Neuropilin-1-VEGFR-2 Complexing Requires the PDZ-binding Domain of Neuropilin-1

Abstract

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF165 binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF165 binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1. Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF165 binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF165 binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1. Angiogenesis, the growth of new blood vessels from a pre-existing vascular network, plays a pivotal role during embryonic development and in numerous diseases including cancer, chronic inflammation, and vision-threatening retinopathies. Vascular endothelial growth factors (VEGF) 2The abbreviations used are: VEGFvascular endothelial growth factorVEGFRVEGF receptorNP-1Neuropilin-1NP-1Neuropilin-2PDZpost-synaptic density, disc large, ZO-1ERKextracellular signal-regulated kinaseECendothelial cellsHUVEChuman umbilical vein endothelial cellsPAECporcine aortic endothelial cellsPTK787/ZK222584PTK/ZKRT-PCRreverse transcription-PCRsiRNAsmall interfering RNAFCSfetal calf serum. are a family of mitogenic and chemotactic factors for endothelial cells (EC), which act as hierarchically high inducers of the angiogenic cascade (1Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4868) Google Scholar). VEGFs signal through the high affinity receptor tyrosine kinases VEGFR-1, VEGFR-2, and VEGFR-3, which are almost exclusively expressed by EC. In addition to the VEGF receptors, two other molecules, namely Neuropilin-1 (NP-1) and Neuropilin-2 (NP-2), have been identified as co-receptors for VEGF (2Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). During development, NP-1 is expressed by arterial EC (3Moyon D. Pardanaud L. Yuan L. Breant C. Eichmann A. Development (Camb.). 2001; 128: 3359-3370Crossref PubMed Google Scholar), whereas NP-2 is expressed by venous and lymphatic EC (4Herzog Y. Kalcheim C. Kahane N. Reshef R. Neufeld G. Mech. Dev. 2001; 109: 115-119Crossref PubMed Scopus (215) Google Scholar). NP-1 enhances the affinity of VEGF165 to VEGFR-2 (2Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar) and increases its phosphorylation, thereby enhancing downstream signaling (5Becker P.M. Waltenberger J. Yachechko R. Mirzapoiazova T. Sham J.S. Lee C.G. Elias J.A. Verin A.D. Circ. Res. 2005; 96: 1257-1265Crossref PubMed Scopus (110) Google Scholar). Correspondingly, overexpression of NP-1 leads to the formation of excess capillaries and blood vessels (6Kitsukawa T. Shimono A. Kawakami A. Kondoh H. Fujisawa H. Development (Camb.). 1995; 121: 4309-4318Crossref PubMed Google Scholar, 7Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar). Targeted disruption of NP-1 results in embryonic lethality and vascular defects such as impairment of neural vascularization, transposition of large vessels, and insufficient development of vascular networks in the yolk sac (7Kitsukawa T. Shimizu M. Sanbo M. Hirata T. Taniguchi M. Bekku Y. Yagi T. Fujisawa H. Neuron. 1997; 19: 995-1005Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar, 8Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development (Camb.). 1999; 126: 4895-4902Crossref PubMed Google Scholar). vascular endothelial growth factor VEGF receptor Neuropilin-1 Neuropilin-2 post-synaptic density, disc large, ZO-1 extracellular signal-regulated kinase endothelial cells human umbilical vein endothelial cells porcine aortic endothelial cells PTK/ZK reverse transcription-PCR small interfering RNA fetal calf serum. The mechanistic analysis of the co-receptor function of neuropilins for VEGFR signaling has concentrated on the extracellular domain of the neuropilins. It was suggested that VEGF165 binds to VEGFR-2 and NP-1 to form a bridge between the extracellular domains, which results in the formation of complexes (9Soker S. Miao H.Q. Nomi M. Takashima S. Klagsbrun M. J. Cell. Biochem. 2002; 85: 357-368Crossref PubMed Scopus (382) Google Scholar). However, recent work employing VEGF variants that either bind only NP-1 or bind only VEGFR-2 has suggested that the extracellular ligand-bridging model is not sufficient to account for a signal enhancing function of NP-1 in the absence of extracellular ligand binding (10Shraga-Heled N. Kessler O. Prahst C. Kroll J. Augustin H. Neufeld G. FASEB J. 2007; 21: 915-926Crossref PubMed Scopus (63) Google Scholar). Likewise, although VEGF121 has been shown to bind NP-1 (11Pan Q. Chathery Y. Wu Y. Rathore N. Tong R.K. Peale F. Bagri A. Tessier-Lavigne M. Koch A.W. Watts R.J. J. Biol. Chem. 2007; 282: 24049-24056Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), stimulation with VEGF121 is not sufficient to promote complex formation between VEGFR-2 and NP-1, suggesting a further mechanism in complex formation between NP-1 and VEGFR-2. Previous research has shown that the intracellular domain of NP-1 does not contain sequences predicted to have enzymatic activities (12Fujisawa H. Kitsukawa T. Curr. Opin. Neurobiol. 1998; 8: 587-592Crossref PubMed Scopus (116) Google Scholar). Nevertheless, the transmembrane and cytoplasmic domains share over 90% amino acid identity across species (13Kawakami A. Kitsukawa T. Takagi S. Fujisawa H. J. Neurobiol. 1996; 29: 1-17Crossref PubMed Scopus (247) Google Scholar, 14Takagi S. Kasuya Y. Shimizu M. Matsuura T. Tsuboi M. Kawakami A. Fujisawa H. Dev. Biol. 1995; 170: 207-222Crossref PubMed Scopus (175) Google Scholar), suggesting an evolutionary conserved role for these domains. We therefore hypothesized that the intracellular domain of NP-1 may be functionally involved in complex formation. This hypothesis was also supported by the observation that blockade of VEGFR-2 phosphorylation negatively interfered with VEGFR-2-NP-1 complex formation. The experiments of this study revealed that the PDZ-binding domain in the cytoplasmic domain of NP-1 is required for stable VEGFR-2-NP-1 complex formation. The identification of Synectin in this intracellular association supports an intracellular VEGFR-2-NP-1 association involving Synectin as a bridging molecule. Materials—Antibodies against NP-1 (C-19), VEGFR-2 (C-1158) and phospho-Tyrosine (PY99) used for Western blot analysis were purchased from Santa Cruz Biotechnology. The antibody directed against NP-1 used in immunofluorescent analysis was obtained from Miltenyi Biotech. The antibody directed against VEGFR-2 (3G2) used for immunoprecipitation of human VEGFR-2 was provided by the Tumor Biology Center Freiburg (Freiburg, Germany), the antibody used for immunoprecipitation of mouse VEGFR-2 was purchased from Santa Cruz Biotechnology (C-1158), and the antibody used for immunofluorescence analysis was obtained from R&D Systems. The low molecular weight VEGFR-inhibitor PTK787/ZK 222584 was kindly provided by Novartis and used at a final concentration of 100 nm. VEGF165 was purchased from R&D Systems and used at a final concentration of 25 ng/ml. Cell Culture—Human umbilical vein endothelial cells (HUVEC), endothelial cell growth medium, endothelial cell basal medium, and corresponding supplements were purchased from Promocell, Heidelberg, Germany. HUVEC were cultured in endothelial cell growth medium containing 10% FCS. For growth factor starvation, cells were incubated in endothelial cell basal medium supplemented with 5% FCS for 16 h. HUVEC were used between passages 2 and 6. Porcine aortic endothelial cells (PAEC) were cultured in Nutmix (Invitrogen) containing 10% FCS. For growth factor starvation, PAEC were incubated in Nutmix without FCS for 16 h. Mouse EC were isolated and cultured as described previously (15Chittenden T.W. Claes F. Lanahan A.A. Autiero M. Palac R.T. Tkachenko E.V. Elfenbein A. Ruiz d.A. Dedkov E. Tomanek R. Li W. Westmore M. Singh J.P. Horowitz A. Mulligan-Kehoe M.J. Moodie K.L. Zhuang Z.W. Carmeliet P. Simons M. Dev. Cell. 2006; 10: 783-795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). siRNA Transfection of HUVEC—The Synectin-specific siRNA (a) sense, 5′-AAAGGAACCCGGAUGAGCUtt-3′ and antisense, 5′-AGCUCAUCCGGGUUCCUUUtg-3′ and (b) sense, 5′-CCUGCUGGAGAGUUACAUGtt-3′ and antisense, 5′-AUGUAACUCUCCAGCAGGtc-3′ and control non-silencing siRNA were purchased from Ambion and transfected into HUVEC as described previously (16Fiedler U. Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. Witzenrath M. Rosseau S. Suttorp N. Sobke A. Herrmann M. Preissner K.T. Vajkoczy P. Augustin H.G. Nat. Med. 2006; 12: 235-239Crossref PubMed Scopus (742) Google Scholar). Down-regulation of Synectin was analyzed by RT-PCR 24 h following transfection. RNA Isolation and RT-PCR—RNA isolation and RT-PCR were performed as described previously (16Fiedler U. Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. Witzenrath M. Rosseau S. Suttorp N. Sobke A. Herrmann M. Preissner K.T. Vajkoczy P. Augustin H.G. Nat. Med. 2006; 12: 235-239Crossref PubMed Scopus (742) Google Scholar). Expression Vector, Retroviral Constructs, and Adenoviral Constructs—PAE cells were transfected using a retroviral expression system pLIB (Clontech), which was modified and kindly provided by Dr. Ralph Graeser (Tumor Biology Center, Freiburg, Germany). Full-length human VEGFR-2, human NP-1, and the human NP-1 lacking the PDZ-binding domain (NP-1ΔPDZ) were cloned into modified pLIB. Together with pVSVG, the constructs were transfected into human embryonic kidney Ampho 293 cells. Virus-containing supernatants were then used for transfection of PAEC. HUVEC were transfected with an adenoviral expression system (Invitrogen). Full-length NP-1 and NP-1ΔPDZ were cloned into pDEST according to the Gateway system manufacturer's protocol, and HUVEC were transfected with 100 infectious units/cell. VEGF165 Binding Assay on PAEC—Specific binding experiments using iodinated VEGF165 binding to PAEC expressing recombinant NP-1 or NP-1ΔPDZ were performed as described previously (10Shraga-Heled N. Kessler O. Prahst C. Kroll J. Augustin H. Neufeld G. FASEB J. 2007; 21: 915-926Crossref PubMed Scopus (63) Google Scholar) and analyzed using the GraphPad prism-4 software. VEGFR-2 Phosphorylation Assay and Co-immunoprecipitation Experiments—HUVEC or PAEC were grown to 90% confluence before being starved overnight. Cells were stimulated with VEGF165 for various time points. After stimulation, cells were washed and lysed (150 mm NaCl, 50 mm Tris/HCl, pH 7.4, 1% Nonidet P-40, 10 mm EDTA, 10% glycerin, 1% protease inhibitor mixture (Serva)). For experiments with VEGFR inhibitor, cells were preincubated with 100 nm PTK787/ZK 222584 1 h prior to stimulation with VEGF165. For immunoprecipitation, extracts were incubated with an antibody against VEGFR-2 and protein-G-Sepharose overnight at 4 °C. The Sepharose beads were spun down and washed twice with ice-cold phosphate-buffered saline, 0.1% Nonidet P-40. The beads were boiled in sample buffer and loaded on an 8% SDS-PAGE, blotted onto a polyvinylidene fluoride membrane, and probed with antibodies directed against phospho-Tyrosine, VEGFR-2, and NP-1. Western blot analysis was performed using horseradish peroxidase conjugated secondary antibodies. Bound antibody was visualized using the ECL kit of Amersham Biosciences according to the instructions of the vendor. VEGFR-2 and NP-1 Associate upon Stimulation with VEGF165—Endothelial cell expressed NP-1 serves as a co-receptor for VEGF165, forming complexes with VEGFR-2 upon stimulation with VEGF165 (2Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2088) Google Scholar). To further characterize the kinetics of this interaction, we performed co-immunoprecipitation experiments following VEGF165 stimulation for various periods of time. VEGFR-2 immunoprecipitation from primary human umbilical vein endothelial cells (HUVEC) followed by Western blot analysis revealed enhanced VEGFR-2-NP-1 complex formation after prolonged stimulation with VEGF165 (30 min versus 5 min). This did not parallel the phosphorylation level of VEGFR-2, which was strongest after 5 min of stimulation (Fig. 1A). Immunocytochemical receptor trafficking analysis showed vesicular co-internalization of VEGFR-2 and NP-1 upon stimulation with VEGF165 (Fig. 1B). VEGFR-2 Phosphorylation Is Required for Association between NP-1 and VEGFR-2—The observed increase of stable VEGFR-2-NP-1 complex formation over time suggested that complex formation itself may be dependent on the activation status of VEGFR-2. We therefore performed VEGF165-dependent complex formation experiments in the presence of the VEGF receptor blocker PTK787/ZK222584 (PTK/ZK). PTK/ZK inhibits VEGFR phosphorylation but does not affect the binding of VEGF165 to its receptor (17Wood J.M. Bold G. Buchdunger E. Cozens R. Ferrari S. Frei J. Hofmann F. Mestan J. Mett H. O'Reilly T. Persohn E. Rosel J. Schnell C. Stover D. Theuer A. Towbin H. Wenger F. Woods-Cook K. Menrad A. Siemeister G. Schirner M. Thierauch K.H. Schneider M.R. Drevs J. Martiny-Baron G. Totzke F. Cancer Res. 2000; 60: 2178-2189PubMed Google Scholar). It thereby facilitated the selective analysis of VEGFR-2-NP-1 complex formation during suppressed VEGFR-2 phosphorylation without affecting the binding of VEGF165. Surprisingly, treatment of primary HUVEC with PTK/ZK prior to stimulation with VEGF165 led to a decrease in VEGFR-2-NP-1 complex formation (Fig. 1C). Western blot analysis of phospho-VEGFR-2 confirmed the effectiveness of PTK/ZK in inhibiting VEGFR-2 phosphorylation. To further validate these findings, we performed the same experiment with NP-1- and VEGFR-2-co-transfected PAEC. As in HUVEC, PTK/ZK inhibited the complex formation in PAEC (Fig. 1D). Taken together, these results indicate that formation of VEGFR-2-NP-1 complexes is not only dependent on ligand binding but also affected by the activation status of VEGFR-2. The Cytoplasmic Domain of NP-1 Contributes to VEGFR-2-NP-1 Complex Formation—The observed contribution of VEGFR-2 phosphorylation suggested an intracellular contribution to VEGFR-2-NP-1 complex formation. Blockade of VEGFR-2 phosphorylation may inhibit receptor internalization, as evidenced by reports showing that VEGFR-2 phosphorylation is required for receptor internalization (18Dougher M. Terman B.I. Oncogene. 1999; 18: 1619-1627Crossref PubMed Scopus (165) Google Scholar, 19Ewan L.C. Jopling H.M. Jia H. Mittar S. Bagherzadeh A. Howell G.J. Walker J.H. Zachary I.C. Ponnambalam S. Traffic. 2006; 7: 1270-1282Crossref PubMed Scopus (150) Google Scholar, 20Kobayashi S. Sawano A. Nojima Y. Shibuya M. Maru Y. FASEB J. 2004; 18: 929-931Crossref PubMed Scopus (77) Google Scholar). In turn, the cytoplasmic domain of NP-1 is highly conserved across species, suggesting an evolutionary pressure to maintain a functional role. To examine whether the cytoplasmic domain of NP-1 is involved in VEGFR-2-NP-1 complex formation, a mutant of NP-1 lacking the cytoplasmic domain (NP-1ΔC) was cloned and expressed in PAEC expressing VEGFR-2. The expression of NP-1ΔC in PAEC was confirmed by Western blot analysis, and live staining for NP-1 validated that NP-1ΔC was expressed at the cell surface (data not shown). NP-1ΔC and VEGFR-2 expressing PAEC were stimulated with VEGF165 and lysed, and complex formation was examined by immunoprecipitation and Western blot analysis. Significantly reduced complex formation of NP-1ΔC and VEGFR-2 was observed upon stimulation with VEGF165 when compared with PAEC co-expressing full-length NP-1 and VEGFR-2 (data not shown). Based on these findings, a series of NP-1 cytoplasmic domain truncation mutants was cloned and co-expressed with VEGFR-2 in PAEC. When analyzing these mutants for VEGF165 ligand-induced complex formation, deletion of just the last three amino acids in the cytoplasmic domain of NP-1 (PDZ-binding domain) proved sufficient to significantly inhibit complex formation between VEGFR-2 and NP-1 (Fig. 2A). Binding experiments confirmed that NP-1ΔPDZ bound VEGF165 with the same affinity as full-length NP-1 (Fig. 2B). To extend these findings to primary EC, NP-1 and NP-1ΔPDZ were expressed in HUVEC. As in PAEC, deletion of the PDZ-binding domain in the C-terminal tail of NP-1 proved sufficient to significantly reduce ligand-induced VEGFR-2-NP-1 complex formation (Fig. 2C). Collectively, these data solidly a role of the PDZ-binding domain in NP-1 in to VEGFR-2-NP-1 complex formation following VEGF165 of Synectin on the Expression of only binding to the cytoplasmic domain of NP-1 was identified in a H. J. 1999; 19: PubMed Google Scholar) and is also as or Synectin is a cytoplasmic that binds the PDZ-binding domain of 25 receptors and including the of signaling interaction L. G. U. S. A. 1998; PubMed Scopus Google Scholar), the receptor tyrosine kinases and H. K. K. K. 2004; PubMed Scopus Google Scholar, H. Lee F. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar), and the H. P. J. H. V. E. E. D. E. A. M. Biol. 2002; 21: PubMed Scopus Google Scholar). recent has an role of Synectin for and formation of (15Chittenden T.W. Claes F. Lanahan A.A. Autiero M. Palac R.T. Tkachenko E.V. Elfenbein A. Ruiz d.A. Dedkov E. Tomanek R. Li W. Westmore M. Singh J.P. Horowitz A. Mulligan-Kehoe M.J. Moodie K.L. Zhuang Z.W. Carmeliet P. Simons M. Dev. Cell. 2006; 10: 783-795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). that Synectin is expressed in cells and the arterial of Synectin-deficient suggested an of or arterial EC expressed interaction The identified contribution of the PDZ-binding domain of NP-1 in VEGFR-2-NP-1 complex formation and the expression of NP-1 (3Moyon D. Pardanaud L. Yuan L. Breant C. Eichmann A. Development (Camb.). 2001; 128: 3359-3370Crossref PubMed Google Scholar) suggested that Synectin may a role in VEGFR-2-NP-1 complex formation. To this HUVEC were transfected with siRNA to Synectin of Synectin expression was confirmed by RT-PCR (Fig. HUVEC were stimulated with VEGF165 for min and lysed, and VEGFR-2 was probed by immunoprecipitation and analyzed by Western blot analysis. of Synectin expression led to an decrease of VEGFR-2-NP-1 complex formation (Fig. However, of VEGFR-2 were also reduced (Fig. suggesting that Synectin may affect VEGFR-2 RT-PCR analysis of VEGFR-2 expression revealed that VEGFR-2 were not suggesting that Synectin may a role in the control of VEGFR-2 (Fig. This to be in to a recent study of Synectin on VEGFR-2 expression in endothelial cells L. T. R. D. 2007; PubMed Scopus Google Scholar), although study has a role of Synectin in the of growth factor receptor L. J.S. R. K. D. Cancer Res. 2006; PubMed Scopus Google Scholar). Taken together, the experiments suggested a role of Synectin for VEGFR-2-NP-1 complex formation. Complex of NP-1 and VEGFR-2 Is on the of the Synectin isolated EC from Synectin-deficient were used to study the role of Synectin in complex formation between NP-1 and VEGFR-2. Synectin-deficient EC expressed VEGFR-2 and NP-1 in as EC (Fig. The between Synectin-deficient EC and EC transfected with Synectin siRNA be to Co-immunoprecipitation experiments upon stimulation with VEGF165 in Synectin-deficient EC when compared with EC revealed reduced VEGFR-2-NP-1 complex formation in mutant EC (Fig. Correspondingly, Synectin-deficient EC to VEGF165, as evidenced by reduced phosphorylation following stimulation with VEGF165 (Fig. The data of this study the that the extracellular VEGF165 ligand-bridging model between VEGFR-2 and NP-1 is not sufficient to account for the of VEGF ligand with the VEGF receptors and the neuropilin co-receptors. the experiments for a functional cytoplasmic interaction between NP-1 and VEGFR-2. The findings in Synectin-deficient mouse EC a role of Synectin in the cytoplasmic contribution to VEGFR-2-NP-1 complex formation and in with the data obtained in HUVEC and PAEC, an model of complex formation and VEGFR-2 phosphorylation. The of a functional role of Synectin in VEGFR-2-NP-1 complex formation also a mechanistic model the arterial expression of NP-1 (3Moyon D. Pardanaud L. Yuan L. Breant C. Eichmann A. Development (Camb.). 2001; 128: 3359-3370Crossref PubMed Google Scholar) with the arterial of Synectin-deficient (15Chittenden T.W. Claes F. Lanahan A.A. Autiero M. Palac R.T. Tkachenko E.V. Elfenbein A. Ruiz d.A. Dedkov E. Tomanek R. Li W. Westmore M. Singh J.P. Horowitz A. Mulligan-Kehoe M.J. Moodie K.L. Zhuang Z.W. Carmeliet P. Simons M. Dev. Cell. 2006; 10: 783-795Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The results of this study are the to a role to the cytoplasmic domain of NP-1 and the a novel of the VEGFR-2 co-receptor role of NP-1. experiments to the contribution of extracellular and intracellular VEGFR-2-NP-1 to downstream signaling and endothelial Previous experiments have at a functional role of the PDZ-binding domain of NP-1. For of NP-1 in leads to vascular which be by NP-1 lacking the last three amino acids of NP-1 L. D. FASEB J. 2006; PubMed Scopus Google Scholar). As the identification of an intracellular association between VEGFR-2 and NP-1 involving a Synectin bridge also that neuropilin vascular a function of VEGFR signaling. We the Center at the of for in analysis.