3):p.263-73.
    4. Foord, S.M., et al., International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol Rev,2005.57(2):p.279-88.
    5. Fredriksson, R., et al., The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol,2003. 63(6):p.1256-72.
    6. Yona, S., et al., Adhesion-GPCRs:emerging roles for novel receptors. Trends Biochem Sci,2008. 33(10):p.491-500.
    7. Gloriam, D.E., R. Fredriksson, and H.B. Schioth, The G protein-coupled receptor subset of the rat genome. BMC Genomics,2007.8:p.338.
    8. Lagerstrom, M.C., et al., The G protein-coupled receptor subset of the chicken genome. PLoS Comput Biol,2006.2(6):p. e54.
    9. Palczewski, K., et al., Crystal structure of rhodopsin:A G protein-coupled receptor. Science, 2000.289(5480):p.739-45.
    10. Rasmussen, S.G., et al., Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature,2007.450(7168):p.383-7.
    11. Cherezov, V., et al., High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science,2007.318(5854):p.1258-65.
    12. Vu, T.K., et al., Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell,1991.64(6):p.1057-68.
    13. Xu, W.F., et al., Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci U S A,1998.95(12):p.6642-6.
    14. Nystedt, S., et al., Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor2. Eur J Biochem,1995.232(1):p.84-9.
    15. shihara, H., et al., Protease-activated receptor 3 is a second thrombin receptor in humans. Nature,1997.386(6624):p.502-6.
    16. Oikonomopoulou, K., et al., Proteinase-mediated cell signalling:targeting proteinase-activated receptors (PARs) by kallikreins and more. Biol Chem,2006.387(6):p. 677-85.
    17. Fan, Q.R. and W.A. Hendrickson, Structure of human follicle-stimulating hormone in complex with its receptor. Nature,2005.433(7023):p.269-77.
    18. Angelova, K., et al., Functional differences of invariant and highly conserved residues in the extracellular domain of the glycoprotein hormone receptors. J Biol Chem,2010.285(45):p. 34813-27.
    19. Bockaert, J. and J.P. Pin, Molecular tinkering of G protein-coupled receptors:an evolutionary success. EMBO J,1999.18(7):p.1723-9.
    20. Lagerstrom, M.C. and H.B. Schioth, Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov,2008.7(4):p.339-57.
    21. Carmon, K.S., et al., R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci USA,2011.108(28):p.11452-7.
    22. Tyndall, J.D. and R. Sandilya, GPCR agonists and antagonists in the clinic. Med Chem,2005. 1(4):p.405-21.
    23. Jacoby, E., et al.. The 7 TM G-protein-coupled receptor target family. ChemMedChem,2006. 1(8):p.761-82.
    24. Harmar, A.J., Family-B G-protein-coupled receptors. Genome Biol,2001.2(12):p. REVIEWS3013.
    25. Stacey, M., et al., LNB-TM7, a group of seven-transmembrane proteins related to family-B G-protein-coupled receptors. Trends Biochem Sci,2000.25(6):p.284-9.
    26. Fredriksson, R. and H.B. Schioth, The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol Pharmacol,2005.67(5):p.1414-25.
    27. Bjarnadottir, T.K., et al., The human and mouse repertoire of the adhesion family of G-protein-coupled receptors. Genomics,2004.84(1):p.23-33.
    28. Baud, V., et al., EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments. Genomics,1995.26(2):p.334-44.
    29. Krasnoperov, V.G., et al., alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron,1997.18(6):p.925-37.
    30. Krasnoperov, V., et al., Post-translational proteolytic processing of the calcium-independent receptor of alpha-latrotoxin (CIRL), a natural chimera of the cell adhesion protein and the G protein-coupled receptor. Role of the G protein-coupled receptor proteolysis site (GPS) motif. J Biol Chem,2002.277(48):p.46518-26.
    31. Lin, H.H., et al., Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif. J Biol Chem,2004.279(30):p.31823-32.
    32. Nechiporuk, T., L.D. Urness, and M.T. Keating, ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily. J Biol Chem,2001. 276(6):p.4150-7.
    33. Lin, H.H., et al., Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions:dissection of the CD97-CD55 complex. J Biol Chem,2001.276(26):p.24160-9.
    34. Little, K.D., M.E. Hemler, and C.S. Stipp, Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells:central role of CD81 in facilitating GPR56-Galpha q/11 association. Mol Biol Cell,2004.15(5):p.2375-87.
    35. Stacey, M., et al., The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans. Blood,2003.102(8): p.2916-24.
    36. Stacey, M., et al., Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils.1 Biol Chem,2001.276(22):p.18863-70.
    37. Stacey, M., et al., EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line A20. J Biol Chem,2002.277(32):p.29283-93.
    38. Hamann, J., et al.. The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF). J Exp Med,1996.184(3):p.1185-9.
    39. Hamann, J., et al., Characterization of the CD55 (DAF)-binding site on the seven-span transmembrane receptor CD97. EurJ Immunol,1998.28(5):p.1701-7.
    40. Xu, L., et al., GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumor growth and metastasis. Proc Natl Acad Sci U S A,2006. 103(24):p.9023-8.
    41. Liu, M., et al., GPR56, a novel secretin-like human G-protein-coupled receptor gene. Genomics, 1999.55(3):p.296-305.
    42. Lelianova, V.G., et al., Alpha-latrotoxin receptor, latrophilin, is a novel member of the secretin family of G protein-coupled receptors. J Biol Chem,1997.272(34):p.21504-8.
    43. Sugita, S., et al., alpha-Latrotoxin receptor CIRL/latrophilin 1 (CL1) defines an unusual family of ubiquitous G-protein-linked receptors. G-protein coupling not required for triggering exocytosis. J Biol Chem,1998.273(49):p.32715-24.
    44. Nishimori, H., et al., A novel brain-specific p53-target gene, BAI1, containing thrombospondin type 1 repeats inhibits experimental angiogenesis. Oncogene,1997.15(18):p.2145-50.
    45. Osterhoff, C., R. Ivell, and C. Kirchhoff, Cloning of a human epididymis-specific mRNA, HE6, encoding a novel member of the seven transmembrane-domain receptor superfamily. DNA Cell Biol,1997.16(4):p.379-89.
    46. Shiratsuchi, T., et al., Cloning and characterization of BAI2 and BAI3, novel genes homologous to brain-specific angiogenesis inhibitor 1 (BAI1). Cytogenet Cell Genet,1997.79(1-2):p. 103-8.
    47. Nikkila, H., et al., Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+exchangers define a cation binding domain. Mol Endocrinol,2000.14(9):p. 1351-64.
    48. Fredriksson, R., et al., Novel human G protein-coupled receptors with long N-terminals containing GPS domains and Ser/Thr-rich regions. FEBS Lett,2002.531(3):p.407-14.
    49. Fredriksson, R., et al., There exist at least 30 human G-protein-coupled receptors with long Ser/Thr-rich N-termini. Biochem Biophys Res Commun,2003.301(3):p.725-34.
    50. LopezJimenez, N.D., et al., Two novel genes, Gpr113, which encodes a family 2 G-protein-coupled receptor, and Trcgl, are selectively expressed in taste receptor cells. Genomics,2005.85(4):p.472-82.
    51. Abe, J., et al., Ig-hepta, a novel member of the G protein-coupled hepta-helical receptor (GPCR) family that has immunoglobulin-like repeats in a long N-terminal extracellular domain and defines a new subfamily of GPCRs. J Biol Chem,1999.274(28):p.19957-64.
    52. Abe, J., T. Fukuzawa, and S. Hirose, Cleavage of Ig-Hepta at a "SEA" module and at a conserved G protein-coupled receptor proteolytic site. J Biol Chem,2002.277(26):p.23391-8.
    53. Wreschner, D.H., et al., Generation of ligand-receptor alliances by "SEA" module-mediated cleavage of membrane-associated mucin proteins. Protein Sci,2002.11(3):p.698-706.
    54. McMillan, D.R., et al., Very large G protein-coupled receptor-1, the largest known cell surface protein, is highly expressed in the developing central nervous system. J Biol Chem,2002. 277(1):p.785-92.
    55. McMillan, D.R. and P.C. White, Loss of the transmembrane and cytoplasmic domains of the very large G-protein-coupled receptor-1 (VLGR1 or Massl) causes audiogenic seizures in mice. Mol Cell Neurosci,2004.26(2):p.322-9.
    56. Hofmann, B.A., et al., Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1. Protein Sci,2001.10(10):p. 2050-62.
    57. Grauschopf, U., et al., The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern. Biochemistry, 2000.39(30):p.8878-87.
    58. Bazarsuren, A., et al., In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor. Biophys Chem,2002.96(2-3):p.305-18.
    59. Grace, C.R., et al., NMR structure and peptide hormone binding site of the first extracellular domain of a type Bl G protein-coupled receptor. Proc Natl Acad Sci U S A,2004.101(35):p. 12836-41.
    60. Godfrey, P., et a I., GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat Genet,1993.4(3):p.227-32.
    61. DeAlmeida, V.I. and K.E. Mayo, Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins. Mol Endocrinol,1998.12(5):p.750-65.
    62. Assil, I.Q., et al., Juxtamembrane region of the amino terminus of the corticotropin releasing factor receptor type 1 is important for ligand interaction. Biochemistry,2001.40(5):p. 1187-95.
    63. Vilardaga, J.P., I. Lin, and R.A. Nissenson, Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/pth-related peptide (PTHrP) receptor on hormone binding and receptor activation. Mol Endocrinol,2001.15(7):p. 1186-99.
    64. Unson, C.G., et al., Roles of specific extracellular domains of the glucagon receptor in ligand binding and signaling. Biochemistry,2002.41(39):p.11795-803.
    65. Pham, V., et al., Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor. J Biol Chem,2004.279(8): p.6720-9.
    66. Dong, M., et al., Spatial approximation between two residues in the mid-region of secretin and the amino terminus of its receptor. Incorporation of seven sets of such constraints into a three-dimensional model of the agonist-bound secretin receptor. J Biol Chem,2003.278(48): p.48300-12.
    67. Dong, M., et al., Importance of the amino terminus in secretin family G protein-coupled receptors. Intrinsic photoaffinity labeling establishes initial docking constraints for the calcitonin receptor. J Biol Chem,2004.279(2):p.1167-75.
    68. Dong, M., et al., Spatial approximation between the amino terminus of a peptide agonist and the top of the sixth transmembrane segment of the secretin receptor. J Biol Chem,2004. 279(4):p.2894-903.
    69. Nauck, M.A. and J.J. Meier, Glucagon-like peptide 1 and its derivatives in the treatment of diabetes. Regul Pept,2005.128(2):p.135-48.
    70. Kunishima, N., et al.. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature,2000.407(6807):p.971-7.
    71. O'Hara, P.J., et al., The ligand-binding domain in metabotropic glutamate receptors is related to bacterialperiplasmic binding proteins. Neuron,1993.11(1):p.41-52.
    72. Muto, T., et al., Structures of the extracellular regions of the group II/III metabotropic glutamate receptors. Proc Natl Acad Sci U S A,2007.104(10):p.3759-64.
    73. D'Souza-Li, L., The calcium-sensing receptor and related diseases. Arq Bras Endocrinol Metabol,2006.50(4):p.628-39.
    74. Brauner-Osborne, H., et al.. The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain. J Biol Chem,1999.274(26):p.18382-6.
    75. Silve, C, et al., Delineating a Ca2+binding pocket within the venus flytrap module of the human calcium-sensing receptor. J Biol Chem,2005.280(45):p.37917-23.
    76. Conigrave, A.D., S.J. Quinn, and E.M. Brown, L-amino acid sensing by the extracellular Ca2+-sensing receptor. Proc Natl Acad Sci U S A,2000.97(9):p.4814-9.
    77. Hoon, M.A., et al., Putative mammalian taste receptors:a class of taste-specific GPCRs with distinct topographic selectivity. Cell,1999.96(4):p.541-51.
    78. Li, X., et al., Human receptors for sweet and umami taste. Proc Natl Acad Sci U S A,2002. 99(7):p.4692-6.
    79. Nelson, G., et al., An amino-acid taste receptor. Nature,2002.416(6877):p.199-202.
    80. Nelson, G., et al., Mammalian sweet taste receptors. Cell,2001.106(3):p.381-90.
    81. Sainz, E., et al., Identification of a novel member of the TIR family of putative taste receptors. J Neurochem,2001.77(3):p.896-903.
    82. Xu, H., et al., Different functional roles of TIR subunits in the heteromeric taste receptors. Proc Natl Acad Sci U S A,2004.101(39):p.14258-63.
    83. Rondard, P., et al., Coupling of agonist binding to effector domain activation in metabotropic glutamate-like receptors. J Biol Chem,2006.281(34):p.24653-61.
    84. Liu, X., et al., NCD3G:a novel nine-cysteine domain in family 3 GPCRs. Trends Biochem Sci, 2004.29(9):p.458-61.
    85. Hu, J., O. Hauache, and A.M. Spiegel, Human Ca2+receptor cysteine-rich domain. Analysis of function of mutant and chimeric receptors. J Biol Chem,2000.275(21):p.16382-9.
    86. Jiang, P., et al., The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J Biol Chem,2004.279(43):p.45068-75.
    87. Galvez, T., et al., Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding. J Biol Chem,1999.274(19):p. 13362-9.
    88. Kaupmann, K., et al., Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors. Nature,1997.386(6622):p.239-46.
    89. Martin, S.C., S.J. Russek, and D.H. Farb, Human GABA(B)R genomic structure:evidence for splice variants in GABA(B)R1 but not GABA(B)R2. Gene,2001.278(1-2):p.63-79.
    90. Shoback, D.M., et al., The calcimimetic cinacalcet normalizes serum calcium in subjects with primary hyperparathyroidism. J Clin Endocrinol Metab,2003.88(12):p.5644-9.
    91. Calver, A.R., et al., Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor. Brain Res Mol Brain Res,2003.110(2):p.305-17.
    92. Brauner-Osborne, H., et al., Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D. Biochim Biophys Acta,2001.1518(3):p.237-48.
    93. Brauner-Osborne, H. and P. Krogsgaard-Larsen, Sequence and expression pattern of a novel human orphan G-protein-coupled receptor, GPRC5B, a family C receptor with a short amino-terminal domain. Genomics,2000.65(2):p.121-8.
    94. Robbins, M.J., et al., Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C). Genomics,2000. 67(1):p.8-18.
    95. Cheng, Y. and R. Lotan, Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor. J Biol Chem,1998. 273(52):p.35008-15.
    96. Bjarnadottir, T.K., R. Fredriksson, and H.B. Schioth, The gene repertoire and the common evolutionary history of glutamate, pheromone (V2R), taste(1) and other related G protein-coupled receptors. Gene,2005.362:p.70-84.
    97. Ota, T., et al., Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat Genet,2004.36(1):p.40-5.
    98. Cismowski, M.J., Non-receptor activators of heterotrimeric G-protein signaling (AGS proteins). Semin Cell Dev Biol,2006.17(3):p.334-44.
    99. Vinson, C.R., S. Conover, and P.N. Adler, A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains. Nature,1989.338(6212):p.263-4.
    100. Wang, Y., et al., A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J Biol Chem,1996.271(8):p.4468-76.
    101. Wang, Y.K., et al., A novel human homologue of the Drosophila frizzled wnt receptor gene binds wingless protein and is in the Williams syndrome deletion at 7q11.23. Hum Mol Genet, 1997.6(3):p.465-72.
    102. Zhao, Z., et al., A human homologue of the Drosophila polarity gene frizzled has been identified and mapped to 17q21.1. Genomics,1995.27(2):p.370-3.
    103. Tokuhara, M., et al., Molecular cloning of human Frizzled-6. Biochem Biophys Res Commun, 1998.243(2):p.622-7.
    104. Sala, C.F., et al., Identification, gene structure, and expression of human frizzled-3 (FZD3). Biochem Biophys Res Commun,2000.273(1):p.27-34.
    105. Sagara, N., et al., Molecular cloning, differential expression, and chromosomal localization of human frizzled-1, frizzled-2, and frizzled-7. Biochem Biophys Res Commun,1998.252(1):p. 117-22.
    106. Saitoh, T., M. Hirai, and M. Katoh, Molecular cloning and characterization of human Frizzled-8 gene on chromosome 10p11.2. Int J Oncol,2001.18(5):p.991-6.
    107. Kirikoshi, H., et al., Molecular cloning and characterization of human Frizzled-4 on chromosome 11q14-q21. Biochem Biophys Res Commun,1999.264(3):p.955-61.
    108. Koike, J., et al., Molecular cloning of Frizzled-10, a novel member of the Frizzled gene family. Biochem Biophys Res Commun,1999.262(1):p.39-43.
    109. Bhanot, P., et al., A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature,1996.382(6588):p.225-30.
    110. Riobo, N.A., et al.. Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A,2006.103(33):p.12607-12.
    111. Barnes, M.R., D.M. Duckworth, and L.J. Beeley, Frizzled proteins constitute a novel family ofG protein-coupled receptors, most closely related to the secretin family. Trends Pharmacol Sci, 1998.19(10):p.399-400.
    112. Carron, C., et al., Frizzled receptor dimerization is sufficient to activate the Wnt/beta-catenin pathway. J Cell Sci,2003.116(Pt 12):p.2541-50.
    113. Murone, M., A. Rosenthal, and F.J. de Sauvage, Sonic hedgehog signaling by the patched-smoothened receptor complex. Curr Biol,1999.9(2):p.76-84.
    114. Chen, C.M., et al., Evidence that the cysteine-rich domain of Drosophila Frizzled family receptors is dispensable for transducing Wingless. Proc Natl Acad Sci U S A,2004.101(45):p. 15961-6.
    115. Xu, Q., et al., Vascular development in the retina and inner ear:control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell,2004.116(6):p.883-95.
    116. Robitaille, J., et al.. Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy. Nat Genet,2002.32(2):p.326-30.
    117. Stone, D.M., et al., The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature,1996.384(6605):p.129-34.
    118. Nakano, Y., et al., Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila. Mech Dev,2004.121(6):p.507-18.
    119. Chen, Y. and G. Struhl, In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex. Development,1998. 125(24):p.4943-8.
    120. Taipale, J., et al.. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature,2000.406(6799):p.1005-9.
    121. Nagayama, S., et al., Therapeutic potential of antibodies against FID 10, a cell-surface protein, for synovial sarcomas. Oncogene,2005.24(41):p.6201-12.
    122. Adler, E., et al., A novel family of mammalian taste receptors. Cell,2000.100(6):p.693-702.
    123. Matsunami, H., J.P. Montmayeur, and L.B. Buck, A family of candidate taste receptors in human and mouse. Nature,2000.404(6778):p.601-4.
    124. Shi, P., et al., Adaptive diversification of bitter taste receptor genes in Mammalian evolution. Mol Biol Evol,2003.20(5):p.805-14.
    125. Go, Y., et al., Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates. Genetics,2005.170(1):p.313-26.
    126. Conte, C., et al., Identification and characterization of human taste receptor genes belonging to the TAS2R family. Cytogenet Genome Res,2002.98(1):p.45-53.
    127. Bufe, B., et al., The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides. Nat Genet,2002.32(3):p.397-401.
    128. Chandrashekar, J., et al., T2Rs function as bitter taste receptors. Cell,2000.100(6):p.703-11.
    129. Miller, T.R. and J.W. Wallis, Clinically important characteristics of maximum-likelihood reconstruction. J Nucl Med,1992.33(9):p.1678-84.
    130. Kuhn, C., et al., Bitter taste receptors for saccharin and acesulfame K. J Neurosci,2004.24(45): p.10260-5.
    131. Pronin, A.N., et al., Identification of ligands for two human bitter T2R receptors. Chem Senses, 2004.29(7):p.583-93.
    132. Wettschureck, N. and S. Offermanns, Mammalian G proteins and their cell type specific functions. Physiol Rev,2005.85(4):p.1159-204.
    133. Brady, A.E. and L.E. Limbird,G protein-coupled receptor interacting proteins:emerging roles in localization and signal transduction. Cell Signal,2002.14(4):p.297-309.
    134. Ambrosio, M., A. Zurn, and M.J. Lohse, Sensing G protein-coupled receptor activation. Neuropharmacology,2011.60(1):p.45-51.
    135. Skalhegg, B.S. and K. Tasken, Specificity in the cAMP/PKA signaling pathway. Differential expression,regulation, and subcellular localization of subunits of PKA. Front Biosci,2000.5:p. D678-93.
    136. Kume, S., T. Inoue, and K. Mikoshiba, Galphas family G proteins activate IP(3)-Ca(2+) signaling via gbetagamma and transduce ventralizing signals in Xenopus. Dev Biol,2000.226(1):p. 88-103.
    137. Kozasa, T., et al., p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science,1998.280(5372):p.2109-11.
    138. Gilman, A.G., G proteins:transducers of receptor-generated signals. Annu Rev Biochem,1987. 56:p.615-49.
    139. Shevtsov, S.P., S. Haq, and T. Force, Activation of beta-catenin signaling pathways by classical G-protein-coupled receptors:mechanisms and consequences in cycling and non-cycling cells. Cell Cycle,2006.5(20):p.2295-300.
    140. Force, T., et al., Molecular scaffolds regulate bidirectional crosstalk between Wnt and classical seven-transmembrane-domain receptor signaling pathways. Sci STKE,2007.2007(397):p. pe41.
    141. Kim, J.Y., P.V. Haastert, and P.N. Devreotes, Social senses:G-protein-coupled receptor signaling pathways in Dictyostelium discoideum. Chem Biol,1996.3(4):p.239-43.
    142. Sun, Y, D. McGarrigle, and X.Y. Huang, When a G protein-coupled receptor does not couple to a G protein. Mol Biosyst,2007.3(12):p.849-54.
    143. Duchene, J., et al., A novel protein-protein interaction between a G protein-coupled receptor and the phosphatase SHP-2 is involved in bradykinin-induced inhibition of cell proliferation. J Biol Chem,2002.277(43):p.40375-83.
    144. Krueger, K.M., et al., The role of sequestration in G protein-coupled receptor resensitization. Regulation of beta2-adrenergic receptor dephosphorylation by vesicular acidification. J Biol Chem,1997.272(1):p.5-8.
    145. Tan, C.M., et al., Membrane trafficking of G protein-coupled receptors. Annu Rev Pharmacol Toxicol,2004.44:p.559-609.
    146. Laporte, S.A., et al., The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits. J Biol Chem,2000.275(30): p.23120-6.
    147. Laporte, S.A., et al.. The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A,1999.96(7):p.3712-7.
    148. Barthet, G., et al., Beta-arrestin 1 phosphorylation by GRK5 regulates G protein-independent 5-HT4 receptor signalling. EMBOJ,2009.28(18):p.2706-18.
    149. Marrero, M.B., et al., Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature,1995.375(6528):p.247-50.
    150. Lukashova, V., et al., G-protein-independent activation of Tyk2 by the platelet-activating factor receptor. J Biol Chem,2001.276(26):p.24113-21.
    151. Mellado, M., et al., The chemokine monocyte chemotactic protein 1 triggers Janus kinase 2 activation and tyrosine phosphorylation of the CCR2B receptor. J Immunol,1998.161(2):p. 805-13.
    152. Wong, M. and E.N. Fish, RANTES and MlP-lalpha activate stats in T cells. J Biol Chem,1998. 273(1):p.309-14.
    153. Vila-Coro, A.J., et al., The chemokine SDF-lalpha triggers CXCR4 receptor dimerization and activates the JAK/STATpathway. FASEBJ,1999.13(13):p.1699-710.
    154. Frank, G.D., et al., Requirement of Ca(2+) and PKCdelta for Janus kinase 2 activation by angiotensin II:involvement of PYK2. Mol Endocrinol,2002.16(2):p.367-77.
    155. Thomas, S.M. and J.S. Brugge, Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol,1997.13:p.513-609.
    156. Ma, Y.C. and X.Y. Huang, Novel regulation and function of Src tyrosine kinase. Cell Mol Life Sci, 2002.59(3):p.456-62.
    157. McGarrigle, D. and X.Y. Huang, GPCRs signaling directly through Src-family kinases. Sci STKE, 2007.2007(392):p. pe35.
    158. Yin, G., C. Yan, and B.C. Berk, Angiotensin II signaling pathways mediated by tyrosine kinases. Int J Biochem Cell Biol,2003.35(6):p.780-3.
    159. Ma, Y.C. and X.Y. Huang, Novel signaling pathway through the beta-adrenergic receptor. Trends Cardiovasc Med,2002.12(1):p.46-9.
    160. Schmitt, J.M. and P.J. Stork, PKA phosphorylation of Src mediates cAMP's inhibition of cell growth via Rapl. Mol Cell,2002.9(1):p.85-94.
    161. Ma, Y.C., et al., Src tyrosine kinase is a novel direct effector of G proteins. Cell,2000.102(5):p. 635-46.
    162. Luttrell, L.M., et al., Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science,1999.283(5402):p.655-61.
    163. Sun, Y, et al., Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR. EMBO J,2007.26(1):p.53-64.
    164. Fan, G., et al., c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization. J Biol Chem,2001.276(16):p.13240-7.
    165. Cao, W., et al.. Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation.1 Biol Chem,2000.275(49):p.38131-4.
    166. Hall, R.A., et al., A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc Natl Acad Sci U S A,1998.95(15):p. 8496-501.
    167. Gorr, T.A., M. Gassmann, and P. Wappner, Sensing and responding to hypoxia via HIF in model invertebrates. J Insect Physiol,2006.52(4):p.349-64.
    168. Semenza, G.L., Vasculogenesis, angiogenesis, and arteriogenesis:mechanisms of blood vessel formation and remodeling. J Cell Biochem,2007.102(4):p.840-7.
    169. Risau, W. and I. Flamme, Vasculogenesis. Annu Rev Cell Dev Biol,1995.11:p.73-91.
    170. Pardanaud, L., et al., Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development,1996.122(5):p.1363-71.
    171. De Val, S. and B.L. Black, Transcriptional control of endothelial cell development. Dev Cell, 2009.16(2):p.180-95.
    172. Olsson, A.K., et al., VEGF receptor signalling-in control of vascular function. Nat Rev Mol Cell Biol,2006.7(5):p.359-71.
    173. Bussmann, J., et al., Zebrafish VEGF receptors:a guideline to nomenclature. PLoS Genet,2008. 4(5):p. e1000064.
    174. Muller, Y.A., et al., Vascular endothelial growth factor:crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci U S A,1997.94(14):p. 7192-7.
    175. De Falco, S., B. Gigante, and M.G. Persico, Structure and function of placental growth factor. Trends Cardiovasc Med,2002.12(6):p.241-6.
    176. Woolard, J., et al., VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression. Cancer Res,2004.64(21):p.7822-35.
    177. Christinger, H.W., et al., The crystal structure of placental growth factor in complex with domain 2 of vascular endothelial growth factor receptor-1. J Biol Chem,2004.279(11):p. 10382-8.
    178. Fuh, G., et al., Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor. J Biol Chem,1998.273(18):p.11197-204.
    179. Kendall, R. L. and K.A. Thomas, Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A,1993.90(22):p. 10705-9.
    180. Ebos, J.M., et al., A naturally occurring soluble form of vascular endothelial growth factor receptor 2 detected in mouse and human plasma. Mol Cancer Res,2004.2(6):p.315-26.
    181. Hughes, D.C., Alternative splicing of the human VEGFGR-3/FLT4 gene as a consequence of an integrated human endogenous retrovirus. J Mol Evol,2001.53(2):p.77-9.
    182. Dosch, D.D. and K. Ballmer-Hofer, Transmembrane domain-mediated orientation of receptor monomers in active VEGFR-2 dimers. FASEB J,2010.24(1):p.32-8.
    183. Stuttfeld, E. and K. Ballmer-Hofer, Structure and function of VEGF receptors. IUBMB Life,2009. 61(9):p.915-22.
    184. Ruch, C., et al., Structure of a VEGF-VEGF receptor complex determined by electron microscopy. Nat Struct Mol Biol,2007.14(3):p.249-50.
    185. Yang, Y., et al., Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling. Proc Natl Acad Sci U S A,2010. 107(5):p.1906-11.
    186. Lemmon, M.A. and J. Schlessinger, Cell signaling by receptor tyrosine kinases. Cell,2010. 141(7):p.1117-34.
    187. Koch, S., et al., Signal transduction by vascular endothelial growth factor receptors. Biochem J, 2011.437(2):p.169-83.
    188. Kappert, K., et al., Tyrosine phosphatases in vessel wall signaling. Cardiovasc Res,2005.65(3): p.587-98.
    189. de Vries, C., et al., The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science,1992.255(5047):p.989-91.
    190. Shibuya, M., et al., Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene,1990.5(4):p.519-24.
    191. Davis-Smyth, T., et al., The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade. EMBOJ,1996.15(18):p.4919-27.
    192. Tanaka, K., et al., Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase). Jpn J Cancer Res,1997.88(9):p.867-76.
    193. Wiesmann, C., et al., Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor. Cell,1997.91(5):p.695-704.
    194. Peters, K.G., C. De Vries, and L.T. Williams, Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc Natl Acad Sci U S A,1993.90(19):p.8915-9.
    195. Jakeman, L.B., et al., Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest,1992.89(1):p.244-53.
    196. Clark, D.E., et al.. Localization of VEGF and expression of its receptors flt and KDR in human placenta throughout pregnancy. Hum Reprod,1996.11(5):p.1090-8.
    197. Podar, K., et al., Vascular endothelial growth factor-induced migration of multiple myeloma cells is associated with beta 1 integrin-and phosphatidylinositol 3-kinase-dependent PKC alpha activation. J Biol Chem,2002.277(10):p.7875-81.
    198. Sawano, A., et al., Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood,2001.97(3):p.785-91.
    199. Schwartz, J.D., et al., Vascular endothelial growth factor receptor-1 in human cancer:concise review and rationale for development of IMC-18F1 (Human antibody targeting vascular endothelial growth factor receptor-1). Cancer,2010.116(4 Suppl):p.1027-32.
    200. Takahashi, T., et al., Protein tyrosine kinases expressed in glomeruli and cultured glomerular cells:Flt-1 and VEGF expression in renal mesangial cells. Biochem Biophys Res Commun,1995. 209(1):p.218-26.
    201. Nomura, M., et al., Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-induced proliferation of endothelial cells and pericytes. J Biol Chem, 1995.270(47):p.28316-24.
    202. Thomas, C.P., et al., Alternate processing of Fltl transcripts is directed by conserved cis-elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation. Nucleic Acids Res,2010.38(15):p.5130-40.
    203. Mezquita, B., et al., A novel intracellular isoform of VEGFR-1 activates Src and promotes cell invasion in MDA-MB-231 breast cancer cells. J Cell Biochem,2010.110(3):p.732-42.
    204. Shinkai, A., et al., Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor. J Biol Chem,1998.273(47):p.31283-8.
    205. Gille, H., et al., A represser sequence in the juxtamembrane domain of Flt-1 (VEGFR-1) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial cell migration. EMBO J,2000.19(15):p.4064-73.
    206. to, N., et al.. Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules. J Biol Chem,1998. 273(36):p.23410-8.
    207. Cunningham, S.A., et al., Interactions of FLT-1 and KDR with phospholipase C gamma: identification of the phosphotyrosine binding sites. Biochem Biophys Res Commun,1997. 240(3):p.635-9.
    208. Yu, Y., et al., Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase binding. Biochem J, 2001.358(Pt 2):p.465-72.
    209. Autiero, M., et al.. Role of PIGF in the intra-and intermoleular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med,2003.9(7):p.936-43.
    210. Sawano, A., et al.. The phosphorylated 1169-tyrosine containing region of flt-1 kinase (VEGFR-1) is a major binding site for PLCgamma. Biochem Biophys Res Commun,1997. 238(2):p.487-91.
    211. Igarashi, K., et al., Tyrosine 1213 of Flt-1 is a major binding site of Nek and SHP-2. Biochem Biophys Res Commun,1998.246(1):p.95-9.
    212. Cunningham, S.A., et al., Interaction of the Flt-1 tyrosine kinase receptor with the p85 subunit of phosphatidylinositol 3-kinase. Mapping of a novel site involved in binding. J Biol Chem, 1995.270(35):p.20254-7.
    213. Gille, H., et al., Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem,2001.276(5):p.3222-30.
    214. Rahimi, N., V. Dayanir, and K. Lashkari, Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J Biol Chem,2000.275(22):p.16986-92.
    215. Waltenberger, J., et al.. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem,1994.269(43):p.26988-95.
    216. Kanno, S., et al., Roles of two VEGF receptors, Flt-1 and KDR, in the signal transduction of VEGF effects in human vascular endothelial cells. Oncogene,2000.19(17):p.2138-46.
    217. Cai, J., et al., Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells. Diabetes,2003.52(12):p.2959-68.
    218. Huang, K., et al.. Signaling properties of VEGF receptor-1 and-2 homo-and heterodimers. Int J Biochem Cell Biol,2001.33(4):p.315-24.
    219. Mac Gabhann, F. and A.S. Popel, Dimerization of VEGF receptors and implications for signal transduction:a computational study. Biophys Chem,2007.128(2-3):p.125-39.
    220. Kou, R., et al., Differential regulation of vascular endothelial growth factor receptors (VEGFR) revealed by RNA interference:interactions of VEGFR-1 and VEGFR-2 in endothelial cell signaling. Biochemistry,2005.44(45):p.15064-73.
    221. Jinnin, M., et al., Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med,2008.14(11):p.1236-46.
    222. Lamalice, L, F. Houle, and J. Huot, Phosphorylation of Tyr1214 within VEGFR-2 triggers the recruitment of Nek and activation of Fyn leading to SAPK2/p38 activation and endothelial cell migration in response to VEGF. J Biol Chem,2006.281(45):p.34009-20.
    223. Ding, Y., et al., NFAT1 mediates placental growth factor-induced myelomonocytic cell recruitment via the induction of TNF-alpha. J Immunol,2010.184(5):p.2593-601.
    224. Tchaikovski, V., G. Fellbrich, and J. Waltenberger, The molecular basis of VEGFR-1 signal transduction pathways in primary human monocytes. Arterioscler Thromb Vase Biol,2008. 28(2):p.322-8.
    225. Bellik, L., et al., Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia. Br J Pharmacol,2005. 146(4):p.568-75.
    226. Lesslie, D.P., et al., Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases. Br J Cancer,2006.94(11): p.1710-7.
    227. Taylor, A.P., E. Leon, and D.M. Goldenberg, Placental growth factor (PIGF) enhances breast cancer cell motility by mobilising ERK1/2 phosphorylation and cytoskeletal rearrangement. Br J Cancer,2010.103(1):p.82-9.
    228. Fong, G.H., et al., Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature,1995.376(6535):p.66-70.
    229. Hiratsuka, S., et al., Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci U S A,1998.95(16):p.9349-54.
    230. Kappas, N.C., et al., The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching. J Cell Biol,2008.181(5):p.847-58.
    231. Hiratsuka, S., et al.,Membrane fixation of vascular endothelial growth factor receptor 1 Iigand-binding domain is important for vasculogenesis and angiogenesis in mice. Mol Cell Biol, 2005.25(1):p.346-54.
    232. Kendall, R.L., et al., Specificity of vascular endothelial cell growth factor receptor ligand binding domains. Biochem Biophys Res Commun,1994.201(1):p.326-30.
    233. Hiratsuka, S., et al., Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res,2001.61(3):p.1207-13.
    234. Hiratsuka, S., et al., MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell,2002.2(4):p.289-300.
    235. Kami, J., et al., Inhibition of choroidal neovascularization by blocking vascular endothelial growth factor receptor tyrosine kinase. Jpn J Ophthalmol,2008.52(2):p.91-8.
    236. Murakami, M., et a I., Signaling of vascular endothelial growth factor receptor-1 tyrosine kinase promotes rheumatoid arthritis through activation of monocytes/macrophages. Blood, 2006.108(6):p.1849-56.
    237. Beck, H., et al., VEGFR-1 signaling regulates the homing of bone marrow-derived cells in a mouse stroke model. J Neuropathol Exp Neurol,2010.69(2):p.168-75.
    238. McColl, B.K., et al., Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D. J Exp Med,2003.198(6):p.863-8.
    239. Albuquerque, R.J., et al., Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med,2009.15(9):p. 1023-30.
    240. Becker, J., et al., Neuroblastoma progression correlates with downregulation of the lymphangiogenesis inhibitor sVEGFR-2. Clin Cancer Res,2010.16(5):p.1431-41.
    241. Lorquet, S., et al., Soluble forms of VEGF receptor-1 and-2 promote vascular maturation via mural cell recruitment. FASEB J,2010.24(10):p.3782-95.
    242. Millauer, B., et al., High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell,1993.72(6):p.835-46.
    243. Oelrichs, R.B., et al., NYK/FLK-1:a putative receptor protein tyrosine kinase isolated from E10 embryonic neuroepithelium is expressed in endothelial cells of the developing embryo. Oncogene,1993.8(1):p.11-8.
    244. Tasaki, Y., et al.. Expression of VEGF and its receptors in the bovine endometrium throughout the estrous cycle:effects of VEGF on prostaglandin production in endometrial cells. J Rep rod Dev,2010.56(2):p.223-9.
    245. Millauer, B., et al., Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature,1994.367(6463):p.576-9.
    246. Plate, K.H., et al., Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res,1993.53(23):p.5822-7.
    247. Youssoufian, H., D.J. Hicklin, and E.K. Rowinsky, Review:monoclonal antibodies to the vascular endothelial growth factor receptor-2 in cancer therapy. Clin Cancer Res,2007.13(18 Pt 2):p.5544s-5548s.
    248. Maharaj, A.S., et al., Vascular endothelial growth factor localization in the adult. Am J Pathol, 2006.168(2):p.639-48.
    249. Takahashi, T., et al., A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J,2001.20(11):p.2768-78.
    250. Matsumoto, T., et al., VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J,2005.24(13):p.2342-53.
    251. Solowiej, J., et al., Characterizing the effects of the juxtamembrane domain on vascular endothelial growth factor receptor-2 enzymatic activity, autophosphorylation, and inhibition byaxitinib. Biochemistry,2009.48(29):p.7019-31.
    252. Wu, L.W., et al., VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor. J Biol Chem,2000.275(9):p.6059-62.
    253. Kendall, R.L., et al., Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues. J Biol Chem,1999. 274(10):p.6453-60.
    254. Meyer, R.D., D.B. Sacks, and N. Rahimi, IQGAPI-dependent signaling pathway regulates endothelial cell proliferation and angiogenesis. PLoS One,2008.3(12):p. e3848.
    255. Holmqvist, K., et al., The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem,2004.279(21):p.22267-75.
    256. Warner, A.J., et al., The She-related adaptor protein, Sck, forms a complex with the vascular-endothelial-growth-factor receptor KDR in transfected cells. Biochem J,2000.347(Pt 2):p.501-9.
    257. Abu-Ghazaleh, R., et al., Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells. Biochem J,2001.360(Pt 1):p.255-64.
    258. Holmqvist, K., et al., The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in murine brain endothelial cells. Cell Signal,2003.15(2):p. 171-9.
    259. Kroll, J. and J. Waltenberger, The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem,1997. 272(51):p.32521-7.
    260. Meadows, K.N., P. Bryant, and K. Pumiglia, Vascular endothelial growth factor induction of the angiogenicphenotype requires Ras activation. J Biol Chem,2001.276(52):p.49289-98.
    261. Zachary, I., Signaling mechanisms mediating vascular protective actions of vascular endothelial growth factor. Am J Physiol Cell Physiol,2001.280(6):p. C1375-86.
    262. Takahashi, T., H. Ueno, and M. Shibuya, VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene,1999.18(13):p.2221-30.
    263. Xia, P., et al., Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest,1996.98(9):p. 2018-26.
    264. Wellner, M., et al., The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta. Arterioscler Thromb Vasc Biol,1999.19(1): p.178-85.
    265. Shu, X., et al., Sphingosine kinase mediates vascular endothelial growth factor-induced activation of ras and mitogen-activated protein kinases. Mol Cell Biol,2002.22(22):p. 7758-68.
    266. Bhattacharya, R., et al., Distinct role of PLCbeta3 in VEGF-mediated directional migration and vascular sprouting. J Cell Sci,2009.122(Pt 7):p.1025-34.
    267. Hatziapostolou, M., et al., Tumor progression locus 2 mediates signal-induced increases in cytoplasmic calcium and cell migration. Sci Signal,2011.4(187):p. ra55.
    268. Wong, C. and Z.G. Jin, Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor. J Biol Chem,2005.280(39):p.33262-9.
    269. Wang, S., et al., Control of endothelial cell proliferation and migration by VEGF signaling to histone deacetylase 7. Proc Natl Acad Sci U S A,2008.105(22):p.7738-43.
    270. Ha, C.H., et al., Protein kinase D-dependent phosphorylation and nuclear export of histone deacetylase 5 mediates vascular endothelial growth factor-induced gene expression and angiogenesis. J Biol Chem,2008.283(21):p.14590-9.
    271. Evans, I.M., G. Britton, and I.C. Zachary, Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase. Cell Signal,2008.20(7):p.1375-84.
    272. Lamalice, L., et al., Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38. Oncogene,2004.23(2):p.434-45.
    273. Laramee, M., et al., The scaffolding adapter Gabl mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation. J Biol Chem,2007.282(11):p.7758-69.
    274. Jin, Z.G., et al., Flow shear stress stimulates Gabl tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells. J Biol Chem, 2005.280(13):p.12305-9.
    275. Datta, S.R., A. Brunet, and M.E. Greenberg, Cellular survival:a play in three Akts. Genes Dev, 1999.13(22):p.2905-27.
    276. Los, M., et al., Apoptin, a tumor-selective killer. Biochim Biophys Acta,2009.1793(8):p. 1335-42.
    277. Gerber, H.P., et al., Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation. J Biol Chem,1998.273(46):p.30336-43.
    278. Kumar, P., A.I. Miller, and P.J. Polverini, p38 MAPK mediates gamma-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway. J Biol Chem,2004.279(41):p. 43352-60.
    279. Deveraux, Q.L., et al., lAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J,1998.17(8):p.2215-23.
    280. Shalaby, F., et al., Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature,1995.376(6535):p.62-6.
    281. Carmeliet, P., et al.. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature,1996.380(6573):p.435-9.
    282. Ferrara, N., et al., Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature,1996.380(6573):p.439-42.
    283. Sakurai, Y., et al., Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice. Proc Natl Acad Sci U S A,2005.102(4):p.1076-81.
    284. Ji, Q.S., et al., Essential role of the tyrosine kinase substrate phospholipase C-gammal in mammalian growth and development. Proc Natl Acad Sci U S A,1997.94(7):p.2999-3003.
    285. Liao, H.J., et al.. Absence of erythrogenesis and vasculogenesis in Plcgl-deficient mice. J Biol Chem,2002.277(11):p.9335-41.
    286. Pajusola, K., et al., Two human FLT4 receptor tyrosine kinase isoforms with distinct carboxy terminal tails are produced by alternative processing of primary transcripts. Oncogene,1993. 8(11):p.2931-7.
    287. Joukov, V., et al., Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J,1997.16(13):p.3898-911.
    288. Stacker, S.A., et al.,Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers. J Biol Chem,1999.274(45):p. 32127-36.
    289. Makinen, T., et al., Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J,2001.20(17):p.4762-73.
    290. Jeltsch, M., et al., Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns. J Biol Chem,2006. 281(17):p.12187-95.
    291. Leppanen, V.M., et al., Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc Natl Acad Sci U S A,2010.107(6):p.2425-30.
    292. Baldwin, M.E., et al., The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man. J Biol Chem,2001.276(22):p.19166-71.
    293. Leppanen, V.M., et al., Structural determinants of vascular endothelial growth factor-D receptor binding and specificity. Blood,2011.117(5):p.1507-15.
    294. Aprelikova, O., et al., FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. Cancer Res,1992.52(3):p.746-8.
    295. Galland, F., et al., The FLT4 gene encodes a transmembrane tyrosine kinase related to the vascular endothelial growth factor receptor. Oncogene,1993.8(5):p.1233-40.
    296. Salameh, A., et al.. Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways. Blood,2005.106(10):p.3423-31.
    297. Fournier, E., et al., Mutation at tyrosine residue 1337 abrogates ligand-dependent transforming capacity of the FLT4 receptor. Oncogene,1995.11(5):p.921-31.
    298. Galvagni, F., et al., Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity. Circ Res,2010.106(12):p.1839-48.
    299. Dixelius, J., et al., Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites. J Biol Chem,2003.278(42):p.40973-9.
    300. Alam, A., et al., Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity. Biochem Biophys Res Commun,2004.324(2):p. 909-15.
    301. Goldman, J., et al., Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis. FASEB J,2007.21(4):p.1003-12.
    302. Nilsson, I., et al., VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts. EMBO J,2010.29(8):p.1377-88.
    303. Ghalamkarpour, A., et al., Hereditary lymphedema type I associated with VEGFR3 mutation: the first de novo case and atypical presentations. Clin Genet,2006.70(4):p.330-5.
    304. rrthum, A., et al., Congenital hereditary lymphedema caused by a mutation that inactivates VEGFR3 tyrosine kinase. Am J Hum Genet,2000.67(2):p.295-301.
    305. Karkkainen, M.J., et al., Missense mutations interfere with VEGFR-3 signalling in primary lymphoedema. Nat Genet,2000.25(2):p.153-9.
    306. Karkkainen, M.J., et al., A model for gene therapy of human hereditary lymphedema. Proc Natl Acad Sci U S A,2001.98(22):p.12677-82.
    307. Dumont, D.J., et al., Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science,1998.282(5390):p.946-9.
    308. Haiko, P., et al., Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos. Mol Cell Biol,2008.28(15):p. 4843-50.
    309. Zhang, L, et al., VEGFR-3 ligand-binding and kinase activity are required for lymphangiogenesis but not for angiogenesis. Cell Res,2010.20(12):p.1319-31.
    310. Tvorogov, D., et al., Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. Cancer Cell,2010.18(6): p.630-40.
    311. Karkkainen, M.J., et al., Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat Immunol,2004.5(1):p.74-80.
    312. Fruman, D.A., et al., Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha. Nat Genet,2000.26(3):p.379-82.
    313. chise, T., N. Yoshida, and H. Ichise, H-, N-and Kras cooperatively regulate lymphatic vessel growth by modulating VEGFR3 expression in lymphatic endothelial cells in mice. Development, 2010.137(6):p.1003-13.
    314. Jakobsson, L., et al., Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis. Dev Cell,2006.10(5):p.625-34.
    315. Favier, B., et al., Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration. Blood,2006.108(4):p.1243-50.
    316. Wang, L., et al., Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration. J Biol Chem,2003.278(49):p.48848-60.
    317. Cal, H. and R.R. Reed, Cloning and characterization of neuropilin-1-interacting protein:a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1. J Neurosci,1999.19(15):p.6519-27.
    318. Roy, A.L., et al., Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF. Nature,1991.354(6350):p.245-8.
    319. Roy, A.L., Biochemistry and biology of the inducible multifunctional transcription factor TFII-I. Gene,2001.274(1-2):p.1-13.
    320. Jackson, T.A., et al., Vascular endothelial growth factor receptor-2:counter-regulation by the transcription factors, TFII-I and TFII-IRD1. J Biol Chem,2005.280(33):p.29856-63.
    321. Liao, E.C., et al., SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev,1998.12(5):p.621-6.
    322. Mammoto, A., et al., A mechanosensitive transcriptional mechanism that controls angiogenesis. Nature,2009.457(7233):p.1103-8.
    323. Kappel, A., et al., Role of SCL/Tal-1, GATA, and ets transcription factor binding sites for the regulation of flk-1 expression during murine vascular development. Blood,2000.96(9):p. 3078-85.
    324. Patterson, C., et al., Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. J Biol Chem,1995.270(39):p.23111-8.
    325. Minami, T, et al., Interaction between hex and GATA transcription factors in vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial growth factor signaling. J Biol Chem,2004.279(20):p.20626-35.
    326. Minami, T., R.D. Rosenberg, and W.C. Aird, Transforming growth factor-beta 1-mediated inhibition of the flk-1/KDR gene is mediated by a 5'-untranslated region palindromic GATA site. J Biol Chem,2001.276(7):p.5395-402.
    327. Elvert, G., et al., Cooperative interaction of hypoxia-inducible factor-2alpha (HIF-2alpha) and Ets-1 in the transcriptional activation of vascular endothelial growth factor receptor-2 (Flk-1). J Biol Chem,2003.278(9):p.7520-30.
    328. Hata, Y., et al., Transcription factors Spl and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence. J Biol Chem,1998.273(30):p. 19294-303.
    329. Illi, B., et al., Nuclear factor-kappaB and cAMP response element binding protein mediate opposite transcriptional effects on the Flk-1/KDR gene promoter. Circ Res,2000.86(12):p. E110-7.
    330. Offermanns, S., et al., Vascular system defects and impaired cell chemokinesis as a result of Galphal3 deficiency. Science,1997.275(5299):p.533-6.
    331. Zeng, H., et al., Heterotrimeric G. alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGFsignaling. J Biol Chem,2003.278(23):p.20738-45.
    332. Liu, Y., et al., Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest,2000.106(8):p.951-61.
    333. Yang, L.V., et al., Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor. Mol Cell Biol,2007.27(4):p.1334-47.
    334. Kim, K.S., et al., GPR4 plays a critical role in endothelial celt function and mediates the effects ofsphingosylphosphorylcholine. FASEB J,2005.19(7):p.819-21.
    335. Sumida, H., et al., LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis. Blood,2010.116(23):p.5060-70.
    336. Wang, T., et al., CD97, an adhesion receptor on inflammatory cells, stimulates angiogenesis through binding integrin counterreceptors on endothelial cells. Blood,2005.105(7):p. 2836-44.
    337. Kang, X., et al., Antiangiogenic activity ofBAll in vivo:implications for gene therapy of human glioblastomas. Cancer Gene Ther,2006.13(4):p.385-92.
    338. Kuhnert, F., et al., Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124. Science,2010.330(6006):p.985-9.
    339. Cullen, M., et al., GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier. Proc Natl Acad Sci U S A,2011. 108(14):p.5759-64.
    340. Anderson, K.D., et al., Angiogenic sprouting into neural tissue requires Gprl24, an orphan G protein-coupled receptor. Proc Natl Acad Sci U S A,2011.108(7):p.2807-12.
    341. Leung, T., et al., Zebrafish G protein gamma2 is required for VEGF signaling during angiogenesis. Blood,2006.108(1):p.160-6.
    342. Coughlin, S.R., Thrombin signalling and protease-activated receptors. Nature,2000. 407(6801):p.258-64.
    343. Even-Ram, S., et al., Thrombin receptor overexpression in malignant and physiological invasion processes. Nat Med,1998.4(8):p.909-14.
    344. Liu, Y., et al., Expression of protease-activated receptor 1 in oral squamous cell carcinoma. Cancer Lett,2001.169(2):p.173-80.
    345. Daaka, Y., G proteins in cancer:the prostate cancer paradigm. Sci STKE,2004.2004(216):p.
    346. Dorsam, R.T. and J.S. Gutkind, G-protein-coupled receptors and cancer. Nat Rev Cancer,2007. 7(2):p.79-94.
    347. Mills, G.B. and W.H. Moolenaar, The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer,2003.3(8):p.582-91.
    348. Lee, Z., et al., Lysophosphatidic acid is a major regulator of growth-regulated oncogene alpha in ovarian cancer. Cancer Res,2006.66(5):p.2740-8.
    349. Rozengurt, E., S. Guha, and J. Sinnett-Smith, Gastrointestinal peptide signalling in health and disease. Eur J Surg Suppl,2002(587):p.23-38.
    350. Heasley, L.E., Autocrine and paracrine signaling through neuropeptide receptors in human cancer. Oncogene,2001.20(13):p.1563-9.
    351. Taub, J.S., et al., Bradykinin receptor subtype 1 expression and function in prostate cancer. Cancer Res,2003.63(9):p.2037-41.
    352. Ariazi, E.A., et al., Estrogen receptors as therapeutic targets in breast cancer. Curr Top Med Chem,2006.6(3):p.181-202.
    353. Feldman, B.J. and D. Feldman, The development of androgen-independent prostate cancer. Nat Rev Cancer,2001.1(1):p.34-45.
    354. McDonnell, D.P. and J.D. Norris, Connections and regulation of the human estrogen receptor. Science,2002.296(5573):p.1642-4.
    355. Filardo, E.J., et al.. Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol,2000.14(10):p.1649-60.
    356. Carmeci, C., et al., Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics, 1997.45(3):p.607-17.
    357. Marinissen, M.J. and J.S. Gutkind, G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci,2001.22(7):p.368-76.
    358. Brown, J.R. and R.N. DuBois, COX-2:a molecular target for colorectal cancer prevention. J Clin Oncol,2005.23(12):p.2840-55.
    359. Gupta, R.A. and R.N. Dubois, Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nat Rev Cancer,2001.1(1):p.11-21.
    360. Hull, M.A., S.C. Ko, and G. Hawcroft, Prostaglandin EP receptors:targets for treatment and prevention of colorectal cancer? Mol Cancer Ther,2004.3(8):p.1031-9.
    361. Hansen-Petrik, M.B., et al., Prostaglandin E(2) protects intestinal tumors from nonsteroidal anti-inflammatory drug-induced regression in Apc(Min/+) mice. Cancer Res,2002.62(2):p. 403-8.
    362. Sonoshita, M., et al., Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice. Nat Med,2001.7(9):p.1048-51.
    363. Vogelstein, B. and K.W. Kinzler, Cancer genes and the pathways they control. Nat Med,2004. 10(8):p.789-99.
    364. Kikuchi, A., Tumor formation by genetic mutations in the components of the Wnt signaling pathway. Cancer Sci,2003.94(3):p.225-9.
    365. Castellone, M.O., et al., Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science,2005.310(5753):p.1504-10.
    366. Shao, J., et al., Prostaglandin E2 Stimulates the beta-catenin/T cell factor-dependent transcription in colon cancer. J Biol Chem,2005.280(28):p.26565-72.
    367. Holla, V.R., et al., Prostaglandin E2 regulates the nuclear receptor NR4A2 in colorectal cancer. J Biol Chem,2006.281(5):p.2676-82.
    368. He, T.C., et al., PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell,1999.99(3):p.335-45.
    369. Kim, T.H., et al., beta-Catenin activates the growth factor endothelin-1 in colon cancer cells. Oncogene,2005.24(4):p.597-604.
    370. Mazhar, D., R. Ang, and J. Waxman, COX inhibitors and breast cancer. Br J Cancer,2006.94(3): p.346-50.
    371. Liu, C.H., et al., Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem,2001.276(21):p.18563-9.
    372. Chang, S.H., et al., The prostaglandin E2 receptor EP2 is required for cyclooxygenase 2-mediated mammary hyperplasia. Cancer Res,2005.65(11):p.4496-9.
    373. Hida, T., et al., Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res,1998.58(17):p.3761-4.
    374. Moriguchi, T., et al., DREG, a developmentally regulated G protein-coupled receptor containing two conservedproteolytic cleavage sites. Genes Cells,2004.9(6):p.549-60.
    375. Bjarnadottir, T.K., et al., Identification of novel splice variants of Adhesion G protein-coupled receptors. Gene,2007.387(1-2):p.38-48.
    376. Huang, T.S., et al., Functional network reconstruction reveals somatic sternness genetic maps and dedifferentiation-like transcriptome reprogramming induced by GATA2. Stem Cells,2008. 26(5):p.1186-201.
    377. Schotta, G., et al., A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes Dev,2008.22(15):p. 2048-61.
    378. Koirala, S. and G. Corfas, Identification of novel glial genes by single-cell transcriptional profiling of Bergmann glial cells from mouse cerebellum. PLoS One,2010.5(2):p. e9198.
    379. Waller-Evans, H., et al., The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse. PLoS One,2010.5(11):p. e14047.
    380. Monk, K.R., et al., A G protein-coupled receptor is essential for Schwann cells to initiate myelination. Science,2009.325(5946):p.1402-5.
    381. Monk, K.R., et al., Gpr126 is essential for peripheral nerve development and myelination in mammals. Development,2011.138(13):p.2673-80.
    382. Hancock, D.B., et al., Meta-analyses of genome-wide association studies identify multiple loci associated with pulmonary function. Nat Genet,2010.42(1):p.45-52.
    383. Lettre, G., et al.. Identification of ten loci associated with height highlights new biological pathways in human growth. Nat Genet,2008.40(5):p.584-91.
    384. Weedon, M.N., et al., Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet,2008.40(5):p.575-83.
    385. Koirala, S., et al., GPR56-regulated granule cell adhesion is essential for rostral cerebellar development. J Neurosci,2009.29(23):p.7439-49.
    386. Bjarnadottir, T.K., R. Fredriksson, and H.B. Schioth, The adhesion GPCRs:a unique family of G protein-coupled receptors with important roles in both central and peripheral tissues. Cell Mol Life Sci,2007.64(16):p.2104-19.
    387. Weston, M.D., et al., Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of Usher syndrome type II. Am J Hum Genet,2004.74(2):p.357-66.
    388. Piao, X., et al., G protein-coupled receptor-dependent development of human frontal cortex. Science,2004.303(5666):p.2033-6.
    389. Lawrence, P.A., G. Struhl, and J. Casal, Planar cell polarity:one or two pathways? Nat Rev Genet,2007.8(7):p.555-63.
    390. Strutt, D., The planar polarity pathway. Curr Biol,2008.18(19):p. R898-902.
    391. Tissir, F., et al., Protocadherin Celsr3 is crucial in axonal tract development. Nat Neurosci, 2005.8(4):p.451-7.
    392. Langenhan, T., et al., Latrophilin signaling links anterior-posterior tissue polarity and oriented cell divisions in the C. elegans embryo. Dev Cell,2009.17(4):p.494-504.
    393. Carmeliet, P. and M. Tessier-Lavigne, Common mechanisms of nerve and blood vessel wiring. Nature,2005.436(7048):p.193-200.
    394. Eichmann, A., T. Makinen, and K. Alitalo, Neural guidance molecules regulate vascular remodeling and vessel navigation. Genes Dev,2005.19(9):p.1013-21.
    395. Larrivee, B., et al., Guidance of vascular development:lessons from the nervous system. Circ Res,2009.104(4):p.428-41.
    396. Lok, J., et al., Neuregulin-1 signaling in brain endothelial cells. J Cereb Blood Flow Metab, 2009.29(1):p.39-43.
    397. Lyons, D.A., et al., erbb3 and erbb2 are essential for schwann cell migration and myelination in zebrafish. Curr Biol,2005.15(6):p.513-24.
    398. Wang, B., et al., Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell,2003.4(1):p.19-29.
    399. Chalasani, S.H., et al., Stromal cell-derived factor-1 antagonizes slit/robo signaling in vivo. J Neurosci,2007.27(5):p.973-80.
    400. Wang, S., et al., Notch receptor activation inhibits oligodendrocyte differentiation. Neuron, 1998.21(1):p.63-75.
    401. Benedito, R., et al., The notch ligands DII4 and Jaggedl have opposing effects on angiogenesis. Cell,2009.137(6):p.1124-35.
    402. Tsai, H.H., M. Tessier-Lavigne, and R.H. Miller, Netrin 1 mediates spinal cord oligodendrocyte precursor dispersal. Development,2003.130(10):p.2095-105.
    403. Nguyen, A. and H. Cai, Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism. Proc Natl Acad Sci U S A,2006.103(17):p.6530-5.
    404. Stehlik, C., et al., VIGR-a novel inducible adhesion family G-protein coupled receptor in endothelial cells. FEBS Lett,2004.569(1-3):p.149-55.
    405. Wang, Y., et al., Angiomotin-like2 is required for migration and proliferation of endothelial cells during angiogenesis. J Biol Chem,2011.
    406. Tanaka, A., et al., Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis. Blood,2010.115(20):p.4138-47.
    407. Li, D., et al., SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin. Nucleic Acids Res,2009.37(21):p.7059-71.
    408. Fish, J.E., et al., miR-126 regulates angiogenic signaling and vascular integrity. Dev Cell,2008. 15(2):p.272-84.
    409. Hidaka, M., W.L. Stanford, and A. Bernstein, Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells. Proc Natl Acad Sci U S A,1999.96(13):p. 7370-5.
    410. Vittet, D., et al., Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood,1996.88(9):p.3424-31.
    411. Overington, J.P., B. Al-Lazikani, and A.L. Hopkins, How many drug targets are there? Nat Rev Drug Discov,2006.5(12):p.993-6.
    412. Potente, M., et al., Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J Clin Invest,2005.115(9):p.2382-92.
    413. Connor, K.M., et al., Quantification of oxygen-induced retinopathy in the mouse:a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protoc,2009.4(11):p. 1565-73.
    414. Schuringa, J.J., et al., Enforced activation of STAT5A facilitates the generation of embryonic stem-derived hematopoietic stem cells that contribute to hematopoiesis in vivo. Stem Cells, 2004.22(7):p.1191-204.
    415. Yang, X., et al., Signal transducers and activators of transcription mediate fibroblast growth factor-induced vascular endothelial morphogenesis. Cancer Res,2009.69(4):p.1668-77.
    416. Boer, A.K., et al., Prostaglandin-E2 enhances EPO-mediated STAT5 transcriptional activity by serine phosphorylation of CREB. Blood,2002.100(2):p.467-73.
    417. Yamashita, J., et al., Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature,2000.408(6808):p.92-6.
    418. Stainier, D.Y. and M.C. Fishman, The zebrafish as a model system to study cardiovascular development. Trends Cardiovasc Med,1994.4(5):p.207-12.
    419. Childs, S., et al., Patterning of angiogenesis in the zebrafish embryo. Development,2002. 129(4):p.973-82.
    420. Tsopanoglou, N.E. and M.E. Maragoudakis, On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem,1999.274(34):p.23969-76.
    421. Yamamizu, K., et al., Enhancement of vascular progenitor potential by protein kinase A through dual induction of Flk-1 and Neuropilin-1. Blood,2009.114(17):p.3707-16.
    422. Pimanda, J.E., et al., Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development. Proc Natl Acad Sci U S A,2007.104(45):p.17692-7.
    423. Zeoli, A., et al., interleukin-3 promotes expansion of hemopoietic-derived CD45+angiogenic cells and their arterial commitment via STAT5 activation. Blood,2008.112(2):p.350-61.
    424. Dudley, A.C., et al., A VEGF/JAK2/STAT5 axis may partially mediate endothelial cell tolerance to hypoxia. Biochem J,2005.390(Pt 2):p.427-36.
    425. Korpelainen, E.I., et al., Endothelial receptor tyrosine kinases activate the STAT signaling pathway:mutant Tie-2 causing venous malformations signals a distinct STAT activation response. Oncogene,1999.18(1):p.1-8.
    426. Sarbassov, D.D., et al., Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science,2005.307(5712):p.1098-101.
    427. Fujishita, T., et al., Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcDelta716 mice. Proc Natl Acad Sci U S A,2008.105(36):p. 13544-9.
    428. Wander, S.A., B.T. Hennessy, and J.M. Slingerland, Next-generation mTOR inhibitors in clinical oncology:how pathway complexity informs therapeutic strategy. J Clin Invest,2011.121(4):p. 1231-41.
    429. Martelli, A.M., et al., Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway. Leukemia,2011.25(7):p.1064-79.
    430. Choo, A.Y. and J. Blenis, Not all substrates are treated equally:implications for mTOR, rapamycin-resistance and cancer therapy. Cell Cycle,2009.8(4):p.567-72.
    431. Zhang, Y.J., et al., mTOR signaling pathway is a target for the treatment ofcolorectal cancer. Ann Surg Oncol,2009.16(9):p.2617-28.
    432. Feng, Z., et al., The regulation ofAMPK betal, TSC2, and PTEN expression by p53:stress, cell and tissue specificity, and the role of these gene products in modulating the IGF-1-AKT-mTOR pathways. Cancer Res,2007.67(7):p.3043-53.
    433. Rostaing, L. and N. Kamar, mTOR inhibitor/proliferation signal inhibitors:entering or leaving the field?} Nephrol,2010.23(2):p.133-42.
    434. Aoki, K., et al., Colonic polyposis caused by mTOR-mediated chromosomal instability in Apc+/Delta716 Cdx2+/-compound mutant mice. Nat Genet,2003.35(4):p.323-30.
    435. Fujishita, T, M. Aoki, and M.M. Taketo, The role of mTORCl pathway in intestinal tumorigenesis. Cell Cycle,2009.8(22):p.3684-7.
    436. Cirulli, E.T., et al., Screening the human exome:a comparison of whole genome and whole transcriptome sequencing. Genome Biol,2010.11(5):p.R57.