EP1281088A2 - Identification of novel pro-and anti-angiogenic agents - Google Patents

Identification of novel pro-and anti-angiogenic agents

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Publication number
EP1281088A2
EP1281088A2 EP00975419A EP00975419A EP1281088A2 EP 1281088 A2 EP1281088 A2 EP 1281088A2 EP 00975419 A EP00975419 A EP 00975419A EP 00975419 A EP00975419 A EP 00975419A EP 1281088 A2 EP1281088 A2 EP 1281088A2
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Prior art keywords
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ser
leu
gly
glu
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German (de)
French (fr)
Inventor
Jan Susan Rosenbaum
George Brian Whitaker
Brian Joseph Limberg
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Procter and Gamble Co
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Procter and Gamble Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • VEGF vascular endothelial growth factor
  • VEGF ⁇ 65 in the physiological angiogenesis that occurs during the female reproductive cycle, longitudinal bone growth and endochondral bone formation, and wound healing has been documented, as has involvement in pathological (dysregulated) angiogenesis such as that which occurs in tumor growth and metastases, rheumatoid arthritis, endometriosis, psoriasis, proliferative retinopathy, and atherosclerosis (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22; M.
  • the VEGF receptor system provides an attractive target for the identification of novel compounds exhibiting both pro- and anti-angiogenic activity.
  • Pro-angiogenic agents are considered useful for the treatment of cardiac ischemia and peripheral vascular disease (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; T.D. Henry, Brit. Med. J. (1999), 318:(7197)1536-1539) in which new blood vessel formation needs to be stimulated, whereas anti-angiogenic agents are considered useful for the treatment of diseases in which dysregulated angiogenesis is a characteristic pathology.
  • the VEGF ligand family consists of VEGF and its various splice variants (discussed below), VEGF-B, VEGF-C, VEGF-D, P1GF and its splice variants (discussed below), and the viral VEGF, VEGF-E (reviewed in T. Veikkola and K. Alitalo, Semin.Cancer Biol. (1999), 9(3): 211-220).
  • the VEGF family members exert their physiological and pharmacological effects through the interaction with three different VEGF receptor tyrosine kinases, termed VEGFR- 1/FLT-l, VEGFR-2/FLK-l/KDR, and VEGFR-3/FLT-4 (for reviews, see T.
  • NP-1 Neuropilin-1
  • NP-2 Neuropilin-2
  • VEGFR-3/FLT-4 is the primary receptor for VEGF-C and VEGF-D and is considered to be responsible for the lymphatic angiogenic activities of these ligands, whereas the blood vessel angiogenic activities of these ligands is thought to be mediated in part through binding to VEGFR-2 (M.G. Achen et al., Proc Natl Acad Sci USA (1998), 95(2): 548-553; Y.H. Cao, et al., Proc.Natl.Acad.Sci. USA (1998), 95(24): 14389-14394; D.J. Dumont et al., Science (1998), 282(5390): 946-949; B. Enholm, L.
  • P1GF-1, P1GF-2, and the various forms of VEGF-B do not bind to either VEGFR-2 or VEGFR-3, are specific ligands for VEGFR-1, and do not exert appreciable mitogenic or angiogenic effects on endothelial cells (for reviews see T. Veikkola and K. Alitalo, Semin. Cancer Biol. (1999), 9(3): 21 1-220; M.G. Achen, S.A. Stacker,. InU.Exp.Pathol. (1998), 79(5): 255-265; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22); N. Ferrara, J. Molec.
  • VEGFR-1 in angiogenesis is incompletely understood, although transgenic and knockout experiments implicate a role for this receptor in endothelial cell and monocyte chemotaxis as well as vascular organization (S. Hiratsuka, O. Minowa, J. Kuno, T. Noda, and M. Shibuya, Proc Natl Acad Sci USA (1998), 95(16): 9349-9354; G. H. Fong, L. Y. Zhang, D. M. Bryce, and J. Peng, Development (1999), 126(13): 3015-3025; G.H. Fong, J. Rossant, M.
  • VEGFR-2 in vasculogenesis and angiogenesis is established, as activation of this receptor is required for both vasculogenesis (F. Shalaby, J. Rossant, T. Yamaguchi, M. Gertsenstein, X. Wu, M. Breitman, and A.C. Schuh, Nature (1995) 376: 62-66) and angiogenesis (N. Ortega et al., Am J Pathol (1997), 151(5): 1215- 1224; B.
  • VEGF exists in multiple protein isoforms with different heparin proteoglycan and extracellular matrix binding properties. These isoforms (VEGF 1 65, VEGF 121 , VEGFus, VEGF ⁇ 8 9, VEGF 206 ) arise from alternate splicing of the VEGF gene. VEGF 16 5 is the predominant isoform and has limited heparin-binding activity, whereas the VEGF 121 isoform is freely soluble and is devoid of heparin-binding activity. Similarly, PLGF exists in three different isoforms, which also exhibit differential heparin binding ability (for reviews see G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J.
  • VEGF 1 65 and VEGF 121 bind to VEGFR-2 with equal affinity, their activity in biochemical assays that rely on activation of VEGFR-2 is not equivalent (B.A. Keyt et al., J. Biol. Chem. (1996), 271 : 7788-7795; S. Soker, S. Gollamudi-Payne, H. Fidder, H. Charmahelli, M. Klagsbrun, J5 o/. Chem. (1997), 272(50): 31582-31588); S. Ogawa et al., J. Biol. Chem.
  • Neuropilin- 1 (NP- 1 ) and Neuropilin-2 (NP-2) were identified as receptors that bind VEGFi 65 and PLGF-2 but do not bind either VEGF 121 or the non-heparin binding form of PLGF, PLGF-1 (S. Soker, S. Takashima, H.Q. Miao, G. Neufeld, and M. Klagsbrun, Cell (1998), 92:(6): 735-745; Migdal, et al., J Biol Chem (1998), 273(35): 22272-22278; R. Tordjman, N. Ortega, L. Coulombel, j. Plouet, P. H. Romeo, and V.
  • NP-1 and NP-2 also bind various semaphorin ligands to mediate repulsive guidance activity in certain neuronal populations (R.J. Giger, et al., Neuron (1998), 21(5): 1079-1092; T. Takahashi, F. Nakamura, Z. Jin, R.G. Kalb, S.M. Strittmatter, Nat.Neurosci. (1998), 1(6): 487- 493; F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100; D. Bagnard, M.
  • NP-1 and NP-2 are not required for semaphorin signaling (F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100) and they do not contain sequences predictive of enzymatic activity nor sequences predicted to be involved in coupling to intracellular signaling molecules, the NP-1 and NP-2 proteins appear to function as part of a signaling receptor complex for mediating semaphorin signals (T. Takahashi, A. Foumier, F. Nakamura, L-H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M.
  • NP-1 and NP-2 are capable of stimulating signal transduction pathways, although it appears that at least some of the semaphorin-mediated signaling capability of the neuropilins requires interaction with the Plexins as a co-receptor complex (T. Takahashi, A. Fournier, F. Nakamura, L-H. Wang, Y.
  • VEGFR-2 Since binding to VEGFR-2 alone does not explain the differential activities of VEGF 121 vs. VEGFi 65 in various endothelial cell assays, and since only VEGF ⁇ 65 is capable of binding to NP-1, we postulate that the ability of VEGF ! 65 to signal through NP-1 in the presence of VEGFR- 2 may be responsible for the increased potency of VEGF 165 vs. VEGF 121 in endothelial cells co- expressing VEGFR-2 and NP-1. In order for this to be true, it is necessary to postulate the existence of a unique VEGFR-2 + NP-1 co-receptor complex to which VEGF 165 has access, but has limited availability to bind VEGF 121 .
  • VEGFR-2 + NP-1 Using a panel of polyclonal antibodies that specifically recognize either VEGFR-2 or NP-1, we demonstrate that such a receptor complex does indeed exist in the HUVEC cells in which VEGF 165 is more potent at stimulating activation of VEGFR-2 than is VEGF 12 _. Using a heterologous expression system, we further demonstrate that this VEGFR-2 + NP-1 complex has the potential to form in the absence of VEGF ligand, and that once formed, the complex binds VEGF ⁇ 65 but has a reduced ability to bind VEGF 12 ⁇ .
  • VEGFR-2 + NP-1 complex appears to be responsible for the enhanced activity of VEGF ⁇ 65 relative to VEGF 12 ⁇ .
  • agents that bind to the VEGFR-2 + NP-1 complex or stabilize the pre-existing VEGFR-2 + NP-1 complex have the potential to be superior angiogenic agents, since signaling through VEGFR-2 is enhanced in the presence of the NP-1 co-receptor.
  • agents that antagonize binding to the VEGFR-2 + NP-1 complex or agents which antagonize formation of the VEGFR-2 + NP-1 complex have the potential to be superior anti-angiogenic agents relative to agents that disrupt binding solely to VEGFR-2.
  • the present invention relates to a method for determining whether a compound is capable of binding to a receptor protein complex comprising Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Neuroplin-1 (NP-1), which receptor protein complex is hereafter referred to as "VEGFR-2 + NP-1 protein complex” or "complex", the method comprising introducing a sample comprising the compound to the VEGFR-2 + NP-1 protein complex and allowing the compound to bind to the complex.
  • This method may utilize the full length proteins, the soluble form of either protein or a combination of full length and soluble proteins.
  • the invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the VEGFR-2 protein and an expression vector comprising a DNA sequence that codes for the NP-1 protein.
  • the invention further relates to a method for determining whether a test compound produces a signal upon binding to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to a composition containing a test compound and (ii) a second set of the cells to a composition lacking the test compound; (c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b); and (d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells.
  • the invention further relates to a method for determining whether a test compound blocks a signal produced by binding of VEGF ⁇ 65 (or another heparin-binding or NP-1 binding VEGF family member) to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to VEGF ⁇ 65 (or another heparin-binding VEGF family member) and a composition comprising a test compound and (ii) a second set of the cells to VEGF 165 (or another heparin-binding VEGF family member) and the composition without the test compound; (c) quantitative
  • VEGFR-2 receptor protein and a recombinant NP-1 receptor protein
  • Figure 1 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of the VEGFR-2 probe used for hybridization screening of a placenta gtlO ⁇ cDNA library to obtain a full-length VEGFR-2 cDNA.
  • the nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G respectively.
  • the primers are derived from the sequence of the human VEGFR-2 receptor (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)).
  • Figure 2 shows the construct pJFE.HFLKl, used for transient mammalian expression of human VEGFR-2.
  • SR-alpha promoter/enhancer
  • ApR ampicillin resistance marker
  • hFLKl human VEGFR-2 gene
  • BstXI restriction site
  • Xbal restriction site.
  • Figure 3 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of PGneuropilin-1.
  • the gene was isolated in two gene fragments which were ligated together forming the full-length human Neuropilin-1 coding sequence.
  • the nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G, respectively.
  • the primers are derived from the sequence of the human Neuropilin-1 receptor (Soker, S., Takashima, S., Miao, H.Q., Neufeld, G., and Klagsbrun, M., Cell, 92: 735-745 (1998)).
  • Figure 4 shows the construct PGNP-l/pJFE14, used for transient mammalian expression of human Neuropilin-1.
  • SR-alpha promoter/enhancer
  • ApR ampicillin resistance marker
  • Neuropilin-1 human gene
  • EcoRI restriction site
  • Xbal restriction site
  • BstXI restriction site.
  • Figure 5 shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or Mock vector, and crosslinked to 261 pM [ I25 I]-VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes of these two receptors crosslinked to [ 125 I]-VEGFi 65 .
  • VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. From left to right: VEGFR-2 immunoprecipitates of COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock), followed by Neuropilin-1 immunoprecipitates in COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock).
  • Figure 6A shows an immunoprecipitation of VEGFR-2 in HUVEC cells.
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 or Neuropilin- 1 receptors crosslinked to 379 pM [ 125 I]-VEGF
  • unlabeled ligand was used to demonstrate the specificity of the immunoprecipitated bands. From left to right: VEGFR-2 immunoprecipitates with no competitor, 30 nM unlabeled VEGF 165 , and 100 nM VEGF 12. .
  • Figure 6B shows an immunoprecipitation of Neuropilin-1 in HUVEC cells. Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of the Neuropilin-1 receptor crosslinked to 379 pM [ 125 I]-VEGF ⁇ 65 . From left to right: Neuropilin-1 immunoprecipitates with no competitor, 30 nM unlabeled VEGF 165 , and 100 nM VEGF 121 .
  • FIG. 7A shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C).
  • Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the VEGFR-2 in the cell ly sates.
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1 , Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 or Neuropilin-1 in concert with VEGFR-2 are also present to demonstrate the ability to detect VEGFR-2 in the cell lysates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed.
  • the final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing NP1 or VEGFR-2 in concert with Neuropilin-1 respectively.
  • Figure 7C shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates.
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or empty vector (Mock); immunoprecipitated using the VEGFR-2 antibody (R2.2C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing empty vector (Mock) or VEGFR-2 respectively.
  • Figure 7D shows an immunoprecipitation/Western blot.
  • COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 only and VEGFR-2 in concert with Neuropilin-1, are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates.
  • NP1ECD4C Neuropilin-1 antibody
  • NP1ECD1A Neuropilin-1 antibody
  • Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, or empty vector (Mock); immunoprecipitated using the Neuropilin-1 antibody (NP1ECD4C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 ⁇ g of total cell lysate from COS-1 cells expressing Neuropilin-1 or VEGFR-2 in concert with Neuropilin-1, respectively.
  • Figure 8A shows a whole cell binding competition of 287 pM [125 vEGF ⁇ 65 with either
  • VEGF, 2 i (-•-) or VEGF, 65 (- ⁇ -) in COS-1 cells overexpressing VEGFR-2 The Y axis is in total DPM, the X axis is in log units of the molar concentration of competing unlabeled ligand used.
  • IC 50 3.19 x 10 " " M vs. 6.85 x 10 " " M for VEGF, 2 ⁇ and VEGF, 65 , repectively).
  • Figure 8B shows whole cell binding competition of 320 pM [12 vEGF ⁇ 65 with either VEGF, 2 i (-•-) or VEGF I65 (- ⁇ -) in HUVEC cells.
  • VEGF 121 and VEGF )65 to compete for [125i]VEGF
  • Figure 9 A shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 and crosslinked to 420 pM [ 125 I]VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 crosslinked to [ l25 I]-VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. In this experiment binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGF 165 , or 100 nM VEGF 121 .
  • Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGF ⁇ 65 , or 100 nM VEGF, 2 ..
  • Figure 9B shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 in concert with Neuropilin-1, and crosslinked to 420 pM [ 125 I]VEGFi 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes involving these two receptors, crosslinked to [ 125 I]VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immuno-precipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody.
  • binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF ⁇ 65 , or 100 nM VEGF .
  • Neuropilin- 1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF, 65 , or 100 nM VEGF .
  • Figure 9C shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing Neuropilin-1, and crosslinked to 420 pM [ 125 I]VEGF ⁇ 65 .
  • Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, or complexes involving this receptor, crosslinked to [ 125 I]VEGF ⁇ 65 .
  • the VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody.
  • binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands.
  • VEGFR-2 from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF
  • Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF, 65 , or 100 nM VEGF, 2 ⁇ .
  • Figure 10A shows an anti-phosphotyrosine Western blot performed in HUVEC cells.
  • the upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower panel shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization.
  • VEGF 165 From left to right, the lanes are treated with: no VEGF 165 , 0.1 pM VEGF ⁇ 65 , 1 pM VEGF ⁇ 65 , 3 pM VEGF 165 , 10 pM VEGF 165 , 30 pM VEGF 165 , 100 pM VEGF 165 , 300 pM VEGF ⁇ 65 , 1,000 pM VEGF 165 , or 10,000 pM VEGF i65 .
  • Only the most mature form of VEGFR-2 top band in lower panel
  • Figure 10B shows an anti-phosphotyrosine Western blot performed in HUVEC cells.
  • the upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization.
  • the lanes are treated with: no VEGFm, 100 pM VEGF 12 ⁇ , 300 pM VEGF , 1,000 pM VEGFm, 3,000 pM VEGF 121 , 10,000 pM VEGF 12 ⁇ , 30,000 pM VEGFm, 100,000 pM VEGFm, 300,000 pM VEGF 121 , or 1,000,000 pM VEGF, 21 .
  • Only the most mature form of VEGFR-2 (top band in lower panel) is phosphorylated on tyrosine in response to ligand.
  • ATCC American Type Culture Collection, Rockville, Maryland.
  • biologically active means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of VEGF ⁇ 65 or another heparin-binding VEGF or NP- 1 -binding family member, or transmitting a VEGF ⁇ 65 stimulus to a cell, e.g., as a component of a hybrid receptor construct.
  • a biologically active VEGFR-2 + NP-1 receptor protein complex within the scope of the present invention means the receptor protein complex is capable of binding and can be immunoprecipitated with antibodies generated against either VEGFR-2 or NP-1.
  • host cell means a cell comprising a recombinant expression vector described herein. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector.
  • the host cells include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf-9 and Sf-21, and permanent mammalian cell lines such as Chinese hamster ovary (CHO), SV40-transformed African green monkey kidney cells (COS), Balb/c3T3 A31 cells, or any other cell line known to those skilled in the art.
  • prokaryotic cells such as Escherichia coli
  • fungal systems such as Saccharomyces cerevisiae
  • permanent cell lines derived from insects such as Sf-9 and Sf-21
  • permanent mammalian cell lines such as Chinese hamster ovary (CHO),
  • isolated in reference to the receptor protein of the present invention or DNA sequences encoding said protein, means that the protein or DNA sequence is removed from the complex cellular milieu in which it naturally occurs, and said protein is expressible from said DNA sequence in a cell that does not naturally express it when operably linked to the appropriate regulatory sequences.
  • NP-1 means a protein having the amino acid sequence SEQ ID NO. 4, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 4 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGF ⁇ 65 and P1GF-2), as well as various semaphorin family members, or crossreacting with antibodies raised against NP-1 protein, or peptides derived from the protein sequence of NP-1 protein.
  • VEGF family member including, but not limited to VEGF ⁇ 65 and P1GF-2
  • semaphorin family members include antibodies raised against NP-1 protein, or peptides derived from the protein sequence of NP-1 protein.
  • the term NP-1 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with VEGFR-2 have been deleted or modified.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence.
  • DNA for a signal peptide secretory leader
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame.
  • recombinant expression vector refers to a DNA construct used to express DNA which encodes a desired protein (for example, VEGFR-2 or NP-1) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences.
  • recombinant expression vectors of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 (as described in World Patent Publication No. 96/14579, published by J.
  • soluble receptor refers to an amino acid sequence corresponding to the extracellular region of VEGFR-2, or a portion thereof, which is capable of binding VEGF ]65 or another VEGF family member (including, but not limited to, VEGF 165 , VEGFm, VEGF 206 , VEGF 189 , VEGF 145 , VEGF-C, VEGF-D and the viral VEGF ligands).
  • Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with NP-1 have been deleted or modified.
  • Examples of such soluble receptors for VEGFR-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:6 (i.e, amino acid residues 1-760 depicted in SEQ ID NO. 2), especially those encoding amino acid number 124-320 corresponding to the Ig domains 2 and 3 of human VEGFR-2; or polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 5 (i.e., nucleic acid residues 71-2350 depicted in SEQ ID NO. 1), especially base pairs 442-1030 corresponding to the Ig domains 2 and 3 of human VEGFR-2.
  • polypeptides having the amino acid sequences substantially similar to SEQ ID NO:6 i.e, amino acid residues 1-760 depicted in SEQ ID NO. 2
  • polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 5 i.e., nucleic acid residues 71-2350 depicted in SEQ ID NO
  • soluble receptor refers to an amino acid sequence corresponding to the extracellular region of NP-1, or a portion thereof, which is capable of binding VEGF 165 or other heparin-binding VEGF family members.
  • Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with VEGFR-2 have been deleted or modified. Examples of such soluble receptors for NP-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO. 8 (i.e., amino acid residues 1-856 depicted in SEQ ID NO.
  • nucleic acid residues substantially similar to SEQ ID NO. 7 i.e., nucleic acid residues 1-2568 depicted in SEQ ID NO. 3
  • substantially similar when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a sequence altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the protein.
  • nucleic acid sequences and analogs are "substantially similar” to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence.
  • substantially similar means a receptor protein that will react with antibodies generated against the protein or peptides derived from the protein sequence.
  • VEGFR-2 means a protein having the amino acid sequence SEQ ID NO. 2, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 2 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGF 165 , VEGFm, VEGF 206 , VEGF 189 , VEGF ⁇ 45 , VEGF-C, VEGF- D and the viral VEGF ligands), or transducing a biological signal initiated by the VEGF family member binding to a cell expressing VEGFR-2, or crossreacting with antibodies raised against VEGFR-2 protein, or peptides derived from the protein sequence of VEGFR-2 protein.
  • VEGFR-2 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with NP-1 have been deleted or modified.
  • VEGFR-2 and NP-1 receptor proteins For purposes of illustrating the methods and expression systems of the present invention, the following non-limiting examples are discussed in detail. While these examples describe the use of human VEGFR-2 and human NP-1 receptor proteins, the skilled artisan will recognize that sequences from other species are readily obtainable and may be used in place of either or both of the human receptor proteins. Examples of such known sequences include, but are not limited: VEGFR-2 from mouse (GENBANK #X70842), rat (GENBANK #U93306) and quail (GENBANK #X83288); NP-1 from mouse (GENBANK #D50086), rat (GENBANK #AF010296) and chicken (GENBANK #D45416). The skilled artisan will also recognize that VEGFR-2 and NP-1 receptor proteins from other species are obtainable using well known methods. The following abbreviations are used in the Examples:
  • VEGF vascular endothelial growth factor
  • VEGFR-2 VEGF Receptor-2
  • FLK-1 Fetal Liver Kinase- 1
  • KDR Kinase Domain Receptor
  • VEGFR-1 VEGF Receptor 1 FLT-1: Ems-like Tyrosine Kinase- 1
  • BSA Bovine Serum Albumin
  • DPM Disintegrations Per Minute
  • ⁇ CL Enhanced Chemiluminescence HUVEC: Human Umbilical
  • NP-1 Neuropilin-1
  • NP-2 Neuropilin-2
  • PBS Phosphate Buffered Saline
  • TBS Tris Buffered Saline
  • DTT Dithiothreitol DSG: Disuccinimidyl Glutarate P1GF: Placental Growth Factor
  • primers shown in Figure 1 are designed from the GENBANK Accession #X61656 (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)) to generate a hybridization probe by PCR corresponding to the first 347 nucleotides.
  • PCR is performed in a 100 ⁇ l reaction using 1 ⁇ l of human placenta cDNA library (see below) as template, 0.25 ⁇ M primers, 25 ⁇ M dNTPs (dTTP, dGTP, and dATP) (Perkin Elmer Cetus, Foster City, CA), 100 ⁇ Ci P-32 dCTP (Cat# BLU513, Dupont-NEN, Boston, MA), IX polymerase buffer, and 5 U polymerase (TaKaRa Shuzo Panver, Kyoto, Japan).
  • the temperature cycle is carried out as follows for 12 cycles: melting, 95°C for 30 sec; annealing, 50°C for 30 sec; extension, 70°C for 30 sec. After the 12 th cycle, the reaction is held at 70°C for an additional 30 seconds to complete extension.
  • the probe was purified by a Nick Column (Cat# 17-0855-01, Pharmacia-Biotech, Upsala, Sweden) according to the manufacturer's instructions. The probe generated is used to screen a gtlO ⁇ cDNA library (Cat.#HL5014A, Clontech, Palo Alto, CA) and obtain a human VEGFR-2 cDNA.
  • the placenta cDNA library is screened according to the Clontech Lambda Library Protocol Handbook (Clontech, Palo Alto, CA).
  • the primary screen is performed on Optitran membranes (Cat# 68350, Schleicher & Schuell, Keene, NH) at a density of 2.5 x 10 5 plaques/filter.
  • the secondary screen is performed on Optitran membranes (Cat#68320, Schleicher & Schuell, Keene, NH) at a density of 1.0 x 10 4 or 1.0 x 10 5 plaques/filter.
  • Each round of hybridization screening is carried out overnight at 65°C in Phosphate Buffer (0.25M Na2P04, 7% sodium dodecyl sulfate, 1% BSA, 1 mM EDTA, 40 ⁇ g/ml ssDNA) using 1 x 10 6 dpm/ml of radio-labeled probe.
  • the membranes are washed in 2X SSC, 6 times for 10 minutes at room temperature, followed by a 65°C wash in 2X SSC for 15 minutes.
  • LambaFlkl#12 is digested with the restriction enzymes BamHI and Fspl (Stratagene, La Jolla, CA) and subcloned into a pBluescript KS vector (Stratagene, La Jolla, CA) previously digested with BamHI and EcorV (Stratagene, La Jolla, CA).
  • the resulting construct is digested with Hindlll (Stratagene, La Jolla, CA) to excise LambaFlkl#12 and then treated with pfu DNA polymerase (Cat# 600140 Stratagene, La Jolla, CA) according to the manufacturer's instructions, the 2.4kb gene fragment is then digested with BamHI and recovered by gel purification. Additionally, LambdaFlkl#l is digested with Xbal and BamHI (Stratagene, La Jolla, CA ) and the resulting 2.3 kb fragment is recovered by gel purification.
  • the two gene fragments are then ligated into pJFE14 mammalian expression vector previously digested with Xbal and Smal (Stratagene, La Jolla, CA).
  • the pJFE14 expression vector is derived from the pCDL-SR ⁇ 296 vector described by Takebe et al. (Y. Takebe et al., MCB 8: 466-472 (1988)).
  • the resulting mammalian construct containing VEGFR-2 is designated pJFE.HFLKl ( Figure 2).
  • the coding region for hVEGFR-2 is identical to the GENBANK sequence X61656.
  • the VEGFR-2 sequences contained within the pJFE14 expression vector contains 4226 nucleotides encompassing the entire coding region plus 86 nucleotides of 3' untranslated DNA sequence (SEQ ID NO. 1).
  • the cDNA clone described herein differs from GENBANK X61656 as follows: 70 nucleotides of 5' untranslated sequence; and 6 additional nucleotides at the 5' end coding for the first two amino acids (Met, Gin) of human VEGFR-2, as described by GENBANK AF035121 (L.Y. Lin et al., unpublished).
  • primers shown in Figure 3 are designed from the AFO 16050 GENBANK sequence, incorporating the indicated restriction sites utilized for subcloning (described below).
  • the template cDNA is generated from human heart polyA+ RNA (Cat.# 6533-1, Clontech, Palo Alto, CA) by reverse transcription.
  • the reverse transcription carried out in a 20 ⁇ l volume, contains approximately 1 ⁇ g-500 ng human heart polyA+ RNA, 10 ⁇ M random hexamers (Perkin Elmer Cetus, Foster City, CA), 1 mM dNTPs, 0.02 mM DTT, 10 U RNase inhibitors (Boehringer Mannheim Biochemical, Indianapolis, IN), 400 U Superscript II MMLV Reverse transcriptase (Gibco-BRL, Grand Island, NY) at 37°C for 1 hour. The reverse transcriptase is heat killed at 72°C for 10 min and each PCR is performed in a 100 ⁇ l volume.
  • Primers BJL-265 and BJL-238R are used to generate the 910 bp 5' region of NP- 1 while primers BJL-259 and BJL-258 are used to generate the 1862 bp 3' region of NP-1.
  • the temperature cycle is carried out as follows for 35 cycles: melting, 95°C for 1 min; annealing, 53°C for 1 min; extension, 72°C for 3 min. After the 35 th cycle, the reaction is held at 72°C min for an additional 10 minutes to complete extension.
  • the respective PCR reactions are purified using Strataprep PCR purification columns (Stratagene, La Jolla, CA) according to the manufacturer's instructions, then eluted with 50 ⁇ l water.
  • the 910 bp and 1862 bp PCR products are subcloned, respectively, into the PCR-ScriptTM Amp vector (Stratagene, La Jolla, CA) using the PCR-ScriptTM Amp Electroporation-Competent Cell Cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Positive clones are identified by DNA sequencing analysis.
  • NP-1 coding sequence is constructed and subcloned into the pJFE14 mammalian expression vector (discussed above).
  • the NP-1 cDNA fragments are excised from the pCRScript vectors by restriction digestion.
  • the plasmid construct for the 5' region of NP-1 is digested with Xbal and Pstl restriction enzymes (Boehringer Mannheim Biochemical, Indianapolis, IN) while the plasmid construct containing the 1862 bp 3' region of NP-1 is digested with Pstl and EcoRI.
  • QIAEX II Qiagen, Chatsworth, CA; a kit for gel purification of DNA fragments, including activated silica spheres and buffers
  • the resulting DNA is recovered by centrifugation at 14,000xg for 20 min, rinsed briefly with 75% cold ethanol to remove salts, and air-dried 20 min.
  • the DNA is resuspended in 10 ⁇ l water then 5 ⁇ l is transformed into XL-1 Blue E.coli (Stratagene, La Jolla, CA) by electroporation using the GenePulserTM (Bio-Rad, Hercules, CA).
  • the resulting cDNA from this clone is designated PGNP-l/pJFE14 ( Figure 4).
  • DNA sequence analysis is performed on the PGNP-1 cDNA (SEQ ID NO. 3). The DNA sequence is identical to the GENBANK NP-1 sequence AF016050 (S.
  • VEGFR-2 and Neuropilin- 1 Transient expression of the receptors in mammalian cells for the binding and immunoprecipitation studies (Examples 3-5), is carried out in COS-1 cells (ATCC CRL 1650) using Lipofectamine 2000 (LF 2000, Gibco-BRL, Grand Island, NY) and the expression plasmids described above (Example 1).
  • COS-1 cells are grown to approximately 70%-90% confluency in DME high glucose media (Gibco-BRL) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in T-175 flasks (Corning, San Diego, CA).
  • Solution A contains 625 ⁇ l of OptiMEM (Gibco-BRL) and the cDNAs of interest: for VEGFR-2, 5 ⁇ g of pJFE.HFLK-1, for Neuropilin-1, 1.5 ⁇ g of PGNP-1 PJFE14, pre-incubated at room temperature for 10 minutes.
  • Solution B contains 37.5 ⁇ l of LF2000 reagent in 625 ⁇ l OptiMEM.
  • the empty expression vector pJFE14 is substituted for the corresponding receptor cDNA, such that all transfections contain the same amount of total cDNA.
  • the transfected cells are split into 12 well plates 24 hr post transfection for whole cell binding (Example 5) or 100 mm plates for immunoprecipitation/Western Blot (Examples 4 and 6), affinity labeling/immunoprecipitation (Examples 3 and 5), and Western blot experiments (Examples 4 and 6). The cells are suitable for binding analysis 36 to 72 hours after transfection.
  • VEGF 165 Recombinant human VEGF 165 is purchased from R&D Systems, Inc. (Minneapolis, MN) [ 125 I]VEGFi65 is prepared using Chloromine T (Sigma, St. Louis MO), using a modification of methods previously described (K. Pajusola et al., Oncogene, 9: 3545-3555 (1994), B. A. Keyt et al., The Journal of Biological Chemistry 271(13): 7778-7795 (1996)). Lyophilized VEGF, 65 (5 ⁇ g) is taken up in 90 ⁇ l of DPBS (Gibco-BRL) which is then divided equally into two 1.7 ml pre- lubricated conical tubes (VWR, St. Louis MO).
  • DPBS Gibco-BRL
  • the mixture is applied to a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) previously equilibrated in PBS containing 0.5% BSA (Sigma, St. Louis MO) and 0.01% Tween 20 (Sigma, St. Louis MO).
  • the resulting labeled material is >95% precipitable by trichloroacetic acid, indicating that all of the isolated [ 125 I] is protein associated, and has a typical specific activity of 4000 to 12000 Ci/mmol.
  • the Neuropilin-1 receptor has previously been demonstrated to bind VEGF ⁇ 65 (S. Soker, et al., Cell 92(6):735-745, (1998)).
  • COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated at a density of 3 x 10 6 cells/dish into 100 mm dishes (Corning, San Diego, CA), as described in Examples 1-2.
  • the receptors are crosslinked to [ 125 I]VEGFi 65 , then subjected to immunoprecipitation with antibodies specific for either receptor using a modification of the method previously described (B. B.
  • the cells are washed three times at 4°C with 5 ml of binding buffer having the same composition as described above, except that no BSA, heparin, or protease inhibitors are added.
  • To each plate is then added 4 ml of fresh BSA-free binding buffer, followed by freshly prepared DSG (Pierce, Rockford, IL) to a final concentration of 186 ⁇ M. After swirling gently to mix the DSG, the plates are incubated for exactly 15 minutes at 4°C with gentle shaking. The media is then aspirated and the cells washed with 15 ml ice cold PBS (Gibco-BRL).
  • the supernatants are transferred to fresh 1.5 ml conical tubes to which 10 ⁇ g of the antibody of choice (see below), 10 ⁇ l of 10% SDS (Gibco-BRL), and 100 ⁇ l of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added.
  • the samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 ⁇ l of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS- PAGE) electrophoresis.
  • the gels are treated to prevent cracking in gel drying buffer (10% methanol, 40% acetic acid, 50% H 2 O) for 15 minutes, and subsequently dried. Radiolabeled bands that were immunoprecipitated by the antibody of choice are visualized and quantitated on a Storm System (Molecular Dynamics, Sunnyvale, CA) .
  • VEGFR-2 rabbit polyclonal antibody used to precipitate VEGFR-2 is produced by Quality Control Biochemicals, Inc. (Hopkinton, MA).
  • the R2.2C antibody is raised against the peptide sequence Ac-SKRKSRPVSVKTFEDIPLEEPC-amide found in the carboxy- terminus of human VEGFR-2 (identical to AA# 1225-1245, SWISSPROT # P35968, except that a C-terminal Cys is added for conjugation). This sequence is conserved in human, mouse, and rat, and this antibody has demonstrated reactivity with human, mouse, rat, canine and bovine KDR/VEGFR-2/FLK-l (data not shown).
  • R2.2C does not cross-react with the homologous VEGFR-1 receptor protein (data not shown), nor does it cross-react with Neuropilin-1 ( Figure 5).
  • the Neuropilin-1 polyclonal antibody (NP1ECD4C) used to precipitate Neuropilin-1 is also produced by Quality Control Biochemicals (Hopkinton, MA).
  • the NP1ECD4C antibody is raised against the peptide sequence AcDLDKKNPEIKIDETGST-C-amide in the extracellular juxtamembrane region of human Neuropilin-1 (identical to AA# 814-830, GENBANK . # AF016050, AF018956 (S.
  • NP-1 antibody is the anti-neuropilin- 1 polyclonal antibody C-19 from Santa Cruz Biotechnology, Inc., Santa Cruz, California (cat. No. sc-7239).
  • NP1ECD4C precipitates a band of the predicted size (-148 kDa) for the VEGF 165 :Neuropilin-l complex (S. Soker et al., Cell (1998), 92: 735-745) as well as a higher molecular weight band that may represent a Neuropilin-1 homodimeric complex.
  • the R2.2C antibody does not immunoprecipitate Neuropilin-1 in these cells, indicating that it does not cross-react with Neuropilin-1.
  • the R2.2C antibody precipitates an intense band the correct size for VEGFR-2 (-230 kDa) as well as a smaller band that corresponds to the molecular weight anticipated for VEGF bound to Neuropilin-1 (-148 kDa). Because the R2.2C antibody does not cross-react with Neuropilin-1, this indicates that VEGFR-2 and Neuropilin-1 are forming a complex in the presence of VEGF ⁇ 65 .
  • the Neuropilin-1 antibody NP1ECD4C precipitates the same bands observed in the COS cells overexpressing only NP-1.
  • the NP1ECD4C antibody does not precipitate a band the size of VEGFR-2 (-230 kDa) which might suggest that Neuropilin-1 is expressed to a much greater extent then is VEGFR-2 at the ratios of cDNA used for this experiment; such that the majority of Neuropilin- 1 that is immunoprecipitated is not in a complex with VEGFR-2.
  • the ability of the R2.2C antibody to co-immunoprecipitate Neuropilin-1 indicates that the majority of VEGFR-2 exists in a complex with Neuropilin-1 in the presence of VEGF ⁇ 65 ligand, under the conditions of this experiment.
  • the NP1ECD4C antibody is unable to co-immunoprecipitate VEGFR-2 in the HUVEC cells; indicating that the majority of NP-1 may not exist in complex with VEGFR-2 in these endothelial cells. Nevertheless, the ability of an antibody that is specific for VEGFR-2 (R2.2C) to co-immunoprecipitate NP-1 in the presence of VEGF ⁇ 65 ligand suggests that the majority of the VEGFR-2 that is present in HUVECs exists in a complex with NP-1 upon ligand addition.
  • VEGF 12 ⁇ is unable to compete for [ ,25 I]VEGF ⁇ 65 binding at either VEGFR-2 or Neuropilin-1 (Figs. 6A and 6B). Since VEGFm exhibits equal affinity at VEGFR-2 as does VEGF 165 (B.A. Keyt et al., J. Biol. Chem. (1996), 271: 7788-7795), these data suggest that VEGF 121 has only limited access to VEGFR-2 when it is in a complex with NP-1 in these endothelial cells.
  • COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated in 100 mM dishes as described in Example 2. 48-68 hours after transfection, the cells are subjected to the binding and affinity labeling protocol described in Example 3, except that [ 125 I]VEGF
  • the supernatants are transferred to fresh 1.5 ml conical tubes to which 10 ⁇ g of the antibody of choice (see below), and 100 ⁇ l of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added.
  • the samples are immunoprecipitated at 4°C with neuration overnight, and subjected to SDS-PAGE as described in Example 3.
  • cell lysates are prepared and analyzed by SDS-PAGE. In this procedure, the cells are seeded 24 hours post transfection at 3 x 10 6 cells into 100 mm plates.
  • the cells are scraped on ice using a standard cell scraper and transferred to 1.5 ml conical tubes where the cells are collected by centrifugation (13,000xg, 5 min., 4°C). After centrifugation the cells are re-suspended in 80 ⁇ l of RIPA buffer (described above) per plate, and run through a 23 gauge needle to help solubilize the lysates.
  • the lysates are vortexed for 30 minutes at 4°C prior to another centrifugation (13,000xg, 5 min., 4°C).
  • the cleared supernatants are then transferred to fresh tubes where the total protein concentration is determined using the Pierce BCA assay system (Pierce, Rockford, IL).
  • Six micrograms of total protein is then diluted to 20 ⁇ l with water and 5X loading dye (4 ⁇ l, Five Prime Three Prime, Boulder, CO, Cat # 2- 910675).
  • lysates or immunoprecipitated protein is prepared (described above), 20 ⁇ l of the supernatants are loaded onto 8% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis. The gels are then transferred to PDVF membranes (Owl Scientific, Woburn, MA) using standard Western blot transfer techniques. Post transfer the membranes are blocked using Western blocking buffer (5% BSA (Cat# 05479, Pharmacia, Piscataway, NJ), 0.1% Tween 20, TBS (Sigma, St. Louis, MO)) for 30 minutes.
  • Western blocking buffer 5% BSA (Cat# 05479, Pharmacia, Piscataway, NJ), 0.1% Tween 20, TBS (Sigma, St. Louis, MO)
  • the membranes are then washed three times for 5 minutes (recovering and storing the blocking buffer used for re- blocking the following day) in TBST-0.1% (0.1% Tween 20, TBS) and placed at 4°C overnight without agitation. The following morning the membranes are re-blocked for 2 hours in the same blocking buffer (saved from the previous night). Post blocking, the membranes are probed with the R2.2C antibody (1 :5,000, described in Example 3) for detection of VEGFR-2 or NP1ECD1A antibody for detection of NP-1 at (1 :1,000; see below) in primary probe buffer (Western blocking buffer diluted 1:1 with TBST-0.1%) for two hours at room temperature with agitation.
  • the NP1ECD1A antibody is created using the same methodology as described for NP1ECD4C in Example 3, but is generated against the peptide sequence Ac-TEKPTVIDSTIQSEFPTC-amide (identical to AA# 629-645, GENBANK # AF016050, AF018956 (S. Soker et al., Cell (1998), 92: 735-745, Z. He and M. Tessier-Lavigne, Cell (1997), 90: 739-757, except that a C-terminal Cys is added for conjugation) located in the B2-MAM domain linker region of NP-1.
  • the membranes are then washed five times for six minutes in TBST-0.1% at room temperature with agitation prior to the addition of the HRP-labeled Goat anti-Rabbit secondary antibody at 1 :40,000 (Pierce, Rockford, IL).
  • the secondary antibody is incubated on the membranes for one hour.
  • Post secondary incubation the membranes are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS.
  • the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions prior to exposure to Hyperfim (Amersham, Piscataway, NJ).
  • Figure 7 demonstrates the R2.2C antibody clearly immunoprecipitates and detects a band of the correct size for VEGFR-2 (-230 kDa) in COS-1 cells either expressing VEGFR-2 alone or in concert with Neuropilin- 1 , as well as detecting a low level expression of VEGFR-2 in empty vector- (mock-) or NP-1 -transfected COS-1 cells (Figure 7A).
  • Figure 7B demonstrates the ability of VEGFR-2 (-230 kDa Band) to be precipitated by the NP1ECD4C antibody, but only when both VEGFR-2 and Neuropilin-1 are co-expressed.
  • Figures 7C and 7D The reciprocal immunoprecipitation is illustrated in Figures 7C and 7D.
  • Figure 7C demonstrates that the R2.2 antibody can only precipitate Neuropilin-1 (-130 kDa band) when VEGFR-2 and Neuropilin-1 are co-expressed in COS-1 cells.
  • the lack of cross-reactivity of the VEGFR-2 antibody with NP-1 is illustrated by the failure of the R2.2C antibody to immunoprecipitate NP-1 in cells only expressing NP-1 ( Figure 7C), further illustrating that the NP-1 that is immunoprecipitated by the R2.2C antibody in cells co-expressing VEGFR-2 + NP-1 must be in a complex with VEGFR-2.
  • NP-1 expression is demonstrated both in cells expressing NP-1 alone, and in cells co- expressing VEGFR-2 + NP-1 ( Figure 7D).
  • the NP1ECD1A Western antibody appears to preferentially recognize Neuropilin-1 when it is co-expressed with VEGFR-2 vs. when it is expressed alone ( Figure 7D). This conclusion is supported by the data in cell lysates where the NP1ECD1A antibody preferentially recognizes the Neuropilin-1 in the VEGFR-2 + Neuropilin-1 co-transfected cell lysate vs. lysate from cells only overexpressing Neuropilin-1 ( Figure 7D).
  • Neuropilin-1 may be more easily detected by this antibody when Neuropilin-1 is in a complex with VEGFR-2, and further supports the conclusion of the existence of the VEGFR-2 + NP-1 complex in this system.
  • the preferential recognition of the complex by the NP1ECD1A antibody suggests that the B2-MAM-domain linker region epitope recognized by this antibody may undergo a conformational change when VEGFR-2 is in proximity to NP-1 that is maintained even under the SDS-PAGE conditions. This is also consistent with the proposed role of the B domains in binding of VEGF ⁇ 65 to NP-1 (R. J. Giger, et al..
  • NP-1 is expressed more efficiently when it is co-expressed with VEGFR-2, since levels of NP-1 are undetectable by the NP1EDC1A antibody in 6 ⁇ g of total protein lysates from cells solely over expressing NP-1.
  • Figure 7D Such apparent differences in NP-1 expression are not apparent when NP-1 is assessed by affinity labeling with VEGF 165 followed by immunoprecipitation by the NP1ECD4C antibody ( Figure 5), again suggesting that the Western antibody is particularly sensitive to detection of differences of NP-1 expression in the presence or absence of VEGFR-2.
  • FIG. 7 illustrates the ability of the VEGFR-2 + NP-1 complex to form in the absence of ligand. Detection of the co-receptor complex is not an artifact created by use of the crosslinking agent (see Example 3 for methods) as it is readily detectable even in the absence of crosslinker ( Figures 7A through 7D). This suggests that the VEGFR-2 + Neuropilin-1 receptor complex is ligand-independent and may exist as a pre-existing heterodimer on the cell surface under these assay conditions.
  • Example 5 Characterization of the VEGF Isoform Binding Profile of the VEGFR-2 + NP-1 Complex
  • the first is the affinity labeling/immunoprecipitation technique described in Example 3.
  • the second is referred to as whole cell binding competition analysis.
  • COS-1 cells are transfected with cDNA for VEGFR-2 or Neuropilin-1 by the methods described in Example 2, or endogenous HUVEC cells are utilized.
  • VEGF 165 is added to the binding buffer at a final concentration of 10 to 50 nM. The cells are incubated for 4 hr at 4°C with gentle shaking.
  • the buffer is aspirated, and the cells are rinsed 3 times with 1 ml BSA- free binding buffer (Example 3).
  • the solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT).
  • the binding curves are analyzed using the Prizm program (GraphPad, San Diego, CA).
  • Figure 8 demonstrates the results of the whole cell binding assay in either COS-1 cells overexpressing VEGFR-2 alone (Figure 8A) or in HUVECs ( Figure 8B).
  • VEGF, 65 and VEGFm exhibit equivalent binding affinity at VEGFR-2, as indicated by the similar IC 50 values (6.85 x 10 " 1 1 M and 3.19 x 10 "U M for VEGF 1 and VEGF 121 , respectively) in COS-1 cells overexpressing VEGFR-2.
  • VEGF 1 65 and VEGF 121 are not identical in HUVEC cells ( Figure 8B), despite the finding that HUVECs contain a substantial amount of immunoprecipitatable VEGFR-2 that is competent to both bind VEGF 165 and be activated by it ( Figures 6 and 10).
  • NP-1 the binding sites to which VEGFm do not have access may be explained as being NP-1, wherein NP-1 appears to represent the majority of the binding sites observable in HUVEC at the whole cell level.
  • NP-1 Since the expression of NP-1 appears to be in excess of that of VEGFR-2 (defining VEGFR-2 as accessible to both VEGF 165 and VEGFm), and since VEGFR-2 can form a complex with NP-1 in the absence of ligand (Example 4), the inability of VEGFm to compete for a substantial portion of the [ 125 I]VEGFi 65 binding sites at either the whole cell level ( Figure 8B), or in the affinity labeling/immunoprecipitation assay ( Figure 6), can be explained as either an inaccessibility to NP-1 alone, or to VEGFR-2 when it is in a complex with NP-1.
  • Figure 9 illustrates that, while VEGFm is able to completely compete with [ 125 I]VEGF 165 binding to VEGFR-2 when it is expressed alone in COS-1 cells (Figure 9A), it does not compete with [ I25 I]VEGF ⁇ 65 binding to NP-1 when it is expressed alone ( Figure 9C) or in combination with VEGFR-2 ( Figure 9B).
  • VEGFm only partially competes for binding at VEGFR-2 in COS cells co- expressing VEGFR-2 + NP-1 (R2.2C IP, Figure 9B) indicating that VEGFm has only limited access to VEGFR-2 when it is in a complex with NP- 1.
  • HUVEC cells BioWhittaker, Walkersville, MD
  • EGM media BioWhittaker, Walkersville, MD
  • the cells are left undisturbed.
  • the day of stimulation the growth media is removed, the cells are rinsed, and returned to the 37°C incubator in serum-free DMEM (Gibco-BRL).
  • the ligands for stimulation are pre-diluted in 1.5 ml of stimulation buffer (25 mM HEPES, Gibco-BRL, DMEM, Gibco-BRL, 0.2% BSA, Sigma, St. Louis, MO, 1 ⁇ g/ml Heparin cat# H7399, Sigma, St. Louis, MO).
  • the media is quickly removed, the stimulation buffer including the ligand is applied, and the cells are returned to the incubator. After exactly 5 minutes at 37°C the cells are placed on ice and the stimulation buffer is removed. A secondary aspiration is performed to ensure complete removal of the stimulation media.
  • the lysates are then transferred to 1.5 ml conical tubes on ice and further solubilized by 3 passes through a 23 gauge needle.
  • the lysates are then centrifuged (13,000 x g, 5 minutes, 4°C) to clear the supernatant and the supernatants are transferred to fresh 1.5 ml conical tubes.
  • Example 4 10 ⁇ g of the R2.2C antibody, and 100 ⁇ l of a 50:50 slurry of the protein G beads (described in Example 4) is then added to each sample. The samples are then placed at 4°C with neuration overnight, or for up to 60-72 hours. Following the overnight incubation the beads are pelleted at 13,000 x g for 5 minutes at 4°C and the supernatants aspirated. Three successive washes of the beads are performed using the RIPA buffer (described above) with the protease inhibitor cocktail (described in Example 3).
  • the samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 ⁇ l of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis; followed by the Western blotting procedure and blocked as described in Example 4.
  • the blocking buffer contains a form of BSA (FLUKA, obtained from Pharmacia, Piscataway, NJ, Cat # 05479), with low endogenous peroxidase activity to avoid high background with the ECL detection.
  • BSA FLUKA
  • Post blocking the membranes are probed for anti-phosphotyrosine using the 4G10 antibody (1:5,000, Upstate Biotechnology Inc., Lake Placid, NY) in primary probe buffer (Western blocking buffer diluted 1 :1 with TBST-0.1%) for two hours at room temperature with agitation.
  • the membranes Prior to the addition of the HRP-labeled Goat anti-Mouse secondary antibody at 1 :40,000 (Pierce, Rockford, IL), the membranes are washed five times for six minutes in TBST-0.1% at room temperature with agitation. The secondary antibody is incubated on the membranes for one hour, after which they are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS. Following the final wash step the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions, prior to exposure to Hyperf ⁇ m (Amersham, Piscataway, NJ).
  • Re-probing with the VEGFR-2 antibody allows the phosphotyrosine signal to be normalized to the amount of VEGFR-2 present in the immunoprecipitate for quantitation of the response. Quantitation of the signal is achieved using Image Quant software (Molecular Dynamics, Sunnyvale, CA) after scanning the film on a Scanmaster 2500 (Howtek, Hudson, NH). The results of the anti-phosphotyrosine assay are illustrated in Figure 10. In HUVEC cells the activation of VEGFR-2 autophosphorylation by VEGF ⁇ 6 s is readily apparent at a concentration of 100 pM, and is maximal at 300 pM (EC 50 -1.25 x 10 "10 M; Figure 10A).
  • VEGF ⁇ 65 and VEGFm have similar binding affinity to VEGFR-2 (Example 5 and B.A. Keyt et al, (1996), J. Biol. Chem. 271 : 7788-7795), and the response that is measured here is a direct activation of VEGFR-2, rather than a more downstream event that may be subject to post-receptor regulation.
  • VEGFR-2 we postulate that this difference in potency in the ability to activate VEGFR-2 results from an inability of VEGFm to bind to the VEGFR-2 + NP-1 complex, and that the presence of NP-1 in the VEGFR-2 complex augments signaling through VEGFR-2.
  • VEGF 16 s and VEGFm may have similar binding affinity at VEGFR-2
  • the ability of VEGF ⁇ 6 s to bind to the VEGFR-2 + NP-1 complex allows VEGF to signal more efficiently through VEGFR-2, thereby inducing receptor autophosphorylation at lower concentrations than that observed with VEGF 121 .
  • Example 7 Use of the VEGFR-2 + NP-1 Complex in a Receptor Binding Assay for the Identification of VEGF Receptor Agonists and Antagonists
  • Identification of ligands that interact with the VEGFR-2 + NP-1 complex can be achieved through the use of assays that are designed to measure the interaction of the ligands with this VEGF receptor complex.
  • a receptor binding assay that uses the VEGFR-2 + NP-1 complex and is adapted to handle large numbers of samples is carried out as follows:
  • the whole cell binding assay is carried out as described in Example 5, except that either single or increasing concentrations of test compounds are used in place of VEGFm (Example 5, Figure 8).
  • binding can be tested either at NP-1 alone, VEGFR-2 alone, or at the VEGFR-2 + NP-1 complex in COS-1 cells overexpressing these receptors, or in alternate cell lines engineered to stably overexpress VEGFR-2 + NP-1, using methods readily available to those skilled in the art.
  • the binding data is analyzed using the Prizm program (GraphPad, San Diego, CA) as described in Example 5.
  • test compounds which interact with the VEGFR-2 + NP-1 receptor complex are observed to compete for binding to the receptor complex with the [ IjVEGFi ⁇ s tracer, such that less [ I]VEGF 16 s tracer is bound in the presence of the test compound in comparison to the binding observed when
  • the binding assay can be carried out using soluble receptor proteins in an ELISA-based capture assay format, similar to that described in the scientific literature for soluble VEGFR-2 (B.A. Keyt et al, (1996), J. Biol. Chem. 271: 7788-7795; G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J. Biol. Chem. 273: (18):11197-11204) or for soluble NP-1 (L.M. Wise, et al, (1999), Proc. Nat. Acad. Sci.
  • the soluble VEGFR-2 receptor would include the extracellular domains present in SEQ ID NO. 5 and SEQ ID NO. 6, or portions thereof, especially those portions including Ig domains 2 and 3, encompassing amino acids 124 to 320, as Ig domains 2 and 3 have been determined to be necessary for high affinity VEGF )65 binding to VEGFR-2 (G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J Biol Chem 273: (18):11197-11204; A.
  • the soluble NP-1 receptor would include the extracellular domains present in SEQ ID NO. 7 and SEQ ID NO. 8, or portions thereof, especially those portions including the B domains (also known as the coagulation factor domains), encompassing amino acids 274 to 647, as the B domains have been determined to be necessary for VEGF 165 binding to NP-1 (RJ. Giger, et al, (1998), Neuron 21 : (5):1079-1092); see also the PCT publication Number WO 99/29858).
  • B domains also known as the coagulation factor domains
  • VEGF, 65 is either radiolabeled (e.g. as [ l25 I]VEGF, 65 ) or is labeled with a tracer than can then be detected by a fluorometer (e.g., europium-labeled VEGF, 6 s).
  • test compounds which interact with the VEGFR-2 + NP- 1 receptor complex are observed to compete for binding to the receptor complex with the labeled VEGF, 65 tracer, such that less VEGF, 65 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound.
  • a decrease in binding of the VEGF, 65 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the VEGFR-2 + NP-1 receptor complex.
  • VEGFR-2 + NP-1 Use of VEGFR-2 + NP-1 in a Signaling Assay for the Identification of VEGF Receptor Agonists and Antagonists
  • Identification of ligands that signal upon interaction with the VEGFR-2 + NP-1 receptor complex can be achieved through the use of assays that are designed to measure the activation of the receptor protein kinase domain after binding of the ligand to the receptor complex.
  • One assay that is used is similar to that described for measuring the relative potency of VEGF ,65 vs. VEGF ,2, alone (Example 6), except that in this case test compounds would be added in the absence of VEGF, 65 or VEGFm, at either a single concentration or at multiple concentrations.
  • a test compound that measurably increases the phosphotyrosine content of the immunoprecipitated VEGFR-2 above that observed in the absence of compound is considered an agonist of either VEGFR-2 or of the VEGFR-2 + NP-1 receptor complex.
  • a compound that is an antagonist at the VEGFR-2 + NP-1 complex can be detected using an anti-phosphotyrosine Western blot assay similar to that previously described in Example 6 ( Figure 10), with the following modifications: (1) In one assay format, increasing concentrations of either VEGF, 6 s or VEGF, 2 , are used alone, or in the presence of a single concentration of the test compound. A compound is determined to be an antagonist of the VEGFR-2 + NP- 1 complex if the concentration-response curve is shifted to the right for VEGF, 65 , but the concentration- response curve for VEGFm is not shifted substantially to the right.
  • a “substantial" shift is one that results in an increase in the EC 5 o of VEGF, 65 or VEGFm by a factor > 5.
  • antagonistic activity can be detected using a single concentration of either VEGF, 65 or VEGF 12 , (e.g., a concentration that is > EC 5 0 for increasing phosphotyrosine content of the immunoprecipitated VEGFR-2 receptor), in the presence of increasing concentrations of the test compound.
  • the response of VEGF,65 should be decreased by at least 30%, whereas a substantial decrease in the VEGFm response would not be obtained.
  • the anti-phosphotyrosine assay described above can be performed in HUVECs or any other cell line determined to express VEGFR-2 + NP-1, in which VEGF, 65 is a more potent agent than is VEGFm in signaling assays that are dependent on activation of VEGFR-2.
  • the assay can also be performed in cells engineered to overexpress VEGFR-2 + NP-1, using methodology common to those skilled in the art.
  • a stable cell line (generated from Balb/c 3T3 clone A31 ; ATCC # CCL-163) exhibiting these characteristics, named D7R2/NP1#4, has been deposited with the ATCC on September 21, 2000, and assigned ATCC Designation No. .
  • the human NP-1 gene (SEQ ID NO. 3) was incorporated into the vector pBluescript II SK+, which was deposited with the ATCC on October 20, 1999, and assigned ATCC Designation No. PTA-858.
  • the vector is commercially available from Stratagene (La Jolla, CA). The gene was sub-cloned into the EcoRI and Xbal restriction sites of the vector.
  • AAA TCT CTA ATC TCT CCT GTG GAT TCC TAC CAG TAC GGC ACC ACT CAA TTT AGA GAT TAG AGA GGA CAC CTA AGG ATG GTC ATG CCG TGG TGA GTT Lys Ser Leu He Ser Pro Val Asp Ser Tyr Gin Tyr Gly Thr Thr Gln>
  • 2690 2700 2710 2720 2730 CTG GAG AAC TAT AAC TTT GAA CTT GTG GAT GGT GTG AAG TTG
  • GCC ATT CCT CCC CCG CAT CAC ATC CAC TGG TAT TGG CAG TTG GAG GAA CGG TAA GGA GGG GGC GTA GTG TAG GTG ACC ATA ACC GTC AAC CTC CTT Ala He Pro Pro Pro His His He His Trp Tyr Trp Gin Leu Glu Glu>
  • GTC TGC CTT GCT CAA GAC AGG AAG ACC AAG AAA AGA CAT TGC GTG GTC CAG ACG GAA CGA GTT CTG TCC TTC TGG TTC TTT TCT GTA ACG CAC CAG Val Cys Leu Ala Gin Asp Arg Lys Thr Lys Lys Arg His Cys Val Val>
  • TCA TCC AAC CAA GGG GAC AGA AAC TGG ATG CCT GAA AAC ATC 1485 AGT AGG TTG GTT CCC CTG TCT TTG ACC TAC GGA CTT TTG TAG
  • SEQUENCE ID NO:8 (i) SEQUENCE CHARACTERISTICS: 1665 (A) LENGTH: 856 amino acids

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Abstract

Disclosed is a method for determining whether a compound is capable of binding to a receptor protein complex comprising Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Neuropilin-1 (NP-1), the method comprising introducing a sample comprising the compound to the VEGFR-2 + NP-1 protein complex. It is also an object of the present invention to provide a complex formed by the interaction of a recombinant VEGFR-2 receptor protein and a recombinant NP-1 receptor protein. Further disclosed are method for determining whether a test compound produces a signal upon binding to a VEGFR-2 + NP-1 protein complex (i.e. is an agonist for the complex), or blocks a signal upon VEGF165 binding to the complex (i.e. is an antagonist for the complex).

Description

THE USE OF A COMPLEX OF VEGFR-2 AND NEUROPILIN-1 FOR IDENTIFICATION OF NOVEL PRO- AND ANTI-ANGIOGENIC ACTIVES
BACKGROUND The involvement of members of the Vascular Endothelial Growth Factor (hereafter
"VEGF") family in embryonic angiogenesis and vasculogenesis is now well established (M.G. Achen, S.A. Stacker, InU.Exp.Pathol (1998), 79:(5):255-265; N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22). In addition to playing a role in the development of the embryonic vasculature through an influence on endothelial cell differentiation and organization (e.g. vasculogenesis), VEGF and its family members have also been implicated in physiological and pathological angiogenesis (e.g. the formation of new blood vessels from pre-existing vessels). The involvement of VEGFι65 in the physiological angiogenesis that occurs during the female reproductive cycle, longitudinal bone growth and endochondral bone formation, and wound healing has been documented, as has involvement in pathological (dysregulated) angiogenesis such as that which occurs in tumor growth and metastases, rheumatoid arthritis, endometriosis, psoriasis, proliferative retinopathy, and atherosclerosis (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22; M. Inoue et al., Circulation (1998), 98(20): 2108-2116)). As such, the VEGF receptor system provides an attractive target for the identification of novel compounds exhibiting both pro- and anti-angiogenic activity. Pro-angiogenic agents are considered useful for the treatment of cardiac ischemia and peripheral vascular disease (N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; T.D. Henry, Brit. Med. J. (1999), 318:(7197)1536-1539) in which new blood vessel formation needs to be stimulated, whereas anti-angiogenic agents are considered useful for the treatment of diseases in which dysregulated angiogenesis is a characteristic pathology.
The VEGF ligand family consists of VEGF and its various splice variants (discussed below), VEGF-B, VEGF-C, VEGF-D, P1GF and its splice variants (discussed below), and the viral VEGF, VEGF-E (reviewed in T. Veikkola and K. Alitalo, Semin.Cancer Biol. (1999), 9(3): 211-220). The VEGF family members exert their physiological and pharmacological effects through the interaction with three different VEGF receptor tyrosine kinases, termed VEGFR- 1/FLT-l, VEGFR-2/FLK-l/KDR, and VEGFR-3/FLT-4 (for reviews, see T. Veikkola and K. Alitalo, Semin.Cancer Biol. (1999), 9(3): 21 1-220; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22), as well as the non-kinase receptors Neuropilin-1 (hereafter "NP-1") and Neuropilin-2 ("NP-2") (Migdal et al., J Biol Chem (1998), 273(35): 22272-22278; RJ. Giger et al., Neuron (1998), 21(5): 1079-1092; T. Makinen et al., J.Biol.Chem. (1999), 274(30): 21217-21222.; H.Q. Miao et al., J.Cell Biol. (1999), 146(1): 233-241; S. Soker, S. Takashima, H.Q. Miao, G. Neufeld, and M. Klagsbrun, Cell (1998), 92(6): 735-745; and reviewed in G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9- 22). VEGFR-3/FLT-4 is the primary receptor for VEGF-C and VEGF-D and is considered to be responsible for the lymphatic angiogenic activities of these ligands, whereas the blood vessel angiogenic activities of these ligands is thought to be mediated in part through binding to VEGFR-2 (M.G. Achen et al., Proc Natl Acad Sci USA (1998), 95(2): 548-553; Y.H. Cao, et al., Proc.Natl.Acad.Sci. USA (1998), 95(24): 14389-14394; D.J. Dumont et al., Science (1998), 282(5390): 946-949; B. Enholm, L. Jussila, M. Karkkainen, K. Alitalo, Trend. Cardiovasc. Med. (1998), 8(7): 292-297; M. Jeltsch, et al., Science (1997), 276(5317): 1423-1425; V. Joukov et al., J Cell Physiol (1997), 173(2): 21 1-215; E. Kukk et al., Development (1996), 122(12): 3829-3837; J. Oh et al., Dev.Biol. (1997), 188(1): 96-109; R. Valtola et al., Amer.J.Pathol. (1999), 154(5): 1381-1390). P1GF-1, P1GF-2, and the various forms of VEGF-B do not bind to either VEGFR-2 or VEGFR-3, are specific ligands for VEGFR-1, and do not exert appreciable mitogenic or angiogenic effects on endothelial cells (for reviews see T. Veikkola and K. Alitalo, Semin. Cancer Biol. (1999), 9(3): 21 1-220; M.G. Achen, S.A. Stacker,. InU.Exp.Pathol. (1998), 79(5): 255-265; G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22); N. Ferrara, J. Molec. Med. (1999). 77(7): 527-543). For this reason, the role of VEGFR-1 in angiogenesis is incompletely understood, although transgenic and knockout experiments implicate a role for this receptor in endothelial cell and monocyte chemotaxis as well as vascular organization (S. Hiratsuka, O. Minowa, J. Kuno, T. Noda, and M. Shibuya, Proc Natl Acad Sci USA (1998), 95(16): 9349-9354; G. H. Fong, L. Y. Zhang, D. M. Bryce, and J. Peng, Development (1999), 126(13): 3015-3025; G.H. Fong, J. Rossant, M. Gertsenstein, and M.L. Breitman, Nature (1995), 376: 66-70). In contrast, the role of VEGFR-2 in vasculogenesis and angiogenesis is established, as activation of this receptor is required for both vasculogenesis (F. Shalaby, J. Rossant, T. Yamaguchi, M. Gertsenstein, X. Wu, M. Breitman, and A.C. Schuh, Nature (1995) 376: 62-66) and angiogenesis (N. Ortega et al., Am J Pathol (1997), 151(5): 1215- 1224; B. Malavaud et al., Cardiovasc Res (1997), 36(2): 276-281), as well as the pathological angiogenesis associated with tumor growth (M.R. Machein, W. Risau, K.H. Plate, Hum. Gene Ther. (1999), 10(7): 1117-1128; P.N. Lin et al., Cell Growth Differ (1998), 9(1): 49-58; B. Millauer, et al., Cancer-Res. (1996), 56: 1615-1620; B. Millauer, L. Shawver, K. Plate, W. Risau, A. Ullrich, Nature (1994), 367: 576-579; T.A. Fong et al., Cancer Res. (1999), 59(1): 99- 106). Hence, drugs that are capable of interacting with VEGFR-2 as receptor agonists have the potential to be angiogenic agents, whereas antagonists at the VEGFR-2 receptor have the potential to be anti-angiogenic agents.
VEGF exists in multiple protein isoforms with different heparin proteoglycan and extracellular matrix binding properties. These isoforms (VEGF165, VEGF121, VEGFus, VEGFι89, VEGF206) arise from alternate splicing of the VEGF gene. VEGF165 is the predominant isoform and has limited heparin-binding activity, whereas the VEGF121 isoform is freely soluble and is devoid of heparin-binding activity. Similarly, PLGF exists in three different isoforms, which also exhibit differential heparin binding ability (for reviews see G. Neufeld, T. Cohen, S. Gengrinovitch, and Z. Poltorak, FASEB J. (1999), 13(1): 9-22; T. Veikkola and K. Alitalo, Semin. Cancer Biol. (1999), 9(3): 211-220; G. Neufeld et al., Cancer Metastasis Rev (1996), 15: 153- 158; N. Ferrara, J. Molec. Med. (1999), 77(7): 527-543; and Y.H. Cao, W.D.R. Ji, P. Qi, A. Rosin, Y.M. Cao, Biochem. Biophys. Res. Commun. (1997), 235(3): 493-498; and references therein). Although VEGF165 and VEGF121 bind to VEGFR-2 with equal affinity, their activity in biochemical assays that rely on activation of VEGFR-2 is not equivalent (B.A. Keyt et al., J. Biol. Chem. (1996), 271 : 7788-7795; S. Soker, S. Gollamudi-Payne, H. Fidder, H. Charmahelli, M. Klagsbrun, J5 o/. Chem. (1997), 272(50): 31582-31588); S. Ogawa et al., J. Biol. Chem. (1998), 273(47): 31273-31282); implying that their ability to activate VEGFR-2 is not solely dependent on VEGFR-2 binding affinity. Indeed, mice engineered to express only VEGF12ι exhibit defects in cardiac vascularization and suffer from ischemic cardiomyopathy, implying that VEGF121 cannot substitute for VEGF|65 in development of the cardiac vasculature during the period of postnatal cardiac development (P. Carmeliet, et al., Nature Med. (1999), 5(5):495-502).
Recently, Neuropilin- 1 (NP- 1 ) and Neuropilin-2 (NP-2) were identified as receptors that bind VEGFi65 and PLGF-2 but do not bind either VEGF121 or the non-heparin binding form of PLGF, PLGF-1 (S. Soker, S. Takashima, H.Q. Miao, G. Neufeld, and M. Klagsbrun, Cell (1998), 92:(6): 735-745; Migdal, et al., J Biol Chem (1998), 273(35): 22272-22278; R. Tordjman, N. Ortega, L. Coulombel, j. Plouet, P. H. Romeo, and V. Lemarchandel, Blood (1999), 94(7): 2301- 2309). NP-1 and NP-2 also bind various semaphorin ligands to mediate repulsive guidance activity in certain neuronal populations (R.J. Giger, et al., Neuron (1998), 21(5): 1079-1092; T. Takahashi, F. Nakamura, Z. Jin, R.G. Kalb, S.M. Strittmatter, Nat.Neurosci. (1998), 1(6): 487- 493; F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100; D. Bagnard, M. Lohrum, D. Uzeil, A.W. Puschel, J. Bolz, Development (1998), 125(24): 5043-5053; A. Chedotal, et al., Development (1998), 125(21): 4313-4323; H. Chen, A. Chedotal, Z. He, C.S. Goodman, L.M. Tessier-Lavigne, Neuron (1997), 19547-559; A.L. Kolodkin, et al., Cell (1997), 90: 753-762; Z. He, L.M. Tessier-Lavigne, Cell (1997), 90739-751). Although the cytoplasmic domains of NP-1 and NP-2 are not required for semaphorin signaling (F. Nakamura, M. Tanaka, T. Takahashi, R.G. Kalb, S.M. Strittmatter, Neuron (1998), 21(5): 1093-1100) and they do not contain sequences predictive of enzymatic activity nor sequences predicted to be involved in coupling to intracellular signaling molecules, the NP-1 and NP-2 proteins appear to function as part of a signaling receptor complex for mediating semaphorin signals (T. Takahashi, A. Foumier, F. Nakamura, L-H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M. Strittmatter Cell (1999), 99: 59-69; L. Tamagnone, S. Artigiani, H. Chen, Z. He, G-L Ming, H-J. Song, A. Chedotal, M.L. Winberg, C.S. Goodman, M-M. Poo, M. Tessier- Lavigne, and P.M. Comoglio, Cell (1999), 99: 71-80). Potential signals that lie downstream of the NP receptors in the semaphorin signaling pathway include activation of a pertussis toxin sensitive G-protein (Y. Goshima, F. Nakamura, P. Strittmatter, S.M. Strittmatter, Nature (1995), 376: 509-514), modulation of G-protein activity via a PDZ domain-containing RGS protein (H.B. Cai, R.R. Reed, J. Neurosci. (1999), 19(15): 6519-6527; V.L. De Vries, X. Lou, G. Zhao, B. Zheng, M.G. Farquhar, Proc.Natl.Acad.Sci. U.S.A. (1998), 95: 12340-12345), and activation of racl, a small molecular weight G-protein involved in cytoskeletal rearrangement (Z. Jin, S.M. Strittmatter, J.Neurosci. (1997), 17: 6256-6263; and T. Takahashi, A. Fournier, F. Nakamura, L- H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M. Strittmatter, Cell (1999), 99: 59-69). The requirement of NP-1 and NP-2 for semaphorin signaling implies that NP-1 and NP-2 are capable of stimulating signal transduction pathways, although it appears that at least some of the semaphorin-mediated signaling capability of the neuropilins requires interaction with the Plexins as a co-receptor complex (T. Takahashi, A. Fournier, F. Nakamura, L-H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M. Strittmatter, Cell (1999), 99: 59-69; L. Tamagnone, S. Artigiani, H. Chen, Z. He, G-L Ming, H-J. Song, A. Chedotal, M.L. Winberg, C.S. Goodman, M- M. Poo, M. Tessier-Lavigne, and P.M. Comoglio, Cell (1999), 99: 71-80).
Since binding to VEGFR-2 alone does not explain the differential activities of VEGF121 vs. VEGFi65 in various endothelial cell assays, and since only VEGFι65 is capable of binding to NP-1, we postulate that the ability of VEGF! 65 to signal through NP-1 in the presence of VEGFR- 2 may be responsible for the increased potency of VEGF165 vs. VEGF121 in endothelial cells co- expressing VEGFR-2 and NP-1. In order for this to be true, it is necessary to postulate the existence of a unique VEGFR-2 + NP-1 co-receptor complex to which VEGF165 has access, but has limited availability to bind VEGF121. Using a panel of polyclonal antibodies that specifically recognize either VEGFR-2 or NP-1, we demonstrate that such a receptor complex does indeed exist in the HUVEC cells in which VEGF 165 is more potent at stimulating activation of VEGFR-2 than is VEGF12_. Using a heterologous expression system, we further demonstrate that this VEGFR-2 + NP-1 complex has the potential to form in the absence of VEGF ligand, and that once formed, the complex binds VEGFι65 but has a reduced ability to bind VEGF12ι. Since the binding affinity of VEGF165 and VEGF12ι is identical at VEGFR-2, the VEGFR-2 + NP-1 complex appears to be responsible for the enhanced activity of VEGFι65 relative to VEGF12ι. Hence, agents that bind to the VEGFR-2 + NP-1 complex or stabilize the pre-existing VEGFR-2 + NP-1 complex have the potential to be superior angiogenic agents, since signaling through VEGFR-2 is enhanced in the presence of the NP-1 co-receptor. Similarly, agents that antagonize binding to the VEGFR-2 + NP-1 complex, or agents which antagonize formation of the VEGFR-2 + NP-1 complex have the potential to be superior anti-angiogenic agents relative to agents that disrupt binding solely to VEGFR-2.
Accordingly, it is an object of the present invention to provide a method for identifying compounds capable of binding to a VEGFR-2 + NP-1 protein complex.
It is also an object of the present invention to provide a method for determining the amount of a compound capable of binding a VEGFR-2 + NP- 1 protein complex in a sample.
It is also an object of the present invention to provide a host cell comprising a recombinant expression vector encoding a VEGFR-2 protein and a recombinant expression vector encoding a NP- 1 protein.
It is also an object of the present invention to provide a complex formed by the interaction of a recombinant VEGFR-2 receptor protein and a recombinant NP- 1 receptor protein.
It is also an object of the present invention to provide a method for determining whether a test compound produces a signal upon binding to a VEGFR-2 + NP-1 protein complex, or antagonizes the signal produced by VEGFι65 binding to a VEGFR-2 + NP-1 protein complex.
SUMMARY OF THE INVENTION
The present invention relates to a method for determining whether a compound is capable of binding to a receptor protein complex comprising Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and Neuroplin-1 (NP-1), which receptor protein complex is hereafter referred to as "VEGFR-2 + NP-1 protein complex" or "complex", the method comprising introducing a sample comprising the compound to the VEGFR-2 + NP-1 protein complex and allowing the compound to bind to the complex. This method may utilize the full length proteins, the soluble form of either protein or a combination of full length and soluble proteins.
The invention further relates to a host cell co-transfected with an expression vector comprising a DNA sequence that codes for the VEGFR-2 protein and an expression vector comprising a DNA sequence that codes for the NP-1 protein.
The invention further relates to a method for determining whether a test compound produces a signal upon binding to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to a composition containing a test compound and (ii) a second set of the cells to a composition lacking the test compound; (c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b); and (d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells.
The invention further relates to a method for determining whether a test compound blocks a signal produced by binding of VEGFι65 (or another heparin-binding or NP-1 binding VEGF family member) to a VEGFR-2 + NP-1 protein complex, the method comprising: (a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both of these receptors (e.g., HUVEC) and/or wherein the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptors; (b) exposing (i) a first set of the cells to VEGFι65 (or another heparin-binding VEGF family member) and a composition comprising a test compound and (ii) a second set of the cells to VEGF165 (or another heparin-binding VEGF family member) and the composition without the test compound; (c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b); and (d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells. The invention further relates to a complex formed by the interaction of a recombinant
VEGFR-2 receptor protein and a recombinant NP-1 receptor protein
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of the VEGFR-2 probe used for hybridization screening of a placenta gtlOλ cDNA library to obtain a full-length VEGFR-2 cDNA. The nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G respectively. The primers are derived from the sequence of the human VEGFR-2 receptor (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)).
Figure 2 shows the construct pJFE.HFLKl, used for transient mammalian expression of human VEGFR-2. SR-alpha, promoter/enhancer; ApR, ampicillin resistance marker; hFLKl, human VEGFR-2 gene; BstXI, restriction site; Xbal, restriction site.
Figure 3 shows the DNA sequence of the oligonucleotide primers used in the PCR amplification of PGneuropilin-1. The gene was isolated in two gene fragments which were ligated together forming the full-length human Neuropilin-1 coding sequence. The nucleotide bases adenine, thymine, cytosine, and guanine are represented by A, T, C, and G, respectively. The primers are derived from the sequence of the human Neuropilin-1 receptor (Soker, S., Takashima, S., Miao, H.Q., Neufeld, G., and Klagsbrun, M., Cell, 92: 735-745 (1998)).
Figure 4 shows the construct PGNP-l/pJFE14, used for transient mammalian expression of human Neuropilin-1. SR-alpha, promoter/enhancer; ApR, ampicillin resistance marker; Neuropilin-1, human gene; EcoRI, restriction site; Xbal, restriction site; BstXI, restriction site.
Figure 5 shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or Mock vector, and crosslinked to 261 pM [I25I]-VEGFι65. Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes of these two receptors crosslinked to [125I]-VEGFi65. The VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. From left to right: VEGFR-2 immunoprecipitates of COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock), followed by Neuropilin-1 immunoprecipitates in COS-1 cells expressing VEGFR-2 only, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock).
Figure 6A shows an immunoprecipitation of VEGFR-2 in HUVEC cells. Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 or Neuropilin- 1 receptors crosslinked to 379 pM [125I]-VEGF|65. In this experiment unlabeled ligand was used to demonstrate the specificity of the immunoprecipitated bands. From left to right: VEGFR-2 immunoprecipitates with no competitor, 30 nM unlabeled VEGF165, and 100 nM VEGF12..
Figure 6B shows an immunoprecipitation of Neuropilin-1 in HUVEC cells. Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of the Neuropilin-1 receptor crosslinked to 379 pM [125I]-VEGFι65. From left to right: Neuropilin-1 immunoprecipitates with no competitor, 30 nM unlabeled VEGF165, and 100 nM VEGF121.
Figure 7A shows an immunoprecipitation/Western blot. Here COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C). Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the VEGFR-2 in the cell ly sates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, and empty vector (Mock); immunoprecipitated using the VEGFR-2 antibody (R2.2C) and detected using the VEGFR-2 antibody (R2.2C). The final two lanes contain 6 μg of total cell lysate from COS-1 cells expressing empty vector (Mock) or VEGFR-2, respectively. Figure 7B shows an immunoprecipitation/Western blot. Here COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1 , Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) crosslinker and, after transfer to PDVF membrane, detected using the VEGFR-2 antibody (R2.2C). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 or Neuropilin-1 in concert with VEGFR-2 are also present to demonstrate the ability to detect VEGFR-2 in the cell lysates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, or empty vector (Mock); immunoprecipitated using the Neuropilin-1 antibody (NP1ECD4C) and detected using the VEGFR-2 antibody (R2.2C). The final two lanes contain 6 μg of total cell lysate from COS-1 cells expressing NP1 or VEGFR-2 in concert with Neuropilin-1 respectively.
Figure 7C shows an immunoprecipitation/Western blot. Here COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the VEGFR-2 antibody (R2.2C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing empty vector (Mock) or VEGFR-2 are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only or empty vector (Mock); immunoprecipitated using the VEGFR-2 antibody (R2.2C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 μg of total cell lysate from COS-1 cells expressing empty vector (Mock) or VEGFR-2 respectively.
Figure 7D shows an immunoprecipitation/Western blot. Here COS-1 cells overexpressing either VEGFR-2 alone, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 alone, or empty vector (Mock) are immunoprecipitated with the Neuropilin-1 antibody (NP1ECD4C) in the absence of ligand, in the presence (+) or absence (-) of crosslinker and, after transfer to PDVF membrane, detected using the Neuropilin-1 antibody (NP1ECD1A). Lysate lanes of COS-1 cells overexpressing Neuropilin-1 only and VEGFR-2 in concert with Neuropilin-1, are also present to demonstrate the ability to detect the Neuropilin-1 in the cell lysates. Molecular weight standards are shown on the left; the relevant area of the detected Western blot is displayed. From left to right: COS-1 cells expressing VEGFR-2, VEGFR-2 in concert with Neuropilin-1, Neuropilin-1 only, or empty vector (Mock); immunoprecipitated using the Neuropilin-1 antibody (NP1ECD4C) and detected using the Neuropilin-1 antibody (NP1ECD1A). The final two lanes contain 6 μg of total cell lysate from COS-1 cells expressing Neuropilin-1 or VEGFR-2 in concert with Neuropilin-1, respectively.
Figure 8A shows a whole cell binding competition of 287 pM [125 vEGFι65 with either
VEGF, 2i (-•-) or VEGF,65 (-■-) in COS-1 cells overexpressing VEGFR-2. The Y axis is in total DPM, the X axis is in log units of the molar concentration of competing unlabeled ligand used. In this experiment the ability of VEGF121 and VEGFι65 to compete for [125 vEGFι65 binding in these cells is very similar (IC50= 3.19 x 10"" M vs. 6.85 x 10"" M for VEGF,2ι and VEGF,65, repectively).
Figure 8B shows whole cell binding competition of 320 pM [12 vEGFι65 with either VEGF,2i (-•-) or VEGFI65 (-■-) in HUVEC cells. The Y axis in total DPM, the X axis is in log units of the molar concentration of competing unlabeled ligand used. In this experiment the ability of VEGF121 and VEGF)65 to compete for [125i]VEGF|65 binding in these cells is distinctly different (VEGFι65 competes for all binding sites with an IC50= 5.61 x 10"" M, whereas VEGF competes for only -46% of the binding sites with an IC5o= 2.64 x 10"12 M). Figure 9 A shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 and crosslinked to 420 pM [125I]VEGFι65. Molecular weight standards are shown on the left; area shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2 crosslinked to [l25I]-VEGFι65. The VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. In this experiment binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands. From left to right on left panel: VEGFR-2 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGF165, or 100 nM VEGF121. From left to right on right panel: Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitor or in the presence of either 30 nM unlabeled VEGFι65, or 100 nM VEGF,2..
Figure 9B shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing VEGFR-2 in concert with Neuropilin-1, and crosslinked to 420 pM [125I]VEGFi65. Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, Neuropilin-1, or potential complexes involving these two receptors, crosslinked to [125I]VEGFι65. The VEGFR-2 antibody R2.2C was used for the VEGFR-2 immuno-precipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. In this experiment binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands. From left to right on left panel: VEGFR-2 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGFι65, or 100 nM VEGF . From left to right on right panel: Neuropilin- 1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF,65, or 100 nM VEGF .
Figure 9C shows an immunoprecipitation of VEGFR-2 or Neuropilin-1 from COS-1 cells over-expressing Neuropilin-1, and crosslinked to 420 pM [125I]VEGFι65. Molecular weight standards are shown on the left; areas shown at the right indicate labeled protein bands migrating at the predicted molecular weight of VEGFR-2, or complexes involving this receptor, crosslinked to [125I]VEGFι65. The VEGFR-2 antibody R2.2C was used for the VEGFR-2 immunoprecipitates, and the Neuropilin-1 immunoprecipitations were performed with the NP1ECD4C antibody. In this experiment binding was performed in the presence of unlabeled ligand to demonstrate the binding specificity of the immunoprecipitated bands. From left to right on left panel: VEGFR-2 from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF|65, or 100 nM VEGF . From left to right on right panel: Neuropilin-1 immunoprecipitates from cells incubated in the absence of competitors, or in the presence of either 30 nM unlabeled VEGF,65, or 100 nM VEGF,2ι.
Figure 10A shows an anti-phosphotyrosine Western blot performed in HUVEC cells. The upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower panel shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization. From left to right, the lanes are treated with: no VEGF165, 0.1 pM VEGFι65, 1 pM VEGFι65, 3 pM VEGF165, 10 pM VEGF165, 30 pM VEGF165, 100 pM VEGF165, 300 pM VEGFι65, 1,000 pM VEGF165, or 10,000 pM VEGFi65. Only the most mature form of VEGFR-2 (top band in lower panel) is phosphorylated on tyrosine in response to ligand. When normalized for the amount of VEGFR-2 in the immunoprecipitate, VEGF165 exhibits an EC50= 1.25 x 10"10 M.
Figure 10B shows an anti-phosphotyrosine Western blot performed in HUVEC cells. The upper panel shows the anti-phosphotyrosine Western blot of VEGFR-2 immunoprecipitates (detection via the 4G10 anti-phosphotyrosine antibody), and the lower shows the same blot stripped and re-probed with the VEGFR-2 antibody (R2.2C) for purpose of normalization. From left to right, the lanes are treated with: no VEGFm, 100 pM VEGF12ι, 300 pM VEGF , 1,000 pM VEGFm, 3,000 pM VEGF121, 10,000 pM VEGF12ι, 30,000 pM VEGFm, 100,000 pM VEGFm, 300,000 pM VEGF121, or 1,000,000 pM VEGF,21. Only the most mature form of VEGFR-2 (top band in lower panel) is phosphorylated on tyrosine in response to ligand. When normalized for the amount of VEGFR-2 in the immunoprecipitate, VEGFm exhibits an ECso=
2.68 x l0"9 M.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "ATCC" means American Type Culture Collection, Rockville, Maryland.
As used herein, "biologically active" means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of binding detectable quantities of VEGFι65 or another heparin-binding VEGF or NP- 1 -binding family member, or transmitting a VEGFι65 stimulus to a cell, e.g., as a component of a hybrid receptor construct. Preferably, a biologically active VEGFR-2 + NP-1 receptor protein complex within the scope of the present invention means the receptor protein complex is capable of binding and can be immunoprecipitated with antibodies generated against either VEGFR-2 or NP-1.
As used herein, "host cell" means a cell comprising a recombinant expression vector described herein. Host cells may be stably transfected or transiently transfected within a recombinant expression plasmid or infected by a recombinant virus vector. The host cells include prokaryotic cells, such as Escherichia coli, fungal systems such as Saccharomyces cerevisiae, permanent cell lines derived from insects such as Sf-9 and Sf-21, and permanent mammalian cell lines such as Chinese hamster ovary (CHO), SV40-transformed African green monkey kidney cells (COS), Balb/c3T3 A31 cells, or any other cell line known to those skilled in the art.
As used herein, "isolated", in reference to the receptor protein of the present invention or DNA sequences encoding said protein, means that the protein or DNA sequence is removed from the complex cellular milieu in which it naturally occurs, and said protein is expressible from said DNA sequence in a cell that does not naturally express it when operably linked to the appropriate regulatory sequences.
As used herein, "NP-1" means a protein having the amino acid sequence SEQ ID NO. 4, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 4 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGFι65 and P1GF-2), as well as various semaphorin family members, or crossreacting with antibodies raised against NP-1 protein, or peptides derived from the protein sequence of NP-1 protein. The term NP-1 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with VEGFR-2 have been deleted or modified.
As used herein, "operably linked" refers to a condition in which portions of a linear DNA sequence are capable of influencing the activity of other portions of the same linear DNA sequence. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous in reading frame.
As used herein, "recombinant expression vector" refers to a DNA construct used to express DNA which encodes a desired protein (for example, VEGFR-2 or NP-1) and which includes a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and 3) appropriate transcription and translation initiation and termination sequences. Using methodology well known in the art, recombinant expression vectors of the present invention can be constructed. Possible vectors for use in the present invention include, but are not limited to: for mammalian cells, pJT4 (as described in World Patent Publication No. 96/14579, published by J. Rosenbaum on May 17, 1996), pJFE14 (described below), pcDNA-1 (Invitrogen, San Diego, Ca) and pSV- SPORT 1 (Gibco-BRL, Grand Island, NY); for insect cells, pBlueBac III or pBlueBacHis baculovirus vectors (Invitrogen, San Diego, CA); and for bacterial cells, pET-3 (Novagen, Madison, WI). The DNA sequence coding for either VEGFR-2 or NP-1 can be present in the vector operably linked to regulatory elements. As used herein, "soluble receptor" with respect to VEGFR-2 refers to an amino acid sequence corresponding to the extracellular region of VEGFR-2, or a portion thereof, which is capable of binding VEGF]65 or another VEGF family member (including, but not limited to, VEGF165, VEGFm, VEGF206, VEGF189, VEGF145, VEGF-C, VEGF-D and the viral VEGF ligands). Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with NP-1 have been deleted or modified. Examples of such soluble receptors for VEGFR-2 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO:6 (i.e, amino acid residues 1-760 depicted in SEQ ID NO. 2), especially those encoding amino acid number 124-320 corresponding to the Ig domains 2 and 3 of human VEGFR-2; or polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 5 (i.e., nucleic acid residues 71-2350 depicted in SEQ ID NO. 1), especially base pairs 442-1030 corresponding to the Ig domains 2 and 3 of human VEGFR-2.
As used herein, "soluble receptor" with respect to NP- 1 refers to an amino acid sequence corresponding to the extracellular region of NP-1, or a portion thereof, which is capable of binding VEGF165 or other heparin-binding VEGF family members. Soluble receptors include truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding or complex formation with VEGFR-2 have been deleted or modified. Examples of such soluble receptors for NP-1 include, but are not limited to, polypeptides having the amino acid sequences substantially similar to SEQ ID NO. 8 (i.e., amino acid residues 1-856 depicted in SEQ ID NO. 4), especially those encoding amino acid number 274-647 corresponding to the coagulation factor domains of human NP-1; or polypeptides encoded by nucleic acid residues substantially similar to SEQ ID NO. 7 (i.e., nucleic acid residues 1-2568 depicted in SEQ ID NO. 3), especially base pairs 822-1941 corresponding to the coagulation factor domains of human NP- 1. As used herein, "substantially similar" when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, a sequence altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the protein. Alternatively, nucleic acid sequences and analogs are "substantially similar" to the specific DNA sequence disclosed herein if the DNA sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence. In addition, "substantially similar" means a receptor protein that will react with antibodies generated against the protein or peptides derived from the protein sequence.
As used herein, "VEGFR-2" means a protein having the amino acid sequence SEQ ID NO. 2, as well as proteins having amino acid sequences substantially similar to SEQ ID NO. 2 and which are biologically active in that they are capable of binding a VEGF family member (including, but not limited to VEGF165, VEGFm, VEGF206, VEGF189, VEGFι45, VEGF-C, VEGF- D and the viral VEGF ligands), or transducing a biological signal initiated by the VEGF family member binding to a cell expressing VEGFR-2, or crossreacting with antibodies raised against VEGFR-2 protein, or peptides derived from the protein sequence of VEGFR-2 protein. The term VEGFR-2 includes truncated and/or mutated proteins wherein regions of the receptor molecule not required for VEGF binding, signaling, or complex formation with NP-1 have been deleted or modified.
For purposes of illustrating the methods and expression systems of the present invention, the following non-limiting examples are discussed in detail. While these examples describe the use of human VEGFR-2 and human NP-1 receptor proteins, the skilled artisan will recognize that sequences from other species are readily obtainable and may be used in place of either or both of the human receptor proteins. Examples of such known sequences include, but are not limited: VEGFR-2 from mouse (GENBANK #X70842), rat (GENBANK #U93306) and quail (GENBANK #X83288); NP-1 from mouse (GENBANK #D50086), rat (GENBANK #AF010296) and chicken (GENBANK #D45416). The skilled artisan will also recognize that VEGFR-2 and NP-1 receptor proteins from other species are obtainable using well known methods. The following abbreviations are used in the Examples:
VEGF: vascular endothelial growth factor VEGFR-2: VEGF Receptor-2 FLK-1: Fetal Liver Kinase- 1 KDR: Kinase Domain Receptor
VEGFR-1: VEGF Receptor 1 FLT-1: Ems-like Tyrosine Kinase- 1
BSA: Bovine Serum Albumin DPM: Disintegrations Per Minute
ΕCL: Enhanced Chemiluminescence HUVEC: Human Umbilical
Vein Endothelial Cells NP-1: Neuropilin-1 NP-2: Neuropilin-2
PBS: Phosphate Buffered Saline TBS: Tris Buffered Saline
DTT: Dithiothreitol DSG: Disuccinimidyl Glutarate P1GF: Placental Growth Factor
Example 1 Cloning of human VEGFR-2 and human Neuropilin-1
In order to generate the human VEGFR-2 cDNA, primers shown in Figure 1 are designed from the GENBANK Accession #X61656 (Terman et al., Oncogene 6 (9):2 1677-1683 (1991)) to generate a hybridization probe by PCR corresponding to the first 347 nucleotides. PCR is performed in a 100 μl reaction using 1 μl of human placenta cDNA library (see below) as template, 0.25 μM primers, 25 μM dNTPs (dTTP, dGTP, and dATP) (Perkin Elmer Cetus, Foster City, CA), 100 μCi P-32 dCTP (Cat# BLU513, Dupont-NEN, Boston, MA), IX polymerase buffer, and 5 U polymerase (TaKaRa Shuzo Panver, Kyoto, Japan). After an initial melting period of 10 seconds at 95°C, the temperature cycle is carried out as follows for 12 cycles: melting, 95°C for 30 sec; annealing, 50°C for 30 sec; extension, 70°C for 30 sec. After the 12th cycle, the reaction is held at 70°C for an additional 30 seconds to complete extension. The probe was purified by a Nick Column (Cat# 17-0855-01, Pharmacia-Biotech, Upsala, Sweden) according to the manufacturer's instructions. The probe generated is used to screen a gtlOλ cDNA library (Cat.#HL5014A, Clontech, Palo Alto, CA) and obtain a human VEGFR-2 cDNA. The placenta cDNA library is screened according to the Clontech Lambda Library Protocol Handbook (Clontech, Palo Alto, CA). The primary screen is performed on Optitran membranes (Cat# 68350, Schleicher & Schuell, Keene, NH) at a density of 2.5 x 105 plaques/filter. The secondary screen is performed on Optitran membranes (Cat#68320, Schleicher & Schuell, Keene, NH) at a density of 1.0 x 104 or 1.0 x 105 plaques/filter. Each round of hybridization screening is carried out overnight at 65°C in Phosphate Buffer (0.25M Na2P04, 7% sodium dodecyl sulfate, 1% BSA, 1 mM EDTA, 40 μg/ml ssDNA) using 1 x 106 dpm/ml of radio-labeled probe. The membranes are washed in 2X SSC, 6 times for 10 minutes at room temperature, followed by a 65°C wash in 2X SSC for 15 minutes.
Two independent clones are obtained containing partial but overlapping inserts for VEGFR-2. The insert LambdaFlkl#l encodes nucleotides 1-2840 of the VEGFR-2 sequence (GENBANK Accession #X61656) and the insert LambaFlkl#12 clone encodes nucleotides 276- 4226 of VEGFR-2 (GENBANK Accession #X61656). These two clones are used to generate single, full-length, VEGFR-2 cDNA.
To generate a full-length VEGFR-2 cDNA, LambaFlkl#12 is digested with the restriction enzymes BamHI and Fspl (Stratagene, La Jolla, CA) and subcloned into a pBluescript KS vector (Stratagene, La Jolla, CA) previously digested with BamHI and EcorV (Stratagene, La Jolla, CA). The resulting construct is digested with Hindlll (Stratagene, La Jolla, CA) to excise LambaFlkl#12 and then treated with pfu DNA polymerase (Cat# 600140 Stratagene, La Jolla, CA) according to the manufacturer's instructions, the 2.4kb gene fragment is then digested with BamHI and recovered by gel purification. Additionally, LambdaFlkl#l is digested with Xbal and BamHI (Stratagene, La Jolla, CA ) and the resulting 2.3 kb fragment is recovered by gel purification. The two gene fragments are then ligated into pJFE14 mammalian expression vector previously digested with Xbal and Smal (Stratagene, La Jolla, CA). The pJFE14 expression vector is derived from the pCDL-SRα296 vector described by Takebe et al. (Y. Takebe et al., MCB 8: 466-472 (1988)). The resulting mammalian construct containing VEGFR-2 is designated pJFE.HFLKl (Figure 2). The coding region for hVEGFR-2 is identical to the GENBANK sequence X61656. The VEGFR-2 sequences contained within the pJFE14 expression vector contains 4226 nucleotides encompassing the entire coding region plus 86 nucleotides of 3' untranslated DNA sequence (SEQ ID NO. 1). The cDNA clone described herein differs from GENBANK X61656 as follows: 70 nucleotides of 5' untranslated sequence; and 6 additional nucleotides at the 5' end coding for the first two amino acids (Met, Gin) of human VEGFR-2, as described by GENBANK AF035121 (L.Y. Lin et al., unpublished). In order to generate the PCR fragments for human Neuropilin-1, primers shown in Figure 3 are designed from the AFO 16050 GENBANK sequence, incorporating the indicated restriction sites utilized for subcloning (described below). The template cDNA is generated from human heart polyA+ RNA (Cat.# 6533-1, Clontech, Palo Alto, CA) by reverse transcription. The reverse transcription, carried out in a 20 μl volume, contains approximately 1 μg-500 ng human heart polyA+ RNA, 10 μM random hexamers (Perkin Elmer Cetus, Foster City, CA), 1 mM dNTPs, 0.02 mM DTT, 10 U RNase inhibitors (Boehringer Mannheim Biochemical, Indianapolis, IN), 400 U Superscript II MMLV Reverse transcriptase (Gibco-BRL, Grand Island, NY) at 37°C for 1 hour. The reverse transcriptase is heat killed at 72°C for 10 min and each PCR is performed in a 100 μl volume. Primers BJL-265 and BJL-238R are used to generate the 910 bp 5' region of NP- 1 while primers BJL-259 and BJL-258 are used to generate the 1862 bp 3' region of NP-1. PCR is performed using 10 μl of the resulting reverse transcription reaction, 0.5% DMSO, 0.05 μM primers, 200 μM dNTPs, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KC1 pH = 8.3 at 20°C, and 2.5 U Taq DNA polymerase (Boehringer Mannheim Biochemical, Indianapolis, IN). After an initial melting period of 2 min at 95°C, the temperature cycle is carried out as follows for 35 cycles: melting, 95°C for 1 min; annealing, 53°C for 1 min; extension, 72°C for 3 min. After the 35th cycle, the reaction is held at 72°C min for an additional 10 minutes to complete extension.
The respective PCR reactions are purified using Strataprep PCR purification columns (Stratagene, La Jolla, CA) according to the manufacturer's instructions, then eluted with 50 μl water. The 910 bp and 1862 bp PCR products are subcloned, respectively, into the PCR-Script™ Amp vector (Stratagene, La Jolla, CA) using the PCR-Script™ Amp Electroporation-Competent Cell Cloning kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Positive clones are identified by DNA sequencing analysis.
To perform NP-1 expression studies the full-length NP-1 coding sequence is constructed and subcloned into the pJFE14 mammalian expression vector (discussed above). The NP-1 cDNA fragments are excised from the pCRScript vectors by restriction digestion. The plasmid construct for the 5' region of NP-1 is digested with Xbal and Pstl restriction enzymes (Boehringer Mannheim Biochemical, Indianapolis, IN) while the plasmid construct containing the 1862 bp 3' region of NP-1 is digested with Pstl and EcoRI. The resulting NP-1 fragments are each gel purified from an agarose gel using QIAEX II (Qiagen, Chatsworth, CA; a kit for gel purification of DNA fragments, including activated silica spheres and buffers) according the manufacturer's instructions, then resuspended in 40 μl 10 mM Tris, pH=8.0. Additionally, the pJFE14 vector is digested with Xbal and EcoRI and gel purified from an agarose gel using the QIAEXII kit as stated previously. The ligation for the NP-1 gene fragments and pJFE14 vector containing Xbal and EcoRI overhangs is carried out overnight at 15°C with ~100 ng of each of the DNAs in a 20 μl ligation reaction containing 1.5 mM rATP, 50 mM Tris-HCl (pH=7.5), 7 mM MgCl2, 1 mM DTT, and 8 U ligase (Stratagene, La Jolla, CA). The resulting reaction is precipitated two hours at -80°C in l/10 h volume 3.5 M sodium acetate pH=5.2 and two volumes of 100% ethanol. The resulting DNA is recovered by centrifugation at 14,000xg for 20 min, rinsed briefly with 75% cold ethanol to remove salts, and air-dried 20 min. The DNA is resuspended in 10 μl water then 5 μl is transformed into XL-1 Blue E.coli (Stratagene, La Jolla, CA) by electroporation using the GenePulser™ (Bio-Rad, Hercules, CA). The resulting cDNA from this clone is designated PGNP-l/pJFE14 (Figure 4). DNA sequence analysis is performed on the PGNP-1 cDNA (SEQ ID NO. 3). The DNA sequence is identical to the GENBANK NP-1 sequence AF016050 (S. Soker, et al. (1998), Cell 92: (6):735-745) with the following exceptions: Transitions; G->C at bp 267; C->G at bp 2248 (2245) results in an amino acid change His->Asp at AA#750 (749): Transversions; C->T at bp 1161; C->T at bp 1266; C->T at bp 1587; A->G at bp 1897 results in an amino acid change Thr- >Ala at AA#633; G->A at bp 2200 (2197) results in an amino acid change Val->Ile at AA#734 (733); G->A at bp 2241 (2238); T->C at bp 2271 (2268) results in an amino acid change Ile->Thr at AA#757 (756); C->T at bp 2406 (2403): Insertions; GCA codon at bp 2060-2062, results in an Arg insertion at AA# 688. Parenthesis indicate the corresponding DNA bases or amino acids of the AF016050 DNA sequence as a result of the PGNP-1 codon insertion. Example 2
Mammalian Expression of VEGFR-2 and Neuropilin- 1 Transient expression of the receptors in mammalian cells for the binding and immunoprecipitation studies (Examples 3-5), is carried out in COS-1 cells (ATCC CRL 1650) using Lipofectamine 2000 (LF 2000, Gibco-BRL, Grand Island, NY) and the expression plasmids described above (Example 1). COS-1 cells are grown to approximately 70%-90% confluency in DME high glucose media (Gibco-BRL) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah), nonessential amino acids, and glutamine in T-175 flasks (Corning, San Diego, CA). The cells are washed twice with 37°C serum-free DME media, after which the mixture of solution A and B is diluted to 10 ml in Serum Free DME and added to each T-175 flask for 3-5 hours. Solution A contains 625 μl of OptiMEM (Gibco-BRL) and the cDNAs of interest: for VEGFR-2, 5 μg of pJFE.HFLK-1, for Neuropilin-1, 1.5 μg of PGNP-1 PJFE14, pre-incubated at room temperature for 10 minutes. Solution B contains 37.5 μl of LF2000 reagent in 625 μl OptiMEM. After 3-5 hours an additional 10 ml of DME high glucose media supplemented with 10% fetal bovine serum, nonessential amino acids, and glutamine is added to each T175 flask overnight. When the experimental design requires transfection of a single receptor only, the empty expression vector pJFE14 is substituted for the corresponding receptor cDNA, such that all transfections contain the same amount of total cDNA. The transfected cells are split into 12 well plates 24 hr post transfection for whole cell binding (Example 5) or 100 mm plates for immunoprecipitation/Western Blot (Examples 4 and 6), affinity labeling/immunoprecipitation (Examples 3 and 5), and Western blot experiments (Examples 4 and 6). The cells are suitable for binding analysis 36 to 72 hours after transfection.
Example 3 Demonstration of Complex Formation Between VEGFR-2 and Neuropilin-1 in the Presence of r'25nVEGF.,.s
Recombinant human VEGF165 is purchased from R&D Systems, Inc. (Minneapolis, MN) [125I]VEGFi65 is prepared using Chloromine T (Sigma, St. Louis MO), using a modification of methods previously described (K. Pajusola et al., Oncogene, 9: 3545-3555 (1994), B. A. Keyt et al., The Journal of Biological Chemistry 271(13): 7778-7795 (1996)). Lyophilized VEGF,65 (5 μg) is taken up in 90 μl of DPBS (Gibco-BRL) which is then divided equally into two 1.7 ml pre- lubricated conical tubes (VWR, St. Louis MO). To each tube is added 500 μCi of [l25I]NaI (Amersham, Arlington Heights, IL) (2200 Ci/mmol) in 5 μl, and 20 μl of a 1 μg/μl solution of Chloromine T (Sigma, St. Louis MO) in 0.5M Sodium Phosphate Buffer pH=7.5. The reaction is incubated at room temperature for 1 min. with occasional shaking. The reaction is then terminated by the addition of 25 μl of a 2 μg/μl sodium metabisulfite solution in 0.5 M sodium phosphate buffer pH=7.5. To remove unreacted [125I], the mixture is applied to a PD-10 gel filtration column (Pharmacia, Piscataway, NJ) previously equilibrated in PBS containing 0.5% BSA (Sigma, St. Louis MO) and 0.01% Tween 20 (Sigma, St. Louis MO). The resulting labeled material is >95% precipitable by trichloroacetic acid, indicating that all of the isolated [125I] is protein associated, and has a typical specific activity of 4000 to 12000 Ci/mmol.
The Neuropilin-1 receptor has previously been demonstrated to bind VEGFι65 (S. Soker, et al., Cell 92(6):735-745, (1998)). In order to demonstrate that this receptor forms a complex with VEGFR-2, COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated at a density of 3 x 106 cells/dish into 100 mm dishes (Corning, San Diego, CA), as described in Examples 1-2. The receptors are crosslinked to [125I]VEGFi65, then subjected to immunoprecipitation with antibodies specific for either receptor using a modification of the method previously described (B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)). In this procedure, cells are transfected with the cDNA for VEGFR-2, Neuropilin-1, or a combination of both, as described in Example 2. At 48 - 68 hr after transfection, the cells are washed, pre-equilibrated for 1-2 hrs at 4°C in binding buffer (MEM, 25 mM HEPES, (Gibco- BRL), 0.2% BSA, (Sigma St. Louis MO)), and incubated for 4 hours at 4°C with [l25I]-VEGF,65 alone or in the presence of unlabeled ligand as competitor in a total volume of 4 ml of binding buffer containing the protease inhibitor cocktail previously described (B. B. Koenig et al., Molecular and Cellular Biology, 14: 5961-5974 (1994)) and further supplemented with 1 μg/ml heparin (Sigma Cat # H9399, St. Louis MO). After completion of the 4 hr incubation with ligand, the cells are washed three times at 4°C with 5 ml of binding buffer having the same composition as described above, except that no BSA, heparin, or protease inhibitors are added. To each plate is then added 4 ml of fresh BSA-free binding buffer, followed by freshly prepared DSG (Pierce, Rockford, IL) to a final concentration of 186 μM. After swirling gently to mix the DSG, the plates are incubated for exactly 15 minutes at 4°C with gentle shaking. The media is then aspirated and the cells washed with 15 ml ice cold PBS (Gibco-BRL). RIP Buffer (20 mM Tris-HCl pH=7.4, 100 mM NaCl, 1 mM EDTA, 10 mM Nal, 0.5% NP-40, 0.5% Na- Deoxycholate, 1% BSA, Sigma, St. Louis, MO) (1 ml) is then added to each plate and the cells are allowed to solubilize for 15 minutes at 4°C with shaking. The samples are then transferred from 100 mm dishes to 1.5 ml conical tubes and the samples are centrifuged at 13,000xg for 10 minutes to pellet the cellular debris. The supernatants are transferred to fresh 1.5 ml conical tubes to which 10 μg of the antibody of choice (see below), 10 μl of 10% SDS (Gibco-BRL), and 100 μl of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added. The samples are then neurated overnight at 4°C. Following the overnight incubation, the beads are pelleted at 13,000xg for 5 minutes and the supernatants aspirated. Three successive washes of the beads are performed using TNEN buffer (20 mM Tris-HCl pH=7.4, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, Sigma, St. Louis MO). After washing the pelleted beads are resuspended in 25 μl of IX sample loading buffer (0.25 M TrisCl, pH=6.8, 10% SDS, 0.5 M DTT, 0.5% bromophenol blue, 50% glycerol; diluted from 5X (Five Prime Three Prime, Boulder, CO, Cat # 2-910675). The samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 μl of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS- PAGE) electrophoresis. The gels are treated to prevent cracking in gel drying buffer (10% methanol, 40% acetic acid, 50% H2O) for 15 minutes, and subsequently dried. Radiolabeled bands that were immunoprecipitated by the antibody of choice are visualized and quantitated on a Storm System (Molecular Dynamics, Sunnyvale, CA) .
The VEGFR-2 rabbit polyclonal antibody (R2.2C) used to precipitate VEGFR-2 is produced by Quality Control Biochemicals, Inc. (Hopkinton, MA). The R2.2C antibody is raised against the peptide sequence Ac-SKRKSRPVSVKTFEDIPLEEPC-amide found in the carboxy- terminus of human VEGFR-2 (identical to AA# 1225-1245, SWISSPROT # P35968, except that a C-terminal Cys is added for conjugation). This sequence is conserved in human, mouse, and rat, and this antibody has demonstrated reactivity with human, mouse, rat, canine and bovine KDR/VEGFR-2/FLK-l (data not shown). R2.2C does not cross-react with the homologous VEGFR-1 receptor protein (data not shown), nor does it cross-react with Neuropilin-1 (Figure 5). The Neuropilin-1 polyclonal antibody (NP1ECD4C) used to precipitate Neuropilin-1 is also produced by Quality Control Biochemicals (Hopkinton, MA). The NP1ECD4C antibody is raised against the peptide sequence AcDLDKKNPEIKIDETGST-C-amide in the extracellular juxtamembrane region of human Neuropilin-1 (identical to AA# 814-830, GENBANK . # AF016050, AF018956 (S. Soker et al., Cell (1998), 92: 735-745, Z. He and M. Tessier-Lavigne, Cell (1997), 90: 739-757, except that a C-terminal Cys is added for conjugation). This antibody does not cross-react with VEGFR-2 (Figure 5), and is predicted to not cross-react with Neuropilin-2. The NP1ECD4C antibody is predicted to recognize Neuropilin-1 from human, mouse, and rat species. For all antibodies used throughout the Examples, the peptide sequences used for immunization are KLH-conjugated to a carboxy-terminal cysteine. After harvesting the serum from the rabbits, the serum is affinity purified by Quality Control Biochemicals (Hopkinoton, MA) using standard affinity purification techniques, and supplied in a standard buffer (PBS without Ca2+, Mg2+, pH=7.4). (Another useful NP-1 antibody is the anti-neuropilin- 1 polyclonal antibody C-19 from Santa Cruz Biotechnology, Inc., Santa Cruz, California (cat. No. sc-7239).)
The results of the affinity labeling/immunoprecipitation experiment are shown in Figure 5. When VEGFR-2 is expressed alone in COS-1 cells and immunoprecipitated using the R2.2C antibody for VEGFR-2, a heavy band running the predicted size of VEGFR-2 (-230 kDa) is visualized. In addition several higher molecular weight bands (> 250 kDa) are also visualized. These are most likely VEGFR-2 homodimeric complexes. The NP1ECD4C antibody does not immunoprecipitate VEGFR-2 in COS-1 cells overexpressing VEGFR-2, demonstrating a lack of cross-reactivity of the NP1ECD4C antibody with VEGFR-2. In COS-1 cells overexpressing only Neuropilin-1, NP1ECD4C precipitates a band of the predicted size (-148 kDa) for the VEGF165:Neuropilin-l complex (S. Soker et al., Cell (1998), 92: 735-745) as well as a higher molecular weight band that may represent a Neuropilin-1 homodimeric complex. In contrast, the R2.2C antibody does not immunoprecipitate Neuropilin-1 in these cells, indicating that it does not cross-react with Neuropilin-1. However, when both Neuropilin-1 and VEGFR-2 are co-expressed in COS-1 cells, the R2.2C antibody precipitates an intense band the correct size for VEGFR-2 (-230 kDa) as well as a smaller band that corresponds to the molecular weight anticipated for VEGF bound to Neuropilin-1 (-148 kDa). Because the R2.2C antibody does not cross-react with Neuropilin-1, this indicates that VEGFR-2 and Neuropilin-1 are forming a complex in the presence of VEGFι65. In the COS-1 cells co-expressing VEGFR-2 and Neuropilin-1, the Neuropilin-1 antibody (NP1ECD4C) precipitates the same bands observed in the COS cells overexpressing only NP-1. Interestingly, the NP1ECD4C antibody does not precipitate a band the size of VEGFR-2 (-230 kDa) which might suggest that Neuropilin-1 is expressed to a much greater extent then is VEGFR-2 at the ratios of cDNA used for this experiment; such that the majority of Neuropilin- 1 that is immunoprecipitated is not in a complex with VEGFR-2. In contrast, the ability of the R2.2C antibody to co-immunoprecipitate Neuropilin-1 indicates that the majority of VEGFR-2 exists in a complex with Neuropilin-1 in the presence of VEGFι65 ligand, under the conditions of this experiment.
Complex formation is not limited to over-expression systems as is demonstrated by Figure 6. Here HUVEC cells (passages 1 through 4) are exposed to the procedure described above (with the exception that they are not transfected, and are plated 24-48 hours prior to use in the binding assay) and similar results are obtained. The R2.2C antibody immunoprecipitates VEGFR-2 (-230 kDa band) as well as a lower molecular weight band that corresponds to the predicted molecular weight of VEGFι65 bound to Neuropilin-1 (-148 kDa). (Figure 6A.) The identity of this band is confirmed when these same cells are immunoprecipitated using the NP1ECD4C antibody (Figure 6B). As is observed in the COS-1 cells co-expressing both VEGFR-2 and NP-1 (Figure 5), the NP1ECD4C antibody is unable to co-immunoprecipitate VEGFR-2 in the HUVEC cells; indicating that the majority of NP-1 may not exist in complex with VEGFR-2 in these endothelial cells. Nevertheless, the ability of an antibody that is specific for VEGFR-2 (R2.2C) to co-immunoprecipitate NP-1 in the presence of VEGFι65 ligand suggests that the majority of the VEGFR-2 that is present in HUVECs exists in a complex with NP-1 upon ligand addition. It is important to note that VEGF12ι is unable to compete for [,25I]VEGFι65 binding at either VEGFR-2 or Neuropilin-1 (Figs. 6A and 6B). Since VEGFm exhibits equal affinity at VEGFR-2 as does VEGF165 (B.A. Keyt et al., J. Biol. Chem. (1996), 271: 7788-7795), these data suggest that VEGF 121 has only limited access to VEGFR-2 when it is in a complex with NP-1 in these endothelial cells.
Example 4 Demonstration of Ligand-Independent Complex Formation Between
VEGFR-2 and Neuropilin-1 by Immunoprecipitation Western Blot
In order to demonstrate that Neuropilin-1 (NP-1) forms a ligand-independent complex with VEGFR-2, COS-1 cells are co-transfected with the cDNA for VEGFR-2 and Neuropilin-1 and plated in 100 mM dishes as described in Example 2. 48-68 hours after transfection, the cells are subjected to the binding and affinity labeling protocol described in Example 3, except that [125I]VEGF|65 is eliminated from the binding reaction, and the cells are treated with or without DSG to determine the effect of the crosslinker on the ability to detect the VEGFR-2 + NP-1 complex. After the ice cold PBS wash step described in Example 3, RIPA Buffer with the protease inhibitor cocktail (described in Example 3) (20 mM Tris-HCl pH=7.6, 150 mM NaCl, 50 mM NaF, 1 mM sodium orthovanadate, 5 mM benzamidine, 0.5% NP-40, 0.5% Na- Deoxycholate, Sigma, St. Louis, MO, 0.1% SDS (Gibco-BRL, Grand Island, NY)) (1 ml) is added to each plate and the cells are allowed to solubilize for 15 minutes at 4°C with shaking. The samples are then transferred from 100 mm dishes to 1.5 ml conical tubes and centrifuged at 13,000xg for 10 minutes to pellet the cellular debris. The supernatants are transferred to fresh 1.5 ml conical tubes to which 10 μg of the antibody of choice (see below), and 100 μl of a 50:50 slurry of Fast Flow protein G beads (Pharmacia, Piscataway, NJ) is added. The samples are immunoprecipitated at 4°C with neuration overnight, and subjected to SDS-PAGE as described in Example 3. Alternatively, cell lysates are prepared and analyzed by SDS-PAGE. In this procedure, the cells are seeded 24 hours post transfection at 3 x 106 cells into 100 mm plates. 24 hours post plating the cells are rinsed twice with ice cold PBS (Gibco-BRL) and 1 ml of detachment buffer (10 mM Tris-HCl pH=7.4, 1 mM EDTA, 0.25 M sucrose, 0.3 mM PMSF) is used to remove cells from the plate. The cells are scraped on ice using a standard cell scraper and transferred to 1.5 ml conical tubes where the cells are collected by centrifugation (13,000xg, 5 min., 4°C). After centrifugation the cells are re-suspended in 80 μl of RIPA buffer (described above) per plate, and run through a 23 gauge needle to help solubilize the lysates. The lysates are vortexed for 30 minutes at 4°C prior to another centrifugation (13,000xg, 5 min., 4°C). The cleared supernatants are then transferred to fresh tubes where the total protein concentration is determined using the Pierce BCA assay system (Pierce, Rockford, IL). Six micrograms of total protein is then diluted to 20 μl with water and 5X loading dye (4 μl, Five Prime Three Prime, Boulder, CO, Cat # 2- 910675).
Whether lysates or immunoprecipitated protein is prepared (described above), 20 μl of the supernatants are loaded onto 8% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis. The gels are then transferred to PDVF membranes (Owl Scientific, Woburn, MA) using standard Western blot transfer techniques. Post transfer the membranes are blocked using Western blocking buffer (5% BSA (Cat# 05479, Pharmacia, Piscataway, NJ), 0.1% Tween 20, TBS (Sigma, St. Louis, MO)) for 30 minutes. The membranes are then washed three times for 5 minutes (recovering and storing the blocking buffer used for re- blocking the following day) in TBST-0.1% (0.1% Tween 20, TBS) and placed at 4°C overnight without agitation. The following morning the membranes are re-blocked for 2 hours in the same blocking buffer (saved from the previous night). Post blocking, the membranes are probed with the R2.2C antibody (1 :5,000, described in Example 3) for detection of VEGFR-2 or NP1ECD1A antibody for detection of NP-1 at (1 :1,000; see below) in primary probe buffer (Western blocking buffer diluted 1:1 with TBST-0.1%) for two hours at room temperature with agitation. The NP1ECD1A antibody is created using the same methodology as described for NP1ECD4C in Example 3, but is generated against the peptide sequence Ac-TEKPTVIDSTIQSEFPTC-amide (identical to AA# 629-645, GENBANK # AF016050, AF018956 (S. Soker et al., Cell (1998), 92: 735-745, Z. He and M. Tessier-Lavigne, Cell (1997), 90: 739-757, except that a C-terminal Cys is added for conjugation) located in the B2-MAM domain linker region of NP-1. The membranes are then washed five times for six minutes in TBST-0.1% at room temperature with agitation prior to the addition of the HRP-labeled Goat anti-Rabbit secondary antibody at 1 :40,000 (Pierce, Rockford, IL). The secondary antibody is incubated on the membranes for one hour. Post secondary incubation the membranes are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS. Following the final wash step the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions prior to exposure to Hyperfim (Amersham, Piscataway, NJ). Figure 7 demonstrates the R2.2C antibody clearly immunoprecipitates and detects a band of the correct size for VEGFR-2 (-230 kDa) in COS-1 cells either expressing VEGFR-2 alone or in concert with Neuropilin- 1 , as well as detecting a low level expression of VEGFR-2 in empty vector- (mock-) or NP-1 -transfected COS-1 cells (Figure 7A). Figure 7B demonstrates the ability of VEGFR-2 (-230 kDa Band) to be precipitated by the NP1ECD4C antibody, but only when both VEGFR-2 and Neuropilin-1 are co-expressed. The lack of cross-reactivity of the NP1ECD4C antibody with VEGFR-2 is demonstrated by the inability to immunoprecipitate VEGFR-2 in the COS- R2 only cells, indicating that the VEGFR-2 that appears in the NP1ECD4C immunoprecipitate (Figure 7B) must be in a complex with NP-1.
The reciprocal immunoprecipitation is illustrated in Figures 7C and 7D. Figure 7C demonstrates that the R2.2 antibody can only precipitate Neuropilin-1 (-130 kDa band) when VEGFR-2 and Neuropilin-1 are co-expressed in COS-1 cells. The lack of cross-reactivity of the VEGFR-2 antibody with NP-1 is illustrated by the failure of the R2.2C antibody to immunoprecipitate NP-1 in cells only expressing NP-1 (Figure 7C), further illustrating that the NP-1 that is immunoprecipitated by the R2.2C antibody in cells co-expressing VEGFR-2 + NP-1 must be in a complex with VEGFR-2.
NP-1 expression is demonstrated both in cells expressing NP-1 alone, and in cells co- expressing VEGFR-2 + NP-1 (Figure 7D). However, the NP1ECD1A Western antibody appears to preferentially recognize Neuropilin-1 when it is co-expressed with VEGFR-2 vs. when it is expressed alone (Figure 7D). This conclusion is supported by the data in cell lysates where the NP1ECD1A antibody preferentially recognizes the Neuropilin-1 in the VEGFR-2 + Neuropilin-1 co-transfected cell lysate vs. lysate from cells only overexpressing Neuropilin-1 (Figure 7D). This suggests that Neuropilin-1 may be more easily detected by this antibody when Neuropilin-1 is in a complex with VEGFR-2, and further supports the conclusion of the existence of the VEGFR-2 + NP-1 complex in this system. The preferential recognition of the complex by the NP1ECD1A antibody suggests that the B2-MAM-domain linker region epitope recognized by this antibody may undergo a conformational change when VEGFR-2 is in proximity to NP-1 that is maintained even under the SDS-PAGE conditions. This is also consistent with the proposed role of the B domains in binding of VEGFι65 to NP-1 (R. J. Giger, et al.. Neuron (1998), 21(5): 1079- 1092; PCT publication number W099/29858), and that of the MAM domain in activation of the NP-1 + Plexin co-receptor complex (T. Takahashi, A. Fournier, F. Nakamura, L-H. Wang, Y. Murakami, R.G. Kalb, H. Fujisawa, and S.M. Strittmatter, Cell (1999), 99: 59-69), which may make the B-domain-MAM-domain linker region particularly susceptible to conformational changes invoked by receptor complex formation. However, it is also possible that NP-1 is expressed more efficiently when it is co-expressed with VEGFR-2, since levels of NP-1 are undetectable by the NP1EDC1A antibody in 6 μg of total protein lysates from cells solely over expressing NP-1. (Figure 7D.) Such apparent differences in NP-1 expression are not apparent when NP-1 is assessed by affinity labeling with VEGF165 followed by immunoprecipitation by the NP1ECD4C antibody (Figure 5), again suggesting that the Western antibody is particularly sensitive to detection of differences of NP-1 expression in the presence or absence of VEGFR-2.
Finally, the data presented in Figure 7 illustrates the ability of the VEGFR-2 + NP-1 complex to form in the absence of ligand. Detection of the co-receptor complex is not an artifact created by use of the crosslinking agent (see Example 3 for methods) as it is readily detectable even in the absence of crosslinker (Figures 7A through 7D). This suggests that the VEGFR-2 + Neuropilin-1 receptor complex is ligand-independent and may exist as a pre-existing heterodimer on the cell surface under these assay conditions.
Example 5 Characterization of the VEGF Isoform Binding Profile of the VEGFR-2 + NP-1 Complex In order to characterize the binding properties of VEGFm and VEGF1 5 at the different VEGF receptors and receptor complexes, two separate assay types are employed. The first is the affinity labeling/immunoprecipitation technique described in Example 3. The second is referred to as whole cell binding competition analysis. In the whole cell binding assay, COS-1 cells are transfected with cDNA for VEGFR-2 or Neuropilin-1 by the methods described in Example 2, or endogenous HUVEC cells are utilized. After transfection (COS-1) or not (HUVEC cells, passages 1-4), cells are seeded into 12 well plates at 200,000 cells per well, and the binding experiments are carried out at 24 to 36 hr. after plating. At that time, cells are washed once with 4°C binding buffer (as in Example 3), then equilibrated in the same buffer at 4°C for 30 - 60 min with gentle shaking. The buffer is then aspirated, and to each well is added 500 μl of (4°C) binding buffer containing protease inhibitors and 1 μg/ml Heparin (as in Example 3), containing a single concentration of tracer (100 - 400 pM), as well as varying concentrations of unlabeled VEGFm, VEGFι65, or other unlabeled ligand, depending on the assay. For determination of nonspecific binding, VEGF165 is added to the binding buffer at a final concentration of 10 to 50 nM. The cells are incubated for 4 hr at 4°C with gentle shaking. At the end of the incubation period, the buffer is aspirated, and the cells are rinsed 3 times with 1 ml BSA- free binding buffer (Example 3). After the final wash is aspirated, 200 μl of RIPS buffer (20 mM Tris Base, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% doxycholic acid, 0.1% SDS, 10 mM Nal, 1% BSA, pH=8.0) is added to each well and incubated at room temperature for 15 - 30 min. The solubilized cells are then transferred to fresh tubes and counted in a Packard Model 5005 COBRA Gamma Counter (Packard Instruments, Meriden, CT). The binding curves are analyzed using the Prizm program (GraphPad, San Diego, CA).
Figure 8 demonstrates the results of the whole cell binding assay in either COS-1 cells overexpressing VEGFR-2 alone (Figure 8A) or in HUVECs (Figure 8B). VEGF,65 and VEGFm exhibit equivalent binding affinity at VEGFR-2, as indicated by the similar IC50 values (6.85 x 10" 1 1 M and 3.19 x 10"UM for VEGF1 and VEGF 121, respectively) in COS-1 cells overexpressing VEGFR-2. These data are in agreement with reported assays using a soluble VEGFR-2 ectodomain, in which VEGFm and VEGFι65 also have similar binding affinities (B.A. Keyt et al., J. Biol. Chem. (1996), 271 : 7788-7795), and supports the assertion that sequences contained the Exon 7 region are not necessary for interaction with VEGFR-2 (B.A. Keyt et al, (1996), J. Biol. Chem. 271: 7788-7795 and R. Tordjman et al., (1999), Blood, 94: (7): 2301-2309)). In contrast to the data obtained in COS-1 cells overexpressing VEGFR-2, the binding profiles of VEGF165 and VEGF121 are not identical in HUVEC cells (Figure 8B), despite the finding that HUVECs contain a substantial amount of immunoprecipitatable VEGFR-2 that is competent to both bind VEGF165 and be activated by it (Figures 6 and 10). In the HUVECs, VEGFι65 appears to bind with high affinity (IC50 = 5.61 x 10"" M), whereas VEGFm only competes for approximately 46% of the [125I]VEGFi65 binding sites (IC50 = 2.64 x 10"12 M). These data indicate that the majority of [l25I]VEGF165 binding sites in HUVECs are inaccessible for VEGFm binding and must, therefore, be something other than VEGFR-2 or the related receptor tyrosine kinase VEGFR-1, which also binds VEGFm with high affinity (B.A. Keyt et al, (1996), J. Biol. Chem. 271 : 7788-7795). Since the HUVECs also contain NP-1 in addition to VEGFR-2 (Figure 6), and since VEGFm does not bind to NP-1 (Figures 6 and 9; see also S. Soker, S. Takashima, H.Q. Miao, G. Neufeld, and M. Klagsbrun, (1998), Cell 92: (6):735-745 and R. Tordjman et al., (1999), Blood, 94: (7): 2301- 2309), the binding sites to which VEGFm do not have access may be explained as being NP-1, wherein NP-1 appears to represent the majority of the binding sites observable in HUVEC at the whole cell level. Since the expression of NP-1 appears to be in excess of that of VEGFR-2 (defining VEGFR-2 as accessible to both VEGF165 and VEGFm), and since VEGFR-2 can form a complex with NP-1 in the absence of ligand (Example 4), the inability of VEGFm to compete for a substantial portion of the [125I]VEGFi65 binding sites at either the whole cell level (Figure 8B), or in the affinity labeling/immunoprecipitation assay (Figure 6), can be explained as either an inaccessibility to NP-1 alone, or to VEGFR-2 when it is in a complex with NP-1.
This hypothesis can be tested directly in the COS-1 cell system using the affinity labeling/immunoprecipitation assay described in Example 3. Figure 9 illustrates that, while VEGFm is able to completely compete with [125I]VEGF165 binding to VEGFR-2 when it is expressed alone in COS-1 cells (Figure 9A), it does not compete with [I25I]VEGFι65 binding to NP-1 when it is expressed alone (Figure 9C) or in combination with VEGFR-2 (Figure 9B). Furthermore, VEGFm only partially competes for binding at VEGFR-2 in COS cells co- expressing VEGFR-2 + NP-1 (R2.2C IP, Figure 9B) indicating that VEGFm has only limited access to VEGFR-2 when it is in a complex with NP- 1.
Example 6
Demonstration of Differential Activity of VEGF^s vs. VEGFm for Stimulation of VEGFR-2 in HUVEC
In order to demonstrate the differential activation of VEGFR-2 by VEGFι6s and VEGFm binding, an anti-phosphotyrosine Western blot assay is employed. In this procedure, HUVEC cells (BioWhittaker, Walkersville, MD) are plated 7 days prior to stimulation at 500,00 cells per T75 flask (Corning, San Diego, CA) in EGM media (BioWhittaker, Walkersville, MD). With the exception of a single media change on day 3 following seeding, the cells are left undisturbed. The day of stimulation, the growth media is removed, the cells are rinsed, and returned to the 37°C incubator in serum-free DMEM (Gibco-BRL). After two to three hours in the serum-free media the cells are ready for stimulation. The ligands for stimulation are pre-diluted in 1.5 ml of stimulation buffer (25 mM HEPES, Gibco-BRL, DMEM, Gibco-BRL, 0.2% BSA, Sigma, St. Louis, MO, 1 μg/ml Heparin cat# H7399, Sigma, St. Louis, MO). The media is quickly removed, the stimulation buffer including the ligand is applied, and the cells are returned to the incubator. After exactly 5 minutes at 37°C the cells are placed on ice and the stimulation buffer is removed. A secondary aspiration is performed to ensure complete removal of the stimulation media. The cells are then lysed using 1.25 ml of RIPA buffer (20 mM Tris-HCL, pH=7.6, 150 mM NaCl, 50 mM NaF, 1 mM Sodium Orthovanadate, 5 mM Benzamadine, 1.5% NP-40, 0.5% Na- Deoxycholate, 0.1% SDS) supplemented with the protease inhibitor cocktail described in Example 3. The lysates are then transferred to 1.5 ml conical tubes on ice and further solubilized by 3 passes through a 23 gauge needle. The lysates are then centrifuged (13,000 x g, 5 minutes, 4°C) to clear the supernatant and the supernatants are transferred to fresh 1.5 ml conical tubes. 10 μg of the R2.2C antibody, and 100 μl of a 50:50 slurry of the protein G beads (described in Example 4) is then added to each sample. The samples are then placed at 4°C with neuration overnight, or for up to 60-72 hours. Following the overnight incubation the beads are pelleted at 13,000 x g for 5 minutes at 4°C and the supernatants aspirated. Three successive washes of the beads are performed using the RIPA buffer (described above) with the protease inhibitor cocktail (described in Example 3). After the final wash the pelleted beads are resuspended in 25 μl of IX sample loading buffer (0.25 M TrisCl, pH=6.8, 10% SDS, 0.5 M DTT, 0.5% bromophenol blue, 50% glycerol; diluted from 5X (Five Prime Three Prime, Boulder, CO, Cat # 2-910675). The samples are boiled for 2 min and centrifuged (13,0000 x g, 5 min). 20 μl of the supernatants are loaded onto 6% SDS-polyacrylamide gels (Novex, San Diego, CA) and subjected to (SDS-PAGE) electrophoresis; followed by the Western blotting procedure and blocked as described in Example 4. As in all of the Western blocking procedures described herein, the blocking buffer contains a form of BSA (FLUKA, obtained from Pharmacia, Piscataway, NJ, Cat # 05479), with low endogenous peroxidase activity to avoid high background with the ECL detection. Post blocking the membranes are probed for anti-phosphotyrosine using the 4G10 antibody (1:5,000, Upstate Biotechnology Inc., Lake Placid, NY) in primary probe buffer (Western blocking buffer diluted 1 :1 with TBST-0.1%) for two hours at room temperature with agitation. Prior to the addition of the HRP-labeled Goat anti-Mouse secondary antibody at 1 :40,000 (Pierce, Rockford, IL), the membranes are washed five times for six minutes in TBST-0.1% at room temperature with agitation. The secondary antibody is incubated on the membranes for one hour, after which they are washed at room temperature with agitation, successively as follows: 3 X 10 minutes with TBST-0.1%, 2 X 10 minutes with TBST-0.3%, and 3 X 5 minutes with TBS. Following the final wash step the membranes are incubated in the ECL solution for 1 minute according to the manufacturer's instructions, prior to exposure to Hyperfϊm (Amersham, Piscataway, NJ). After the detection of the anti-phosphotyrosine signal has been completed, the blots are re-hydrated with three 5 minute washes in TBS and then stripped overnight at room temperature with agitation in stripping buffer (200 mM Glycine pH=2.8). The following morning the stripping buffer is removed with three 5 minute washes of TBST-0.1% (described above). The membranes are then subjected to the 2 hour blocking, probing, and detection steps described above. During this round of detection the membranes are probed using the R2.2C antibody for VEGFR-2 (1 :5,000) and an HRP-labeled Goat anti-Rabbit secondary antibody at 1 :40,000 (Pierce, Rockford, IL). Re-probing with the VEGFR-2 antibody allows the phosphotyrosine signal to be normalized to the amount of VEGFR-2 present in the immunoprecipitate for quantitation of the response. Quantitation of the signal is achieved using Image Quant software (Molecular Dynamics, Sunnyvale, CA) after scanning the film on a Scanmaster 2500 (Howtek, Hudson, NH). The results of the anti-phosphotyrosine assay are illustrated in Figure 10. In HUVEC cells the activation of VEGFR-2 autophosphorylation by VEGFι6s is readily apparent at a concentration of 100 pM, and is maximal at 300 pM (EC50 -1.25 x 10"10 M; Figure 10A). In contrast, the response to VEGFm is not apparent until a concentration of 1 nM, and is maximal at 10 nM (EC50 -2.68 x 10"9 M; Figure 10B). This result is surprising given that VEGFι65 and VEGFm have similar binding affinity to VEGFR-2 (Example 5 and B.A. Keyt et al, (1996), J. Biol. Chem. 271 : 7788-7795), and the response that is measured here is a direct activation of VEGFR-2, rather than a more downstream event that may be subject to post-receptor regulation. We postulate that this difference in potency in the ability to activate VEGFR-2 results from an inability of VEGFm to bind to the VEGFR-2 + NP-1 complex, and that the presence of NP-1 in the VEGFR-2 complex augments signaling through VEGFR-2. Hence, while VEGF16s and VEGFm may have similar binding affinity at VEGFR-2, the ability of VEGFι6s to bind to the VEGFR-2 + NP-1 complex allows VEGF to signal more efficiently through VEGFR-2, thereby inducing receptor autophosphorylation at lower concentrations than that observed with VEGF 121.
Example 7 Use of the VEGFR-2 + NP-1 Complex in a Receptor Binding Assay for the Identification of VEGF Receptor Agonists and Antagonists
Identification of ligands that interact with the VEGFR-2 + NP-1 complex can be achieved through the use of assays that are designed to measure the interaction of the ligands with this VEGF receptor complex. A receptor binding assay that uses the VEGFR-2 + NP-1 complex and is adapted to handle large numbers of samples is carried out as follows:
The whole cell binding assay is carried out as described in Example 5, except that either single or increasing concentrations of test compounds are used in place of VEGFm (Example 5, Figure 8). In this manner, binding can be tested either at NP-1 alone, VEGFR-2 alone, or at the VEGFR-2 + NP-1 complex in COS-1 cells overexpressing these receptors, or in alternate cell lines engineered to stably overexpress VEGFR-2 + NP-1, using methods readily available to those skilled in the art. The binding data is analyzed using the Prizm program (GraphPad, San Diego, CA) as described in Example 5. When only a single concentration of test compounds is used, test compounds which interact with the VEGFR-2 + NP-1 receptor complex are observed to compete for binding to the receptor complex with the [ IjVEGFiβs tracer, such that less [ I]VEGF16s tracer is bound in the presence of the test compound in comparison to the binding observed when
125 the tracer is incubated in the absence of the novel compound. A decrease in binding of the [ I] VEGF165 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the VEGFR-2 + NP-1 receptor complex.
Alternately, the binding assay can be carried out using soluble receptor proteins in an ELISA-based capture assay format, similar to that described in the scientific literature for soluble VEGFR-2 (B.A. Keyt et al, (1996), J. Biol. Chem. 271: 7788-7795; G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J. Biol. Chem. 273: (18):11197-11204) or for soluble NP-1 (L.M. Wise, et al, (1999), Proc. Nat. Acad. Sci. Usa 96: (6):3071-3076) except that here the soluble VEGFR-2 and soluble NP-1 receptor proteins would be combined prior to incubation with the labeled ligand. In this case the soluble VEGFR-2 receptor would include the extracellular domains present in SEQ ID NO. 5 and SEQ ID NO. 6, or portions thereof, especially those portions including Ig domains 2 and 3, encompassing amino acids 124 to 320, as Ig domains 2 and 3 have been determined to be necessary for high affinity VEGF)65 binding to VEGFR-2 (G. Fuh, B. Li, C. Crowley, B. Cunningham, J.A. Wells, (1998), J Biol Chem 273: (18):11197-11204; A. Shinkai, et al,. (1998), J. Biol. Chem. 273: (47):31283-31288). In this case the soluble NP-1 receptor would include the extracellular domains present in SEQ ID NO. 7 and SEQ ID NO. 8, or portions thereof, especially those portions including the B domains (also known as the coagulation factor domains), encompassing amino acids 274 to 647, as the B domains have been determined to be necessary for VEGF165 binding to NP-1 (RJ. Giger, et al, (1998), Neuron 21 : (5):1079-1092); see also the PCT publication Number WO 99/29858). As in the discussion provided above for the use of VEGFR-2 + NP-1 complex in a whole cell binding assay, either increasing concentrations or a single concentration of test compounds could be added in combination with the labeled VEGF,65. where VEGF,65 is either radiolabeled (e.g. as [l25I]VEGF,65) or is labeled with a tracer than can then be detected by a fluorometer (e.g., europium-labeled VEGF,6s). When only a single concentration of test compounds is used, test compounds which interact with the VEGFR-2 + NP- 1 receptor complex are observed to compete for binding to the receptor complex with the labeled VEGF,65 tracer, such that less VEGF,65 tracer is bound in the presence of the test compound in comparison to the binding observed when the tracer is incubated in the absence of the novel compound. A decrease in binding of the VEGF, 65 tracer by > 30% at the highest concentration of the test compound that is studied demonstrates that the test compound binds to the VEGFR-2 + NP-1 receptor complex. Example 8
Use of VEGFR-2 + NP-1 in a Signaling Assay for the Identification of VEGF Receptor Agonists and Antagonists
Identification of ligands that signal upon interaction with the VEGFR-2 + NP-1 receptor complex can be achieved through the use of assays that are designed to measure the activation of the receptor protein kinase domain after binding of the ligand to the receptor complex. One assay that is used is similar to that described for measuring the relative potency of VEGF ,65 vs. VEGF ,2, alone (Example 6), except that in this case test compounds would be added in the absence of VEGF,65 or VEGFm, at either a single concentration or at multiple concentrations. A test compound that measurably increases the phosphotyrosine content of the immunoprecipitated VEGFR-2 above that observed in the absence of compound is considered an agonist of either VEGFR-2 or of the VEGFR-2 + NP-1 receptor complex.
A compound that is an antagonist at the VEGFR-2 + NP-1 complex can be detected using an anti-phosphotyrosine Western blot assay similar to that previously described in Example 6 (Figure 10), with the following modifications: (1) In one assay format, increasing concentrations of either VEGF,6s or VEGF,2, are used alone, or in the presence of a single concentration of the test compound. A compound is determined to be an antagonist of the VEGFR-2 + NP- 1 complex if the concentration-response curve is shifted to the right for VEGF,65, but the concentration- response curve for VEGFm is not shifted substantially to the right. In this case a "substantial" shift is one that results in an increase in the EC5o of VEGF,65 or VEGFm by a factor > 5. (2) In another assay format, antagonistic activity can be detected using a single concentration of either VEGF,65 or VEGF12, (e.g., a concentration that is > EC50 for increasing phosphotyrosine content of the immunoprecipitated VEGFR-2 receptor), in the presence of increasing concentrations of the test compound. In this case the response of VEGF,65 should be decreased by at least 30%, whereas a substantial decrease in the VEGFm response would not be obtained. This latter method is preferred when the binding affinity of the test compound is unknown, such that multiple concentrations of the test compound would need to be evaluated for antagonistic activity. The anti-phosphotyrosine assay described above can be performed in HUVECs or any other cell line determined to express VEGFR-2 + NP-1, in which VEGF,65 is a more potent agent than is VEGFm in signaling assays that are dependent on activation of VEGFR-2. The assay can also be performed in cells engineered to overexpress VEGFR-2 + NP-1, using methodology common to those skilled in the art. A stable cell line (generated from Balb/c 3T3 clone A31 ; ATCC # CCL-163) exhibiting these characteristics, named D7R2/NP1#4, has been deposited with the ATCC on September 21, 2000, and assigned ATCC Designation No. .
Deposit of Human NP-1 :
The human NP-1 gene (SEQ ID NO. 3) was incorporated into the vector pBluescript II SK+, which was deposited with the ATCC on October 20, 1999, and assigned ATCC Designation No. PTA-858. The vector is commercially available from Stratagene (La Jolla, CA). The gene was sub-cloned into the EcoRI and Xbal restriction sites of the vector.
As is recognized in the art, there are occasionally errors in DNA and amino acid sequencing methods. As a result, the sequence encoded in the deposited material are incorporated herein by reference and controlling in the event of an error in any of the sequences found in the written description of the present invention. It is further noted that one of ordinary skill in the art reproducing Applicants' work from the written disclosure can discover any sequencing errors using routine skill. The deposit of ATCC Nos. PTA-858 and are not to be considered as an admission that the deposited materials are essential to the practice of the present invention.
All publications mentioned hereinabove are hereby incorporated in their entirety by reference. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to one skilled in the art and are to be included in the spirit and purview of this application and scope of the appended claims. SEQUENCE LISTINGS
INFORMATION FOR SEQ ID No. 1 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4226 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(iii) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 70...4140
(iv) SEQUENCE DESCRIPTION: SEQ ID No.1 10 20 30 40
TCT AGA CAG GCG CTG GGA GAA AGA ACC GGC TCC CGA GTT CTG GGC ATT AGA TCT GTC CGC GAC CCT CTT TCT TGG CCG AGG GCT CAA GAC CCG TAA>
50 60 70 80 90 TCG CCC GGC TCG AGG TGC AGG ATG CAG AGC AAG GTG CTG CTG GCC GTC AGC GGG CCG AGC TCC ACG TCC TAC GTC TCG TTC CAC GAC GAC CGG CAG
Met Gin Ser Lys Val Leu Leu Ala Val>
100 110 120 130 140 GCC CTG TGG CTC TGC GTG GAG ACC CGG GCC GCC TCT GTG GGT TTG CCT CGG GAC ACC GAG ACG CAC CTC TGG GCC CGG CGG AGA CAC CCA AAC GGA Ala Leu Trp Leu Cys Val Glu Thr Arg Ala Ala Ser Val Gly Leu Pro>
150 160 170 180 190 AGT GTT TCT CTT GAT CTG CCC AGG CTC AGC ATA CAA AAA GAC ATA CTT TCA CAA AGA GAA CTA GAC GGG TCC GAG TCG TAT GTT TTT CTG TAT GAA Ser Val Ser Leu Asp Leu Pro Arg Leu Ser lie Gin Lys Asp lie Leu>
200 210 220 230 240 ACA ATT AAG GCT AAT ACA ACT CTT CAA ATT ACT TGC AGG GGA CAG AGG
TGT TAA TTC CGA TTA TGT TGA GAA GTT TAA TGA ACG TCC CCT GTC TCC
Thr lie Lys Ala Asn Thr Thr Leu Gin lie Thr Cys Arg Gly Gin Arg>
250 260 270 280 GAC TTG GAC TGG CTT TGG CCC AAT AAT CAG AGT GGC AGT GAG CAA AGG CTG AAC CTG ACC GAA ACC GGG TTA TTA GTC TCA CCG TCA CTC GTT TCC Asp Leu Asp Trp Leu Trp Pro Asn Asn Gin Ser Gly Ser Glu Gin Arg>
290 300 310 320 330 GTG GAG GTG ACT GAG TGC AGC GAT GGC CTC TTC TGT AAG ACA CTC ACA
CAC CTC CAC TGA CTC ACG TCG CTA CCG GAG AAG ACA TTC TGT GAG TGT
Val Glu Val Thr Glu Cys Ser Asp Gly Leu Phe Cys Lys Thr Leu Thr> 340 350 360 370 380 ATT CCA AAA GTG ATC GGA AAT GAC ACT GGA GCC TAC AAG TGC TTC TAC
TAA GGT TTT CAC TAG CCT TTA CTG TGA CCT CGG ATG TTC ACG AAG ATG
He Pro Lys Val He Gly Asn Asp Thr Gly Ala Tyr Lys Cys Phe Tyr>
390 400 410 420 430 CGG GAA ACT GAC TTG GCC TCG GTC ATT TAT GTC TAT GTT CAA GAT TAC GCC CTT TGA CTG AAC CGG AGC CAG TAA ATA CAG ATA CAA GTT CTA ATG Arg Glu Thr Asp Leu Ala Ser Val He Tyr Val Tyr Val Gin Asp Tyr>
440 450 460 470 480 AGA TCT CCA TTT ATT GCT TCT GTT AGT GAC CAA CAT GGA GTC GTG TAC TCT AGA GGT AAA TAA CGA AGA CAA TCA CTG GTT GTA CCT CAG CAC ATG Arg Ser Pro Phe He Ala Ser Val Ser Asp Gin His Gly Val Val Tyr>
490 500 510 520 ATT ACT GAG AAC AAA AAC AAA ACT GTG GTG ATT CCA TGT CTC GGG TCC TAA TGA CTC TTG TTT TTG TTT TGA CAC CAC TAA GGT ACA GAG CCC AGG He Thr Glu Asn Lys Asn Lys Thr Val Val He Pro Cys Leu Gly Ser> 30 540 550 560 570 ATT TCA AAT CTC AAC GTG TCA CTT TGT GCA AGA TAC CCA GAA AAG AGA
TAA AGT TTA GAG TTG CAC AGT GAA ACA CGT TCT ATG GGT CTT TTC TCT
He Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg>
580 590 600 610 620 TTT GTT CCT GAT GGT AAC AGA ATT TCC TGG GAC AGC AAG AAG GGC TTT AAA CAA GGA CTA CCA TTG TCT TAA AGG ACC CTG TCG TTC TTC CCG AAA Phe Val Pro Asp Gly Asn Arg He Ser Trp Asp Ser Lys Lys Gly Phe>
630 640 650 660 670 ACT ATT CCC AGC TAC ATG ATC AGC TAT GCT GGC ATG GTC TTC TGT GAA TGA TAA GGG TCG ATG TAC TAG TCG ATA CGA CCG TAC CAG AAG ACA CTT Thr He Pro Ser Tyr Met He Ser Tyr Ala Gly Met Val Phe Cys Glu>
680 690 700 710 720 GCA AAA ATT AAT GAT GAA AGT TAC CAG TCT ATT ATG TAC ATA GTT GTC CGT TTT TAA TTA CTA CTT TCA ATG GTC AGA TAA TAC ATG TAT CAA CAG Ala Lys He Asn Asp Glu Ser Tyr Gin Ser He Met Tyr He Val Val>
730 740 750 760 GTT GTA GGG TAT AGG ATT TAT GAT GTG GTT CTG AGT CCG TCT CAT GGA CAA CAT CCC ATA TCC TAA ATA CTA CAC CAA GAC TCA GGC AGA GTA CCT Val Val Gly Tyr Arg He Tyr Asp Val Val Leu Ser Pro Ser His Gly> 70 780 790 800 810 ATT GAA CTA TCT GTT GGA GAA AAG CTT GTC TTA AAT TGT ACA GCA AGA TAA CTT GAT AGA CAA CCT CTT TTC GAA CAG AAT TTA ACA TGT CGT TCT He Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg>
820 830 840 850 860 ACT GAA CTA AAT GTG GGG ATT GAC TTC AAC TGG GAA TAC CCT TCT TCG
TGA CTT GAT TTA CAC CCC TAA CTG AAG TTG ACC CTT ATG GGA AGA AGC
Thr Glu Leu Asn Val Gly He Asp Phe Asn Trp Glu Tyr Pro Ser Ser>
870 880 890 900 910 AAG CAT CAG CAT AAG AAA CTT GTA AAC CGA GAC CTA AAA ACC CAG TCT TTC GTA GTC GTA TTC TTT GAA CAT TTG GCT CTG GAT TTT TGG GTC AGA Lys His Gin His Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gin Ser>
920 930 940 950 960 110 GGG AGT GAG ATG AAG AAA TTT TTG AGC ACC TTA ACT ATA GAT GGT GTA CCC TCA CTC TAC TTC TTT AAA AAC TCG TGG AAT TGA TAT CTA CCA CAT Gly Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr He Asp Gly Val>
970 980 990 1000
115 ACC CGG AGT GAC CAA GGA TTG TAC ACC TGT GCA GCA TCC AGT GGG CTG TGG GCC TCA CTG GTT CCT AAC ATG TGG ACA CGT CGT AGG TCA CCC GAC Thr Arg Ser Asp Gin Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu>
1010 1020 1030 1040 1050
120 ATG ACC AAG AAG AAC AGC ACA TTT GTC AGG GTC CAT GAA AAA CCT TTT
TAC TGG TTC TTC TTG TCG TGT AAA CAG TCC CAG GTA CTT TTT GGA AAA
Met Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Pro Phe>
1060 1070 1080 1090 1100
125 GTT GCT TTT GGA AGT GGC ATG GAA TCT CTG GTG GAA GCC ACG GTG GGG CAA CGA AAA CCT TCA CCG TAC CTT AGA GAC CAC CTT CGG TGC CAC CCC Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly>
1110 1120 1130 1140 1150
130 GAG CGT GTC AGA ATC CCT GCG AAG TAC CTT GGT TAC CCA CCC CCA GAA CTC GCA CAG TCT TAG GGA CGC TTC ATG GAA CCA ATG GGT GGG GGT CTT Glu Arg Val Arg He Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro Glu>
1160 1170 1180 1190 1200
135 ATA AAA TGG TAT AAA AAT GGA ATA CCC CTT GAG TCC AAT CAC ACA ATT TAT TTT ACC ATA TTT TTA CCT TAT GGG GAA CTC AGG TTA GTG TGT TAA He Lys Trp Tyr Lys Asn Gly He Pro Leu Glu Ser Asn His Thr He>
1210 1220 1230 1240
140 AAA GCG GGG CAT GTA CTG ACG ATT ATG GAA GTG AGT GAA AGA GAC ACA
TTT CGC CCC GTA CAT GAC TGC TAA TAC CTT CAC TCA CTT TCT CTG TGT
Lys Ala Gly His Val Leu Thr He Met Glu Val Ser Glu Arg Asp Thr>
1250 1260 1270 1280 1290
145 GGA AAT TAC ACT GTC ATC CTT ACC AAT CCC ATT TCA AAG GAG AAG CAG CCT TTA ATG TGA CAG TAG GAA TGG TTA GGG TAA AGT TTC CTC TTC GTC Gly Asn Tyr Thr Val He Leu Thr Asn Pro He Ser Lys Glu Lys Gln>
1300 1310 1320 1330 1340
150 AGC CAT GTG GTC TCT CTG GTT GTG TAT GTC CCA CCC CAG ATT GGT GAG
TCG GTA CAC CAG AGA GAC CAA CAC ATA CAG GGT GGG GTC TAA CCA CTC
Ser His Val Val Ser Leu Val Val Tyr Val Pro Pro Gin He Gly Glu>
1350 1360 1370 1380 1390
155 AAA TCT CTA ATC TCT CCT GTG GAT TCC TAC CAG TAC GGC ACC ACT CAA TTT AGA GAT TAG AGA GGA CAC CTA AGG ATG GTC ATG CCG TGG TGA GTT Lys Ser Leu He Ser Pro Val Asp Ser Tyr Gin Tyr Gly Thr Thr Gln>
1400 1410 1420 1430 1440
160 ACG CTG ACA TGT ACG GTC TAT GCC ATT CCT CCC CCG CAT CAC ATC CAC TGC GAC TGT ACA TGC CAG ATA CGG TAA GGA GGG GGC GTA GTG TAG GTG Thr Leu Thr Cys Thr Val Tyr Ala He Pro Pro Pro His His He His>
1450 1460 1470 1480
165 TGG TAT TGG CAG TTG GAG GAA GAG TGC GCC AAC GAG CCC AGC CAA GCT ACC ATA ACC GTC AAC CTC CTT CTC ACG CGG TTG CTC GGG TCG GTT CGA Trp Tyr Trp Gin Leu Glu Glu Glu Cys Ala Asn Glu Pro Ser Gin Ala>
1490 1500 1510 1520 1530
170 GTC TCA GTG ACA AAC CCA TAC CCT TGT GAA GAA TGG AGA AGT GTG GAG CAG AGT CAC TGT TTG GGT ATG GGA ACA CTT CTT ACC TCT TCA CAC CTC Val Ser Val Thr Asn Pro Tyr Pro Cys Glu Glu Trp Arg Ser Val Glu>
1540 1550 1560 1570 1580
175 GAC TTC CAG GGA GGA AAT AAA ATT GAA GTT AAT AAA AAT CAA TTT GCT CTG AAG GTC CCT CCT TTA TTT TAA CTT CAA TTA TTT TTA GTT AAA CGA Asp Phe Gin Gly Gly Asn Lys He Glu Val Asn Lys Asn Gin Phe Ala>
1590 1600 1610 1620 1630
180 CTA ATT GAA GGA AAA AAC AAA ACT GTA AGT ACC CTT GTT ATC CAA GCG GAT TAA CTT CCT TTT TTG TTT TGA CAT TCA TGG GAA CAA TAG GTT CGC Leu He Glu Gly Lys Asn Lys Thr Val Ser Thr Leu Val He Gin Ala>
1640 1650 1660 1670 1680
185 GCA AAT GTG TCA GCT TTG TAC AAA TGT GAA GCG GTC AAC AAA GTC GGG CGT TTA CAC AGT CGA AAC ATG TTT ACA CTT CGC CAG TTG TTT CAG CCC Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Val Asn Lys Val Gly>
1690 1700 1710 1720
190 AGA GGA GAG AGG GTG ATC TCC TTC CAC GTG ACC AGG GGT CCT GAA ATT TCT CCT CTC TCC CAC TAG AGG AAG GTG CAC TGG TCC CCA GGA CTT TAA Arg Gly Glu Arg Val He Ser Phe His Val Thr Arg Gly Pro Glu He>
1730 1740 1750 1760 1770
195 ACT TTG CAA CCT GAC ATG CAG CCC ACT GAG CAG GAG AGC GTG TCT TTG TGA AAC GTT GGA CTG TAC GTC GGG TGA CTC GTC CTC TCG CAC AGA AAC Thr Leu Gin Pro Asp Met Gin Pro Thr Glu Gin Glu Ser Val Ser Leu>
1780 1790 1800 1810 1820
200 TGG TGC ACT GCA GAC AGA TCT ACG TTT GAG AAC CTC ACA TGG TAC AAG ACC ACG TGA CGT CTG TCT AGA TGC AAA CTC TTG GAG TGT ACC ATG TTC Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu Asn Leu Thr Trp Tyr Lys>
1830 1840 1850 1860 1870
205 CTT GGC CCA CAG CCT CTG CCA ATC CAT GTG GGA GAG TTG CCC ACA CCT
GAA CCG GGT GTC GGA GAC GGT TAG GTA CAC CCT CTC AAC GGG TGT GGA
Leu Gly Pro Gin Pro Leu Pro He His Val Gly Glu Leu Pro Thr Pro>
1880 1890 1900 1910 1920
210 GTT TGC AAG AAC TTG GAT ACT CTT TGG AAA TTG AAT GCC ACC ATG TTC CAA ACG TTC TTG AAC CTA TGA GAA ACC TTT AAC TTA CGG TGG TAC AAG Val Cys Lys Asn Leu Asp Thr Leu Trp Lys Leu Asn Ala Thr Met Phe>
1930 1940 1950 1960
215 TCT AAT AGC ACA AAT GAC ATT TTG ATC ATG GAG CTT AAG AAT GCA TCC AGA TTA TCG TGT TTA CTG TAA AAC TAG TAC CTC GAA TTC TTA CGT AGG Ser Asn Ser Thr Asn Asp He Leu He Met Glu Leu Lys Asn Ala Ser>
1970 1980 1990 2000 2010
220 TTG CAG GAC CAA GGA GAC TAT GTC TGC CTT GCT CAA GAC AGG AAG ACC AAC GTC CTG GTT CCT CTG ATA CAG ACG GAA CGA GTT CTG TCC TTC TGG Leu Gin Asp Gin Gly Asp Tyr Val Cys Leu Ala Gin Asp Arg Lys Thr>
2020 2030 2040 2050 2060
225 AAG AAA AGA CAT TGC GTG GTC AGG CAG CTC ACA GTC CTA GAG CGT GTG TTC TTT TCT GTA ACG CAC CAG TCC GTC GAG TGT CAG GAT CTC GCA CAC Lys Lys Arg His Cys Val Val Arg Gin Leu Thr Val Leu Glu Arg Val>
2070 2080 2090 2100 2110
230 GCA CCC ACG ATC ACA GGA AAC CTG GAG AAT CAG ACG ACA AGT ATT GGG CGT GGG TGC TAG TGT CCT TTG GAC CTC TTA GTC TGC TGT TCA TAA CCC Ala Pro Thr He Thr Gly Asn Leu Glu Asn Gin Thr Thr Ser He Gly>
2120 2130 2140 2150 2160
235 GAA AGC ATC GAA GTC TCA TGC ACG GCA TCT GGG AAT CCC CCT CCA CAG CTT TCG TAG CTT CAG AGT ACG TGC CGT AGA CCC TTA GGG GGA GGT GTC Glu Ser He Glu Val Ser Cys Thr Ala Ser Gly Asn Pro Pro Pro Gln>
2170 2180 2190 2200
240 ATC ATG TGG TTT AAA GAT AAT GAG ACC CTT GTA GAA GAC TCA GGC ATT TAG TAC ACC AAA TTT CTA TTA CTC TGG GAA CAT CTT CTG AGT CCG TAA He Met Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser Gly He>
2210 2220 2230 2240 2250
245 GTA TTG AAG GAT GGG AAC CGG AAC CTC ACT ATC CGC AGA GTG AGG AAG CAT AAC TTC CTA CCC TTG GCC TTG GAG TGA TAG GCG TCT CAC TCC TTC Val Leu Lys Asp Gly Asn Arg Asn Leu Thr He Arg Arg Val Arg Lys>
2260 2270 2280 2290 2300
250 GAG GAC GAA GGC CTC TAC ACC TGC CAG GCA TGC AGT GTT CTT GGC TGT CTC CTG CTT CCG GAG ATG TGG ACG GTC CGT ACG TCA CAA GAA CCG ACA Glu Asp Glu Gly Leu Tyr Thr Cys Gin Ala Cys Ser Val Leu Gly Cys>
2310 2320 2330 2340 2350
255 GCA AAA GTG GAG GCA TTT TTC ATA ATA GAA GGT GCC CAG GAA AAG ACG
CGT TTT CAC CTC CGT AAA AAG TAT TAT CTT CCA CGG GTC CTT TTC TGC
Ala Lys Val Glu Ala Phe Phe He He Glu Gly Ala Gin Glu Lys Thr>
2360 2370 2380 2390 2400
260 AAC TTG GAA ATC ATT ATT CTA GTA GGC ACG GCG GTG ATT GCC ATG TTC TTG AAC CTT TAG TAA TAA GAT CAT CCG TGC CGC CAC TAA CGG TAC AAG Asn Leu Glu He He He Leu Val Gly Thr Ala Val He Ala Met Phe>
2410 2420 2430 2440
265 TTC TGG CTA CTT CTT GTC ATC ATC CTA CGG ACC GTT AAG CGG GCC AAT AAG ACC GAT GAA GAA CAG TAG TAG GAT GCC TGG CAA TTC GCC CGG TTA Phe Trp Leu Leu Leu Val He He Leu Arg Thr Val Lys Arg Ala Asn>
2450 2460 2470 2480 2490
270 GGA GGG GAA CTG AAG ACA GGC TAC TTG TCC ATC GTC ATG GAT CCA GAT CCT CCC CTT GAC TTC TGT CCG ATG AAC AGG TAG CAG TAC CTA GGT CTA Gly Gly Glu Leu Lys Thr Gly Tyr Leu Ser He Val Met Asp Pro Asp>
2500 2510 2520 2530 2540
275 GAA CTC CCA TTG GAT GAA CAT TGT GAA CGA CTG CCT TAT GAT GCC AGC
CTT GAG GGT AAC CTA CTT GTA ACA CTT GCT GAC GGA ATA CTA CGG TCG
Glu Leu Pro Leu Asp Glu His Cys Glu Arg Leu Pro Tyr Asp Ala Ser>
2550 2560 2570 2580 2590
280 AAA TGG GAA TTC CCC AGA GAC CGG CTG AAG CTA GGT AAG CCT CTT GGC TTT ACC CTT AAG GGG TCT CTG GCC GAC TTC GAT CCA TTC GGA GAA CCG Lys Trp Glu Phe Pro Arg Asp Arg Leu Lys Leu Gly Lys Pro Leu Gly>
2600 2610 2620 2630 2640
285 CGT GGT GCC TTT GGC CAA GTG ATT GAA GCA GAT GCC TTT GGA ATT GAC
GCA CCA CGG AAA CCG GTT CAC TAA CTT CGT CTA CGG AAA CCT TAA CTG
Arg Gly Ala Phe Gly Gin Val He Glu Ala Asp Ala Phe Gly He Asp>
2650 2660 2670 2680
290 AAG ACA GCA ACT TGC AGG ACA GTA GCA GTC AAA ATG TTG AAA GAA GGA TTC TGT CGT TGA ACG TCC TGT CAT CGT CAG TTT TAC AAC TTT CTT CCT Lys Thr Ala Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys Glu Gly> 2690 2700 2710 2720 2730
295 GCA ACA CAC AGT GAG CAT CGA GCT CTC ATG TCT GAA CTC AAG ATC CTC CGT TGT GTG TCA CTC GTA GCT CGA GAG TAC AGA CTT GAG TTC TAG GAG Ala Thr His Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys He Leu>
2740 2750 2760 2770 2780
300 ATT CAT ATT GGT CAC CAT CTC AAT GTG GTC AAC CTT CTA GGT GCC TGT TAA GTA TAA CCA GTG GTA GAG TTA CAC CAG TTG GAA GAT CCA CGG ACA He His He Gly His His Leu Asn Val Val Asn Leu Leu Gly Ala Cys>
2790 2800 2810 2820 2830
305 ACC AAG CCA GGA GGG CCA CTC ATG GTG ATT GTG GAA TTC TGC AAA TTT TGG TTC GGT CCT CCC GGT GAG TAC CAC TAA CAC CTT AAG ACG TTT AAA Thr Lys Pro Gly Gly Pro Leu Met Val He Val Glu Phe Cys Lys Phe>
2840 2850 2860 2870 2880
310 GGA AAC CTG TCC ACT TAC CTG AGG AGC AAG AGA AAT GAA TTT GTC CCC CCT TTG GAC AGG TGA ATG GAC TCC TCG TTC TCT TTA CTT AAA CAG GGG Gly Asn Leu Ser Thr Tyr Leu Arg Ser Lys Arg Asn Glu Phe Val Pro>
2890 2900 2910 2920
315 TAC AAG ACC AAA GGG GCA CGA TTC CGT CAA GGG AAA GAC TAC GTT GGA
ATG TTC TGG TTT CCC CGT GCT AAG GCA GTT CCC TTT CTG ATG CAA CCT
Tyr Lys Thr Lys Gly Ala Arg Phe Arg Gin Gly Lys Asp Tyr Val Gly>
2930 2940 2950 2960 2970
320 GCA ATC CCT GTG GAT CTG AAA CGG CGC TTG GAC AGC ATC ACC AGT AGC CGT TAG GGA CAC CTA GAC TTT GCC GCG AAC CTG TCG TAG TGG TCA TCG Ala He Pro Val Asp Leu Lys Arg Arg Leu Asp Ser He Thr Ser Ser>
2980 2990 3000 3010 3020
325 CAG AGC TCA GCC AGC TCT GGA TTT GTG GAG GAG AAG TCC CTC AGT GAT GTC TCG AGT CGG TCG AGA CCT AAA CAC CTC CTC TTC AGG GAG TCA CTA Gin Ser Ser Ala Ser Ser Gly Phe Val Glu Glu Lys Ser Leu Ser Asp>
3030 3040 3050 3060 3070
330 GTA GAA GAA GAG GAA GCT CCT GAA GAT CTG TAT AAG GAC TTC CTG ACC
CAT CTT CTT CTC CTT CGA GGA CTT CTA GAC ATA TTC CTG AAG GAC TGG
Val Glu Glu Glu Glu Ala Pro Glu Asp Leu Tyr Lys Asp Phe Leu Thr>
3080 3090 3100 3110 3120
335 TTG GAG CAT CTC ATC TGT TAC AGC TTC CAA GTG GCT AAG GGC ATG GAG AAC CTC GTA GAG TAG ACA ATG TCG AAG GTT CAC CGA TTC CCG TAC CTC Leu Glu His Leu He Cys Tyr Ser Phe Gin Val Ala Lys Gly Met Glu>
3130 3140 3150 3160
340 TTC TTG GCA TCG CGA AAG TGT ATC CAC AGG GAC CTG GCG GCA CGA AAT AAG AAC CGT AGC GCT TTC ACA TAG GTG TCC CTG GAC CGC CGT GCT TTA Phe Leu Ala Ser Arg Lys Cys He His Arg Asp Leu Ala Ala Arg Asn>
3170 3180 3190 3200 3210
345 ATC CTC TTA TCG GAG AAG AAC GTG GTT AAA ATC TGT GAC TTT GGC TTG TAG GAG AAT AGC CTC TTC TTG CAC CAA TTT TAG ACA CTG AAA CCG AAC He Leu Leu Ser Glu Lys Asn Val Val Lys He Cys Asp Phe Gly Leu>
3220 3230 3240 3250 3260
350 GCC CGG GAT ATT TAT AAA GAT CCA GAT TAT GTC AGA AAA GGA GAT GCT CGG GCC CTA TAA ATA TTT CTA GGT CTA ATA CAG TCT TTT CCT CTA CGA Ala Arg Asp He Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala> 3270 3280 3290 3300 3310
355 CGC CTC CCT TTG AAA TGG ATG GCC CCA GAA ACA ATT TTT GAC AGA GTG GCG GAG GGA AAC TTT ACC TAC CGG GGT CTT TGT TAA AAA CTG TCT CAC Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Thr He Phe Asp Arg Val>
3320 3330 3340 3350 3360
360 TAC ACA ATC CAG AGT GAC GTC TGG TCT TTT GGT GTT TTG CTG TGG GAA ATG TGT TAG GTC TCA CTG CAG ACC AGA AAA CCA CAA AAC GAC ACC CTT Tyr Thr He Gin Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu>
3370 3380 3390 3400
365 ATA TTT TCC TTA GGT GCT TCT CCA TAT CCT GGG GTA AAG ATT GAT GAA
TAT AAA AGG AAT CCA CGA AGA GGT ATA GGA CCC CAT TTC TAA CTA CTT
He Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys He Asp Glu>
3410 3420 3430 3440 3450
370 GAA TTT TGT AGG CGA TTG AAA GAA GGA ACT AGA ATG AGG GCC CCT GAT
CTT AAA ACA TCC GCT AAC TTT CTT CCT TGA TCT TAC TCC CGG GGA CTA
Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp>
3460 3470 3480 3490 3500
375 TAT ACT ACA CCA GAA ATG TAC CAG ACC ATG CTG GAC TGC TGG CAC GGG ATA TGA TGT GGT CTT TAC ATG GTC TGG TAC GAC CTG ACG ACC GTG CCC Tyr Thr Thr Pro Glu Met Tyr Gin Thr Met Leu Asp Cys Trp His Gly>
3510 3520 3530 3540 3550
380 GAG CCC AGT CAG AGA CCC ACG TTT TCA GAG TTG GTG GAA CAT TTG GGA CTC GGG TCA GTC TCT GGG TGC AAA AGT CTC AAC CAC CTT GTA AAC CCT Glu Pro Ser Gin Arg Pro Thr Phe Ser Glu Leu Val Glu His Leu Gly>
3560 3570 3580 3590 3600
385 AAT CTC TTG CAA GCT AAT GCT CAG CAG GAT GGC AAA GAC TAC ATT GTT TTA GAG AAC GTT CGA TTA CGA GTC GTC CTA CCG TTT CTG ATG TAA CAA Asn Leu Leu Gin Ala Asn Ala Gin Gin Asp Gly Lys Asp Tyr He Val>
3610 3620 3630 3640
390 CTT CCG ATA TCA GAG ACT TTG AGC ATG GAA GAG GAT TCT GGA CTC TCT GAA GGC TAT AGT CTC TGA AAC TCG TAC CTT CTC CTA AGA CCT GAG AGA Leu Pro He Ser Glu Thr Leu Ser Met Glu Glu Asp Ser Gly Leu Ser>
3650 3660 3670 3680 3690
395 CTG CCT ACC TCA CCT GTT TCC TGT ATG GAG GAG GAG GAA GTA TGT GAC GAC GGA TGG AGT GGA CAA AGG ACA TAC CTC CTC CTC CTT CAT ACA CTG Leu Pro Thr Ser Pro Val Ser Cys Met Glu Glu Glu Glu Val Cys Asp>
3700 3710 3720 3730 3740
400 CCC AAA TTC CAT TAT GAC AAC ACA GCA GGA ATC AGT CAG TAT CTG CAG
GGG TTT AAG GTA ATA CTG TTG TGT CGT CCT TAG TCA GTC ATA GAC GTC
Pro Lys Phe His Tyr Asp Asn Thr Ala Gly He Ser Gin Tyr Leu Gln>
3750 3760 3770 3780 3790
405 AAC AGT AAG CGA AAG AGC CGG CCT GTG AGT GTA AAA ACA TTT GAA GAT TTG TCA TTC GCT TTC TCG GCC GGA CAC TCA CAT TTT TGT AAA CTT CTA Asn Ser Lys Arg Lys Ser Arg Pro Val Ser Val Lys Thr Phe Glu Asp>
3800 3810 3820 3830 3840
410 ATC CCG TTA GAA GAA CCA GAA GTA AAA GTA ATC CCA GAT GAC AAC CAG TAG GGC AAT CTT CTT GGT CTT CAT TTT CAT TAG GGT CTA CTG TTG GTC He Pro Leu Glu Glu Pro Glu Val Lys Val He Pro Asp Asp Asn Gln>
3850 3860 3870 3880 415 ACG GAC AGT GGT ATG GTT CTT GCC TCA GAA GAG CTG AAA ACT TTG GAA TGC CTG TCA CCA TAC CAA GAA CGG AGT CTT CTC GAC TTT TGA AAC CTT Thr Asp Ser Gly Met Val Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu>
3890 3900 3910 3920 3930
420 GAC AGA ACC AAA TTA TCT CCA TCT TTT GGT GGA ATG GTG CCC AGC AAA CTG TCT TGG TTT AAT AGA GGT AGA AAA CCA CCT TAC CAC GGG TCG TTT Asp Arg Thr Lys Leu Ser Pro Ser Phe Gly Gly Met Val Pro Ser Lys>
3940 3950 3960 3970 3980
425 AGC AGG GAG TCT GTG GCA TCT GAA GGC TCA AAC CAG ACA AGC GGC TAC TCG TCC CTC AGA CAC CGT AGA CTT CCG AGT TTG GTC TGT TCG CCG ATG Ser Arg Glu Ser Val Ala Ser Glu Gly Ser Asn Gin Thr Ser Gly Tyr>
3990 4000 4010 4020 4030
430 CAG TCC GGA TAT CAC TCC GAT GAC ACA GAC ACC ACC GTG TAC TCC AGT GTC AGG CCT ATA GTG AGG CTA CTG TGT CTG TGG TGG CAC ATG AGG TCA Gin Ser Gly Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser>
4040 4050 4060 4070 4080
435 GAG GAA GCA GAA CTT TTA AAG CTG ATA GAG ATT GGA GTG CAA ACC GGT
CTC CTT CGT CTT GAA AAT TTC GAC TAT CTC TAA CCT CAC GTT TGG CCA
Glu Glu Ala Glu Leu Leu Lys Leu He Glu He Gly Val Gin Thr Gly>
4090 4100 4110 4120
440 AGC ACA GCC CAG ATT CTC CAG CCT GAC TCG GGG ACC ACA CTG AGC TCT
TCG TGT CGG GTC TAA GAG GTC GGA CTG AGC CCC TGG TGT GAC TCG AGA
Ser Thr Ala Gin He Leu Gin Pro Asp Ser Gly Thr Thr Leu Ser Ser>
4130 4140 4150 4160 4170
445 CCT CCT GTT TAA AAG GAA GCA TCC ACA CCC CCA ACT CCC GGA CAT CAC GGA GGA CAA ATT TTC CTT CGT AGG TGT GGG GGT TGA GGG CCT GTA GTG Pro Pro Val ***
4180 4190 4200 4210 4220
450 ATG AGA GGT GCT GCT CAG ATT TTC AAG TGT TGT TCT TTC CAC CAG CAG TAC TCT CCA CGA CGA GTC TAA AAG TTC ACA ACA AGA AAG GTG GTC GTC
4226
455 G
C
INFORMATION FOR SEQUENCE ID NO:2:
(i) SEQUENCE CHARACTERISTICS: 460 (A) LENGTH: 1356 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: Linear (ii) MOLECULE TYPE: protein
(iii) SEQUENCE DESCRIPTION: SEQ ID NO:2: 465 10 20 30 40 50 60 MQSKVLLAVA L LCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
70 80 90 100 110 120 LWP NQSGS EQRVEVTECS DGLFCKTLTI P VIGNDTGA YKCFYRETDL ASVIYVYVQD
130 140 150 160 170 180 YRSPFIASVS DQHGWYITE NKNKTWIPC LGSISNLNVS LCARYPEKRF VPDGNRIS D
190 200 210 220 230 240
SKKGFTIPSY MISYAGMVFC EAKINDESYQ SIMYIWWG YRIYDWLSP SHGIELSVGE
250 260 270 280 290 300
KLVLNCTART ELNVGIDFN EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS 310 320 330 340 350 360
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
370 380 390 400 410 420
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HWSLWYVP
430 440 450 460 470 480 PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWY QLEEECANEP SQAVSVTNPY
490 500 510 520 530 540
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
550 560 570 580 590 600
RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT 610 620 630 640 650 660
PVCKNLDTL KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCWRQLT
670 680 690 700 710 720
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
730 740 750 760 770 780 NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFIIEGAQEK TNLEIIILVG TAVIAMFFWL
790 800 810 820 830 840
LLVIILRTVK RANGGELKTG YLSIVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
850 860 870 880 890 900
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNWN 910 920 930 940 950 960
LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK 970 980 990 1000 1010 1020 RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA 1030 1040 1050 1060 1070 1080 500 SRKCIHRDLA ARNILLSEKN WKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR 1090 1100 1110 1120 1130 1140 VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML 1150 1160 1170 1180 1190 1200
DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS 505 1210 1220 1230 1240 1250 1260
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS 1270 1280 1290 1300 1310 1320 GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS 1330 1340 1350 1356 510 SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV*
INFORMATION FOR SEQ ID No.3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2775 base pairs 515 (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA (iii) FEATURE:
520 (A) NAME/KEY: CDS
(B) LOCATION: 1...2775
(iv) SEQUENCE DESCRIPTION: SEQ ID No.3
10 20 30 40 525 ATG GAG AGG GGG CTG CCG CTC CTC TGC GCC GTG CTC GCC CTC TAC CTC TCC CCC GAC GGC GAG GAG ACG CGG CAC GAG CGG GAG Met Glu Arg Gly Leu Pro Leu Leu Cys Ala Val Leu Ala Leu> a a TRANSLATION OF PGNP-1CODING [A] a a a > 530 50 60 70 80
GTC CTC GCC CCG GCC GGC GCT TTT CGC AAC GAT AAA TGT GGC CAG GAG CGG GGC CGG CCG CGA AAA GCG TTG CTA TTT ACA CCG Val Leu Ala Pro Ala Gly Ala Phe Arg Asn Asp Lys Cys Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
535
90 100 110 120
GAT ACT ATA AAA ATT GAA AGC CCC GGG TAC CTT ACA TCT CCT CTA TGA TAT TTT TAA CTT TCG GGG CCC ATG GAA TGT AGA GGA Asp Thr He Lys He Glu Ser Pro Gly Tyr Leu Thr Ser Pro>
540 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
130 140 150 160 GGT TAT CCT CAT TCT TAT CAC CCA AGT GAA AAA TGC GAA TGG CCA ATA GGA GTA AGA ATA GTG GGT TCA CTT TTT ACG CTT ACC 545 Gly Tyr Pro His Ser Tyr His Pro Ser Glu Lys Cys Glu Trp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
170 180 190 200 210 CTG ATT CAG GCT CCG GAC CCA TAC CAG AGA ATT ATG ATC AAC 550 GAC TAA GTC CGA GGC CTG GGT ATG GTC TCT TAA TAC TAG TTG Leu He Gin Ala Pro Asp Pro Tyr Gin Arg He Met He Asn> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
220 230 240 250 555 TTC AAC CCT CAC TTC GAT TTG GAG GAC AGA GAC TGC AAG TAT AAG TTG GGA GTG AAG CTA AAC CTC CTG TCT CTG ACG TTC ATA Phe Asn Pro His Phe Asp Leu Glu Asp Arg Asp Cys Lys Tyr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
560 260 270 280 290
GAC TAC GTG GAA GTC TTC GAT GGA GAA AAT GAA AAT GGA CAT CTG ATG CAC CTT CAG AAG CTA CCT CTT TTA CTT TTA CCT GTA Asp Tyr Val Glu Val Phe Asp Gly Glu Asn Glu Asn Gly His> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
565
300 310 320 330
TTT AGG GGA AAG TTC TGT GGA AAG ATA GCC CCT CCT CCT GTT AAA TCC CCT TTC AAG ACA CCT TTC TAT CGG GGA GGA GGA CAA Phe Arg Gly Lys Phe Cys Gly Lys He Ala Pro Pro Pro Val>
570 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
340 350 360 370 GTG TCT TCA GGG CCA TTT CTT TTT ATC AAA TTT GTC TCT GAC CAC AGA AGT CCC GGT AAA GAA AAA TAG TTT AAA CAG AGA CTG 575 Val Ser Ser Gly Pro Phe Leu Phe He Lys Phe Val Ser Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
380 390 400 410 420 TAC GAA ACA CAT GGT GCA GGA TTT TCC ATA CGT TAT GAA ATT 580 ATG CTT TGT GTA CCA CGT CCT AAA AGG TAT GCA ATA CTT TAA Tyr Glu Thr His Gly Ala Gly Phe Ser He Arg Tyr Glu He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
430 440 450 460 585 TTC AAG AGA GGT CCT GAA TGT TCC CAG AAC TAC ACA ACA CCT AAG TTC TCT CCA GGA CTT ACA AGG GTC TTG ATG TGT TGT GGA Phe Lys Arg Gly Pro Glu Cys Ser Gin Asn Tyr Thr Thr Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
590 470 480 490 500 AGT GGA GTG ATA AAG TCC CCC GGA TTC CCT GAA AAA TAT CCC TCA CCT CAC TAT TTC AGG GGG CCT AAG GGA CTT TTT ATA GGG Ser Gly Val He Lys Ser Pro Gly Phe Pro Glu Lys Tyr Pro> a a TRANSLATION OF PGNP-1CODING [A] a a a >
595
510 520 530 540 AAC AGC CTT GAA TGC ACT TAT ATT GTC TTT GCG CCA AAG ATG TTG TCG GAA CTT ACG TGA ATA TAA CAG AAA CGC GGT TTC TAC Asn Ser Leu Glu Cys Thr Tyr He Val Phe Ala Pro Lys Met> 600 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
550 560 570 580 TCA GAG ATT ATC CTG GAA TTT GAA AGC TTT GAC CTG GAG CCT AGT CTC TAA TAG GAC CTT AAA CTT TCG AAA CTG GAC CTC GGA 605 Ser Glu He He Leu Glu Phe Glu Ser Phe Asp Leu Glu Pro a a TRANSLATION OF PGNP-1CODING [A] _a a a >
590 600 610 620 630 GAC TCA AAT CCT CCA GGG GGG ATG TTC TGT CGC TAC GAC CGG 610 CTG AGT TTA GGA GGT CCC CCC TAC AAG ACA GCG ATG CTG GCC Asp Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr Asp Arg> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
640 650 660 670 615 CTA GAA ATC TGG GAT GGA TTC CCT GAT GTT GGC CCT CAC ATT GAT CTT TAG ACC CTA CCT AAG GGA CTA CAA CCG GGA GTG TAA Leu Glu He Trp Asp Gly Phe Pro Asp Val Gly Pro His He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
620 680 690 700 710
GGG CGT TAC TGT GGA CAG AAA ACA CCA GGT CGA ATC CGA TCC CCC GCA ATG ACA CCT GTC TTT TGT GGT CCA GCT TAG GCT AGG Gly Arg Tyr Cys Gly Gin Lys Thr Pro Gly Arg He Arg Ser> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
625
720 730 740 750
TCA TCG GGC ATT CTC TCC ATG GTT TTT TAC ACC GAC AGC GCG AGT AGC CCG TAA GAG AGG TAC CAA AAA ATG TGG CTG TCG CGC Ser Ser Gly He Leu Ser Met Val Phe Tyr Thr Asp Ser Ala>
630 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
760 770 780 790 ATA GCA AAA GAA GGT TTC TCA GCA AAC TAC AGT GTC TTG CAG TAT CGT TTT CTT CCA AAG AGT CGT TTG ATG TCA CAG AAC GTC 635 He Ala Lys Glu Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
800 810 820 830 840 AGC AGT GTC TCA GAA GAT TTC AAA TGT ATG GAA GCT CTG GGC 640 TCG TCA CAG AGT CTT CTA AAG TTT ACA TAC CTT CGA GAC CCG Ser Ser Val Ser Glu Asp Phe Lys Cys Met Glu Ala Leu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
850 860 870 880 645 ATG GAA TCA GGA GAA ATT CAT TCT GAC CAG ATC ACA GCT TCT TAC CTT AGT CCT CTT TAA GTA AGA CTG GTC TAG TGT CGA AGA Met Glu Ser Gly Glu He His Ser Asp Gin He Thr Ala Ser> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
650 890 900 910 920
TCC CAG TAT AGC ACC AAC TGG TCT GCA GAG CGC TCC CGC CTG AGG GTC ATA TCG TGG TTG ACC AGA CGT CTC GCG AGG GCG GAC Ser Gin Tyr Ser Thr Asn Trp Ser Ala Glu Arg Ser Arg Leu> a a TRANSLATION OF PGNP-1CODING [A] a a a >
655
930 940 950 960 AAC TAC CCT GAG AAT GGG TGG ACT CCC GGA GAG GAT TCC TAC TTG ATG GGA CTC TTA CCC ACC TGA GGG CCT CTC CTA AGG ATG Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp Ser Tyr> 660 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
970 980 990 1000 CGA GAG TGG ATA CAG GTA GAC TTG GGC CTT CTG CGC TTT GTC GCT CTC ACC TAT GTC CAT CTG AAC CCG GAA GAC GCG AAA CAG 665 Arg Glu Trp He Gin Val Asp Leu Gly Leu Leu Arg Phe Val> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1010 1020 1030 1040 1050 ACG GCT GTC GGG ACA CAG GGC GCC ATT TCA AAA GAA ACC AAG 670 TGC CGA CAG CCC TGT GTC CCG CGG TAA AGT TTT CTT TGG TTC Thr Ala Val Gly Thr Gin Gly Ala He Ser Lys Glu Thr Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1060 1070 1080 1090 675 AAG AAA TAT TAT GTC AAG ACT TAC AAG ATC GAC GTT AGC TCC TTC TTT ATA ATA CAG TTC TGA ATG TTC TAG CTG CAA TCG AGG Lys Lys Tyr Tyr Val Lys Thr Tyr Lys He Asp Val Ser Ser> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
680 1100 1110 1120 1130
AAC GGG GAA GAC TGG ATC ACC ATA AAA GAA GGA AAC AAA CCT TTG CCC CTT CTG ACC TAG TGG TAT TTT CTT CCT TTG TTT GGA Asn Gly Glu Asp Trp He Thr He Lys Glu Gly Asn Lys Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
685
1140 1150 1160 1170
GTT CTC TTT CAG GGA AAC ACC AAC CCT ACA GAT GTT GTG GTT CAA GAG AAA GTC CCT TTG TGG TTG GGA TGT CTA CAA CAC CAA Val Leu Phe Gin Gly Asn Thr Asn Pro Thr Asp Val Val Val>
690 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1180 1190 1200 1210 GCA GTA TTC CCC AAA CCA CTG ATA ACT CGA TTT GTC CGA ATC CGT CAT AAG GGG TTT GGT GAC TAT TGA GCT AAA CAG GCT TAG 695 Ala Val Phe Pro Lys Pro Leu He Thr Arg Phe Val Arg He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1220 1230 1240 1250 1260 AAG CCT GCA ACT TGG GAA ACT GGC ATA TCT ATG AGA TTT GAA 700 TTC GGA CGT TGA ACC CTT TGA CCG TAT AGA TAC TCT AAA CTT Lys Pro Ala Thr Trp Glu Thr Gly He Ser Met Arg Phe Glu a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1270 1280 1290 1300 705 GTA TAT GGT TGC AAG ATA ACA GAT TAT CCT TGC TCT GGA ATG CAT ATA CCA ACG TTC TAT TGT CTA ATA GGA ACG AGA CCT TAC Val Tyr Gly Cys Lys He Thr Asp Tyr Pro Cys Ser Gly Met> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
710 1310 1320 1330 1340
TTG GGT ATG GTG TCT GGA CTT ATT TCT GAC TCC CAG ATC ACA AAC CCA TAC CAC AGA CCT GAA TAA AGA CTG AGG GTC TAG TGT Leu Gly Met Val Ser Gly Leu He Ser Asp Ser Gin He Thr> a a TRANSLATION OF PGNP-1CODING [A] a a a >
715
1350 1360 1370 1380 TCA TCC AAC CAA GGG GAC AGA AAC TGG ATG CCT GAA AAC ATC AGT AGG TTG GTT CCC CTG TCT TTG ACC TAC GGA CTT TTG TAG Ser Ser Asn Gin Gly Asp Arg Asn Trp Met Pro Glu Asn He> 720 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1390 1400 1410 1420 CGC CTG GTA ACC AGT CGC TCT GGC TGG GCA CTT CCA CCC GCA GCG GAC CAT TGG TCA GCG AGA CCG ACC CGT GAA GGT GGG CGT 725 Arg Leu Val Thr Ser Arg Ser Gly Trp Ala Leu Pro Pro Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1430 1440 1450 1460 1470 CCT CAT TCC TAC ATC AAT GAG TGG CTC CAA ATA GAC CTG GGG 730 GGA GTA AGG ATG TAG TTA CTC ACC GAG GTT TAT CTG GAC CCC Pro His Ser Tyr He Asn Glu Trp Leu Gin He Asp Leu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1480 1490 1500 1510 735 GAG GAG AAG ATC GTG AGG GGC ATC ATC ATT CAG GGT GGG AAG CTC CTC TTC TAG CAC TCC CCG TAG TAG TAA GTC CCA CCC TTC Glu Glu Lys He Val Arg Gly He He He Gin Gly Gly Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
740 1520 1530 1540 1550
CAC CGA GAG AAC AAG GTG TTC ATG AGG AAG TTC AAG ATC GGG GTG GCT CTC TTG TTC CAC AAG TAC TCC TTC AAG TTC TAG CCC His Arg Glu Asn Lys Val Phe Met Arg Lys Phe Lys He Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
745
1560 1570 1580 1590
TAC AGC AAC AAC GGC TCG GAC TGG AAG ATG ATT ATG GAT GAC ATG TCG TTG TTG CCG AGC CTG ACC TTC TAC TAA TAC CTA CTG Tyr Ser Asn Asn Gly Ser Asp Trp Lys Met He Met Asp Asp>
750 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1600 1610 1620 1630 AGC AAA CGC AAG GCG AAG TCT TTT GAG GGC AAC AAC AAC TAT TCG TTT GCG TTC CGC TTC AGA AAA CTC CCG TTG TTG TTG ATA 755 Ser Lys Arg Lys Ala Lys Ser Phe Glu Gly Asn Asn Asn Tyr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1640 1650 1660 1670 1680 GAT ACA CCT GAG CTG CGG ACT TTT CCA GCT CTC TCC ACG CGA 760 CTA TGT GGA CTC GAC GCC TGA AAA GGT CGA GAG AGG TGC GCT Asp Thr Pro Glu Leu Arg Thr Phe Pro Ala Leu Ser Thr Arg> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1690 1700 1710 1720 765 TTC ATC AGG ATC TAC CCC GAG AGA GCC ACT CAT GGC GGA CTG AAG TAG TCC TAG ATG GGG CTC TCT CGG TGA GTA CCG CCT GAC Phe He Arg He Tyr Pro Glu Arg Ala Thr His Gly Gly Leu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
770 1730 1740 1750 1760
GGG CTC AGA ATG GAG CTG CTG GGC TGT GAA GTG GAA GCC CCT CCC GAG TCT TAC CTC GAC GAC CCG ACA CTT CAC CTT CGG GGA Gly Leu Arg Met Glu Leu Leu Gly Cys Glu Val Glu Ala Pro> TRANSLATION OF PGNP- 1CODING [A] a
775
1770 1780 1790 1800
ACA GCT GGA CCG ACC ACT CCC AAC GGG AAC TTG GTG GAT GAA TGT CGA CCT GGC TGG TGA GGG TTG CCC TTG AAC CAC CTA CTT Thr Ala Gly Pro Thr Thr Pro Asn Gly Asn Leu Val Asp Glu>
780 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1810 1820 1830 1840 TGT GAT GAC GAC CAG GCC AAC TGC CAC AGT GGA ACA GGT GAT ACA CTA CTG CTG GTC CGG TTG ACG GTG TCA CCT TGT CCA CTA 785 Cys Asp Asp Asp Gin Ala Asn Cys His Ser Gly Thr Gly Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1850 1860 1870 1880 1890 GAC TTC CAG CTC ACA GGT GGC ACC ACT GTG CTG GCC ACA GAA 790 CTG AAG GTC GAG TGT CCA CCG TGG TGA CAC GAC CGG TGT CTT Asp Phe Gin Leu Thr Gly Gly Thr Thr Val Leu Ala Thr Glu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1900 1910 1920 1930 795 AAG CCC GCG GTC ATA GAC AGC ACC ATA CAA TCA GAG TTT CCA TTC GGG CGC CAG TAT CTG TCG TGG TAT GTT AGT CTC AAA GGT Lys Pro Ala Val He Asp Ser Thr He Gin Ser Glu Phe Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
800 1940 1950 1960 1970
ACA TAT GGT TTT AAC TGT GAA TTT GGC TGG GGC TCT CAC AAG TGT ATA CCA AAA TTG ACA CTT AAA CCG ACC CCG AGA GTG TTC Thr Tyr Gly Phe Asn Cys Glu Phe Gly Trp Gly Ser His Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
805
1980 1990 2000 2010
ACC TTC TGC CAC TGG GAA CAT GAC AAT CAC GTG CAG CTC AAG TGG AAG ACG GTG ACC CTT GTA CTG TTA GTG CAC GTC GAG TTC Thr Phe Cys His Trp Glu His Asp Asn His Val Gin Leu Lys>
810 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2020 2030 2040 2050 TGG AGT GTG TTG ACC AGC AAG ACG GGA CCC ATT CAG GAT CAC ACC TCA CAC AAC TGG TCG TTC TGC CCT GGG TAA GTC CTA GTG 815 Trp Ser Val Leu Thr Ser Lys Thr Gly Pro He Gin Asp His> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2060 2070 2080 2090 2100 ACA GCA GGA GAT GGC AAC TTC ATC TAT TCC CAA GCT GAC GAA 820 TGT CGT CCT CTA CCG TTG AAG TAG ATA AGG GTT CGA CTG CTT Thr Ala Gly Asp Gly Asn Phe He Tyr Ser Gin Ala Asp Glu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2110 2120 2130 2140 825 AAT CAG AAG GGC AAA GTG GCT CGC CTG GTG AGC CCT GTG GTT TTA GTC TTC CCG TTT CAC CGA GCG GAC CAC TCG GGA CAC CAA Asn Gin Lys Gly Lys Val Ala Arg Leu Val Ser Pro Val Val> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
830 2150 2160 2170 2180
TAT TCC CAG AAC TCT GCC CAC TGC ATG ACC TTC TGG TAT CAC ATA AGG GTC TTG AGA CGG GTG ACG TAC TGG AAG ACC ATA GTG Tyr Ser Gin Asn Ser Ala His Cys Met Thr Phe Trp Tyr His> a a TRANSLATION OF PGNP-1CODING [A] a a a > 835
2190 2200 2210 2220
ATG TCT GGG TCC CAC ATC GGC ACA CTC AGG GTC AAA CTG CGC TAC AGA CCC AGG GTG TAG CCG TGT GAG TCC CAG TTT GAC GCG Met Ser Gly Ser His He Gly Thr Leu Arg Val Lys Leu Arg>
840 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2230 2240 2250 2260 TAC CAG AAG CCA GAA GAG TAC GAT CAG CTG GTC TGG ATG GCC ATG GTC TTC GGT CTT CTC ATG CTA GTC GAC CAG ACC TAC CGG 845 Tyr Gin Lys Pro Glu Glu Tyr Asp Gin Leu Val Trp Met Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2270 2280 2290 2300 2310 CTT GGA CAC CAA GGT GAC CAC TGG AAG GAA GGG CGT GTC TTG 850 GAA CCT GTG GTT CCA CTG GTG ACC TTC CTT CCC GCA CAG AAC Leu Gly His Gin Gly Asp His Trp Lys Glu Gly Arg Val Leu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2320 2330 2340 2350 855 CTC CAC AAG TCT CTG AAA CTT TAT CAG GTG ATT TTC GAG GGC GAG GTG TTC AGA GAC TTT GAA ATA GTC CAC TAA AAG CTC CCG Leu His Lys Ser Leu Lys Leu Tyr Gin Val He Phe Glu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
860 2360 2370 2380 2390
GAA ATC GGA AAA GGA AAC CTT GGT GGG ATT GCT GTG GAT GAC CTT TAG CCT TTT CCT TTG GAA CCA CCC TAA CGA CAC CTA CTG Glu He Gly Lys Gly Asn Leu Gly Gly He Ala Val Asp Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
865
2400 2410 2420 2430
ATT AGT ATT AAT AAC CAC ATT TCA CAA GAA GAT TGT GCA AAA TAA TCA TAA TTA TTG GTG TAA AGT GTT CTT CTA ACA CGT TTT He Ser He Asn Asn His He Ser Gin Glu Asp Cys Ala Lys>
870 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2440 2450 2460 2470 CCA GCA GAC CTG GAT AAA AAG AAC CCA GAA ATT AAA ATT GAT GGT CGT CTG GAC CTA TTT TTC TTG GGT CTT TAA TTT TAA CTA 875 Pro Ala Asp Leu Asp Lys Lys Asn Pro Glu He Lys He Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2480 2490 2500 2510 2520 GAA ACA GGG AGC ACG CCA GGA TAC GAA GGT GAA GGA GAA GGT 880 CTT TGT CCC TCG TGC GGT CCT ATG CTT CCA CTT CCT CTT CCA Glu Thr Gly Ser Thr Pro Gly Tyr Glu Gly Glu Gly Glu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2530 2540 2550 2560 885 GAC AAG AAC ATC TCC AGG AAG CCA GGC AAT GTG TTG AAG ACC CTG TTC TTG TAG AGG TCC TTC GGT CCG TTA CAC AAC TTC TGG Asp Lys Asn He Ser Arg Lys Pro Gly Asn Val Leu Lys Thr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
890 2570 2580 2590 2600
TTA GAC CCC ATC CTC ATC ACC ATC ATA GCC ATG AGT GCC CTG AAT CTG GGG TAG GAG TAG TGG TAG TAT CGG TAC TCA CGG GAC Leu Asp Pro He Leu He Thr He He Ala Met Ser Ala Leu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
895 2610 2620 2630 2640 GGG GTC CTC CTG GGG GCT GTC TGT GGG GTC GTG CTG TAC TGT CCC CAG GAG GAC CCC CGA CAG ACA CCC CAG CAC GAC ATG ACA Gly Val Leu Leu Gly Ala Val Cys Gly Val Val Leu Tyr Cys>
900 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2650 2660 2670 2680 GCC TGT TGG CAT AAT GGG ATG TCA GAA AGA AAC TTG TCT GCC CGG ACA ACC GTA TTA CCC TAC AGT CTT TCT TTG AAC AGA CGG
905 Ala Cys Trp His Asn Gly Met Ser Glu Arg Asn Leu Ser Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2690 2700 2710 2720 2730 CTG GAG AAC TAT AAC TTT GAA CTT GTG GAT GGT GTG AAG TTG
910 GAC CTC TTG ATA TTG AAA CTT GAA CAC CTA CCA CAC TTC AAC Leu Glu Asn Tyr Asn Phe Glu Leu Val Asp Gly Val Lys Leu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2740 2750 2760 2770
915 AAA AAA GAC AAA CTG AAT ACA CAG AGT ACT TAT TCG GAG GCA TTT TTT CTG TTT GAC TTA TGT GTC TCA TGA ATA AGC CTC CGT Lys Lys Asp Lys Leu Asn Thr Gin Ser Thr Tyr Ser Glu Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
920 TGA ACT
925 INFORMATION FOR SEQUENCE ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 925 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: Linear
930 (ii) MOLECULE TYPE: protein
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:4:
10 20 30 40 50
MERGLPLLCA VLALVLAPAG AFRNDKCGDT IKIESPGYLT SPGYPHSYHP
935 60 70 80 90 100
SEKCEWLIQA PDPYQRIMIN FNPHFDLEDR DCKYDYVEVF DGENENGHFR
110 120 130 140 150
GKFCGKIAPP PWSSGPFLF IKFVSDYETH GAGFSIRYEI FKRGPECSQN
940
160 170 180 190 200
YTTPSGVIKS PGFPEKYPNS LECTYIVFAP KMSEIILEFE SFDLEPDSNP
210 220 230 240 250 45 PGGMFCRYDR LEIWDGFPDV GPHIGRYCGQ KTPGRIRSSS GILSMVFYTD
260 270 280 290 300 SAIAKEGFSA NYSVLQSSVS EDFKCMEALG MESGEIHSDQ ITASSQYSTN
950 310 320 330 340 350
WSAERSRLNY PENGWTPGED SYREWIQVDL GLLRFVTAVG TQGAISKETK
360 370 380 390 400 KKYYVKTYKI DVSSNGEDWI TIKEGNKPVL FQGNTNPTDV WAVFPKPLI
955
410 420 430 440 450 TRFVRIKPAT WETGISMRFE VYGCKITDYP CSGMLGMVSG LISDSQITSS
460 470 480 490 500
960 NQGDRNWMPE NIRLVTSRSG WALPPAPHSY INEWLQIDLG EEKIVRGIII
510 520 530 540 550 QGGKHRENKV FMRKFKIGYS NNGSDWKMIM DDSKRKAKSF EGNNNYDTPE
965 560 570 580 590 600
LRTFPALSTR FIRIYPERAT HGGLGLRMEL LGCEVEAPTA GPTTPNGNLV
610 620 630 640 650 DECDDDQANC HSGTGDDFQL TGGTTVLATE KPAVIDSTIQ SEFPTYGFNC
970
660 670 680 690 700 EFGWGSHKTF CHWEHDNHVQ LKWSVLTSKT GPIQDHTAGD GNFIYSQADE
710 720 730 740 750
975 NQKGKVARLV SPWYSQNSA HCMTFWYHMS GSHIGTLRVK LRYQKPEEYD
760 770 780 790 800
QLVWMALGHQ GDHWKEGRVL LHKSLKLYQV IFEGEIGKGN LGGIAVDDIS
980 810 820 830 840 850
INNHISQEDC AKPADLDKKN PEIKIDETGS TPGYEGEGEG DKNISRKPGN
860 870 880 890 900
VLKTLDPILI TIIAMSALGV LLGAVCGWL YCACWHNGMS ERNLSALENY
985
910 920
NFELVDGVKL KKDKLNTQST YSEA*
INFORMATION FOR SEQ ID No. 5:
990 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2280 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear 995 (ii) MOLECULE TYPE: cDNA
(iii) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 71...2350
(iv) SEQUENCE DESCRIPTION: SEQ ID No.5
1000 70 80 90 100 110
ATG CAG AGC AAG GTG CTG CTG GCC GTC GCC CTG TGG CTC TGC GTG GAG TAC GTC TCG TTC CAC GAC GAC CGG CAG CGG GAC ACC GAG ACG CAC CTC Met Gin Ser Lys Val Leu Leu Ala Val Ala Leu Trp Leu Cys Val Glu>
1005 120 130 140 150 160
ACC CGG GCC GCC TCT GTG GGT TTG CCT AGT GTT TCT CTT GAT CTG CCC TGG GCC CGG CGG AGA CAC CCA AAC GGA TCA CAA AGA GAA CTA GAC GGG Thr Arg Ala Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu Pro>
1010 170 180 190 200 210
AGG CTC AGC ATA CAA AAA GAC ATA CTT ACA ATT AAG GCT AAT ACA ACT TCC GAG TCG TAT GTT TTT CTG TAT GAA TGT TAA TTC CGA TTA TGT TGA Arg Leu Ser He Gin Lys Asp He Leu Thr He Lys Ala Asn Thr Thr>
1015 220 230 240 250 260
CTT CAA ATT ACT TGC AGG GGA CAG AGG GAC TTG GAC TGG CTT TGG CCC GAA GTT TAA TGA ACG TCC CCT GTC TCC CTG AAC CTG ACC GAA ACC GGG Leu Gin He Thr Cys Arg Gly Gin Arg Asp Leu Asp Trp Leu Trp Pro>
1020 270 280 290 300
AAT AAT CAG AGT GGC AGT GAG CAA AGG GTG GAG GTG ACT GAG TGC AGC TTA TTA GTC TCA CCG TCA CTC GTT TCC CAC CTC CAC TGA CTC ACG TCG Asn Asn Gin Ser Gly Ser Glu Gin Arg Val Glu Val Thr Glu Cys Ser>
1025 310 320 330 340 350
GAT GGC CTC TTC TGT AAG ACA CTC ACA ATT CCA AAA GTG ATC GGA AAT CTA CCG GAG AAG ACA TTC TGT GAG TGT TAA GGT TTT CAC TAG CCT TTA Asp Gly Leu Phe Cys Lys Thr Leu Thr He Pro Lys Val He Gly Asn>
1030 360 370 380 390 400
GAC ACT GGA GCC TAC AAG TGC TTC TAC CGG GAA ACT GAC TTG GCC TCG CTG TGA CCT CGG ATG TTC ACG AAG ATG GCC CTT TGA CTG AAC CGG AGC Asp Thr Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser>
1035 410 420 430 440 450
GTC ATT TAT GTC TAT GTT CAA GAT TAC AGA TCT CCA TTT ATT GCT TCT CAG TAA ATA CAG ATA CAA GTT CTA ATG TCT AGA GGT AAA TAA CGA AGA Val He Tyr Val Tyr Val Gin Asp Tyr Arg Ser Pro Phe He Ala Ser>
1040 460 470 480 490 500
GTT AGT GAC CAA CAT GGA GTC GTG TAC ATT ACT GAG AAC AAA AAC AAA CAA TCA CTG GTT GTA CCT CAG CAC ATG TAA TGA CTC TTG TTT TTG TTT Val Ser Asp Gin His Gly Val Val Tyr He Thr Glu Asn Lys Asn Lys>
1045 510 520 530 540
ACT GTG GTG ATT CCA TGT CTC GGG TCC ATT TCA AAT CTC AAC GTG TCA TGA CAC CAC TAA GGT ACA GAG CCC AGG TAA AGT TTA GAG TTG CAC AGT Thr Val Val He Pro Cys Leu Gly Ser He Ser Asn Leu Asn Val Ser>
1050 550 560 570 580 590
CTT TGT GCA AGA TAC CCA GAA AAG AGA TTT GTT CCT GAT GGT AAC AGA GAA ACA CGT TCT ATG GGT CTT TTC TCT AAA CAA GGA CTA CCA TTG TCT Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg>
1055 600 610 620 630 640
ATT TCC TGG GAC AGC AAG AAG GGC TTT ACT ATT CCC AGC TAC ATG ATC TAA AGG ACC CTG TCG TTC TTC CCG AAA TGA TAA GGG TCG ATG TAC TAG He Ser Trp Asp Ser Lys Lys Gly Phe Thr He Pro Ser Tyr Met He>
1060 650 660 670 680 690
AGC TAT GCT GGC ATG GTC TTC TGT GAA GCA AAA ATT AAT GAT GAA AGT
TCG ATA CGA CCG TAC CAG AAG ACA CTT CGT TTT TAA TTA CTA CTT TCA
Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys He Asn Asp Glu Ser>
1065 700 710 720 730 740
TAC CAG TCT ATT ATG TAC ATA GTT GTC GTT GTA GGG TAT AGG ATT TAT ATG GTC AGA TAA TAC ATG TAT CAA CAG CAA CAT CCC ATA TCC TAA ATA Tyr Gin Ser He Met Tyr He Val Val Val Val Gly Tyr Arg He Tyr>
1070 750 760 770 780
GAT GTG GTT CTG AGT CCG TCT CAT GGA ATT GAA CTA TCT GTT GGA GAA
CTA CAC CAA GAC TCA GGC AGA GTA CCT TAA CTT GAT AGA CAA CCT CTT
Asp Val Val Leu Ser Pro Ser His Gly He Glu Leu Ser Val Gly Glu>
1075 790 800 810 820 830
AAG CTT GTC TTA AAT TGT ACA GCA AGA ACT GAA CTA AAT GTG GGG ATT TTC GAA CAG AAT TTA ACA TGT CGT TCT TGA CTT GAT TTA CAC CCC TAA Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly He>
1080 840 850 860 870 880
GAC TTC AAC TGG GAA TAC CCT TCT TCG AAG CAT CAG CAT AAG AAA CTT CTG AAG TTG ACC CTT ATG GGA AGA AGC TTC GTA GTC GTA TTC TTT GAA Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gin His Lys Lys Leu>
1085 890 900 910 920 930
GTA AAC CGA GAC CTA AAA ACC CAG TCT GGG AGT GAG ATG AAG AAA TTT
CAT TTG GCT CTG GAT TTT TGG GTC AGA CCC TCA CTC TAC TTC TTT AAA
Val Asn Arg Asp Leu Lys Thr Gin Ser Gly Ser Glu Met Lys Lys Phe>
1090 940 950 960 970 980
TTG AGC ACC TTA ACT ATA GAT GGT GTA ACC CGG AGT GAC CAA GGA TTG AAC TCG TGG AAT TGA TAT CTA CCA CAT TGG GCC TCA CTG GTT CCT AAC Leu Ser Thr Leu Thr He Asp Gly Val Thr Arg Ser Asp Gin Gly Leu>
1095 990 1000 1010 1020
TAC ACC TGT GCA GCA TCC AGT GGG CTG ATG ACC AAG AAG AAC AGC ACA ATG TGG ACA CGT CGT AGG TCA CCC GAC TAC TGG TTC TTC TTG TCG TGT Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr>
1100 1030 1040 1050 1060 1070
TTT GTC AGG GTC CAT GAA AAA CCT TTT GTT GCT TTT GGA AGT GGC ATG AAA CAG TCC CAG GTA CTT TTT GGA AAA CAA CGA AAA CCT TCA CCG TAC Phe Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met>
1105 1080 1090 1100 1110 1120
GAA TCT CTG GTG GAA GCC ACG GTG GGG GAG CGT GTC AGA ATC CCT GCG CTT AGA GAC CAC CTT CGG TGC CAC CCC CTC GCA CAG TCT TAG GGA CGC Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg He Pro Ala>
1110 1130 1140 1150 1160 1170
AAG TAC CTT GGT TAC CCA CCC CCA GAA ATA AAA TGG TAT AAA AAT GGA
TTC ATG GAA CCA ATG GGT GGG GGT CTT TAT TTT ACC ATA TTT TTA CCT
Lys Tyr Leu Gly Tyr Pro Pro Pro Glu He Lys Trp Tyr Lys Asn Gly>
1115 1180 1190 1200 1210 1220
ATA CCC CTT GAG TCC AAT CAC ACA ATT AAA GCG GGG CAT GTA CTG ACG TAT GGG GAA CTC AGG TTA GTG TGT TAA TTT CGC CCC GTA CAT GAC TGC He Pro Leu Glu Ser Asn His Thr He Lys Ala Gly His Val Leu Thr>
1120 1230 1240 1250 1260
ATT ATG GAA GTG AGT GAA AGA GAC ACA GGA AAT TAC ACT GTC ATC CTT TAA TAC CTT CAC TCA CTT TCT CTG TGT CCT TTA ATG TGA CAG TAG GAA He Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val He Leu>
1125 1270 1280 1290 1300 1310
ACC AAT CCC ATT TCA AAG GAG AAG CAG AGC CAT GTG GTC TCT CTG GTT TGG TTA GGG TAA AGT TTC CTC TTC GTC TCG GTA CAC CAG AGA GAC CAA Thr Asn Pro He Ser Lys Glu Lys Gin Ser His Val Val Ser Leu Val>
1130 1320 1330 1340 1350 1360
GTG TAT GTC CCA CCC CAG ATT GGT GAG AAA TCT CTA ATC TCT CCT GTG
CAC ATA CAG GGT GGG GTC TAA CCA CTC TTT AGA GAT TAG AGA GGA CAC
Val Tyr Val Pro Pro Gin He Gly Glu Lys Ser Leu He Ser Pro Val>
1135 1370 1380 1390 1400 1410
GAT TCC TAC CAG TAC GGC ACC ACT CAA ACG CTG ACA TGT ACG GTC TAT
CTA AGG ATG GTC ATG CCG TGG TGA GTT TGC GAC TGT ACA TGC CAG ATA
Asp Ser Tyr Gin Tyr Gly Thr Thr Gin Thr Leu Thr Cys Thr Val Tyr>
1140 1420 1430 1440 1450 1460
GCC ATT CCT CCC CCG CAT CAC ATC CAC TGG TAT TGG CAG TTG GAG GAA CGG TAA GGA GGG GGC GTA GTG TAG GTG ACC ATA ACC GTC AAC CTC CTT Ala He Pro Pro Pro His His He His Trp Tyr Trp Gin Leu Glu Glu>
1145 1470 1480 1490 1500
GAG TGC GCC AAC GAG CCC AGC CAA GCT GTC TCA GTG ACA AAC CCA TAC
CTC ACG CGG TTG CTC GGG TCG GTT CGA CAG AGT CAC TGT TTG GGT ATG
Glu Cys Ala Asn Glu Pro Ser Gin Ala Val Ser Val Thr Asn Pro Tyr>
1150 1510 1520 1530 1540 1550
CCT TGT GAA GAA TGG AGA AGT GTG GAG GAC TTC CAG GGA GGA AAT AAA GGA ACA CTT CTT ACC TCT TCA CAC CTC CTG AAG GTC CCT CCT TTA TTT Pro Cys Glu Glu Trp Arg Ser Val Glu Asp Phe Gin Gly Gly Asn Lys>
1155 1560 1570 1580 1590 1600
ATT GAA GTT AAT AAA AAT CAA TTT GCT CTA ATT GAA GGA AAA AAC AAA TAA CTT CAA TTA TTT TTA GTT AAA CGA GAT TAA CTT CCT TTT TTG TTT He Glu Val Asn Lys Asn Gin Phe Ala Leu He Glu Gly Lys Asn Lys>
1160 1610 1620 1630 1640 1650
ACT GTA AGT ACC CTT GTT ATC CAA GCG GCA AAT GTG TCA GCT TTG TAC TGA CAT TCA TGG GAA CAA TAG GTT CGC CGT TTA CAC AGT CGA AAC ATG Thr Val Ser Thr Leu Val He Gin Ala Ala Asn Val Ser Ala Leu Tyr>
1165 1660 1670 1680 1690 1700
AAA TGT GAA GCG GTC AAC AAA GTC GGG AGA GGA GAG AGG GTG ATC TCC TTT ACA CTT CGC CAG TTG TTT CAG CCC TCT CCT CTC TCC CAC TAG AGG Lys Cys Glu Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val He Ser>
1170 1710 1720 1730 1740
TTC CAC GTG ACC AGG GGT CCT GAA ATT ACT TTG CAA CCT GAC ATG CAG
AAG GTG CAC TGG TCC CCA GGA CTT TAA TGA AAC GTT GGA CTG TAC GTC
Phe His Val Thr Arg Gly Pro Glu He Thr Leu Gin Pro Asp Met Gln>
1175 1750 1760 1770 1780 1790
CCC ACT GAG CAG GAG AGC GTG TCT TTG TGG TGC ACT GCA GAC AGA TCT GGG TGA CTC GTC CTC TCG CAC AGA AAC ACC ACG TGA CGT CTG TCT AGA Pro Thr Glu Gin Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser> 1180 1800 1810 1820 1830 1840
ACG TTT GAG AAC CTC ACA TGG TAC AAG CTT GGC CCA CAG CCT CTG CCA TGC AAA CTC TTG GAG TGT ACC ATG TTC GAA CCG GGT GTC GGA GAC GGT Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gin Pro Leu Pro>
1185 1850 1860 1870 1880 1890
ATC CAT GTG GGA GAG TTG CCC ACA CCT GTT TGC AAG AAC TTG GAT ACT TAG GTA CAC CCT CTC AAC GGG TGT GGA CAA ACG TTC TTG AAC CTA TGA He His Val Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr>
1190 1900 1910 1920 1930 1940
CTT TGG AAA TTG AAT GCC ACC ATG TTC TCT AAT AGC ACA AAT GAC ATT GAA ACC TTT AAC TTA CGG TGG TAC AAG AGA TTA TCG TGT TTA CTG TAA Leu Trp Lys Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp He>
1195 1950 1960 1970 1980
TTG ATC ATG GAG CTT AAG AAT GCA TCC TTG CAG GAC CAA GGA GAC TAT
AAC TAG TAC CTC GAA TTC TTA CGT AGG AAC GTC CTG GTT CCT CTG ATA
Leu He Met Glu Leu Lys Asn Ala Ser Leu Gin Asp Gin Gly Asp Tyr>
1200 1990 2000 2010 2020 2030
GTC TGC CTT GCT CAA GAC AGG AAG ACC AAG AAA AGA CAT TGC GTG GTC CAG ACG GAA CGA GTT CTG TCC TTC TGG TTC TTT TCT GTA ACG CAC CAG Val Cys Leu Ala Gin Asp Arg Lys Thr Lys Lys Arg His Cys Val Val>
1205 2040 2050 2060 2070 2080
AGG CAG CTC ACA GTC CTA GAG CGT GTG GCA CCC ACG ATC ACA GGA AAC TCC GTC GAG TGT CAG GAT CTC GCA CAC CGT GGG TGC TAG TGT CCT TTG Arg Gin Leu Thr Val Leu Glu Arg Val Ala Pro Thr He Thr Gly Asn>
1210 2090 2100 2110 2120 2130
CTG GAG AAT CAG ACG ACA AGT ATT GGG GAA AGC ATC GAA GTC TCA TGC GAC CTC TTA GTC TGC TGT TCA TAA CCC CTT TCG TAG CTT CAG AGT ACG Leu Glu Asn Gin Thr Thr Ser He Gly Glu Ser He Glu Val Ser Cys>
1215 2140 2150 2160 2170 2180
ACG GCA TCT GGG AAT CCC CCT CCA CAG ATC ATG TGG TTT AAA GAT AAT
TGC CGT AGA CCC TTA GGG GGA GGT GTC TAG TAC ACC AAA TTT CTA TTA
Thr Ala Ser Gly Asn Pro Pro Pro Gin He Met Trp Phe Lys Asp Asn>
1220 2190 2200 2210 2220
GAG ACC CTT GTA GAA GAC TCA GGC ATT GTA TTG AAG GAT GGG AAC CGG CTC TGG GAA CAT CTT CTG AGT CCG TAA CAT AAC TTC CTA CCC TTG GCC Glu Thr Leu Val Glu Asp Ser Gly He Val Leu Lys Asp Gly Asn Arg>
1225 2230 2240 2250 2260 2270
AAC CTC ACT ATC CGC AGA GTG AGG AAG GAG GAC GAA GGC CTC TAC ACC TTG GAG TGA TAG GCG TCT CAC TCC TTC CTC CTG CTT CCG GAG ATG TGG Asn Leu Thr He Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr>
1230 2280 2290 2300 2310 2320
TGC CAG GCA TGC AGT GTT CTT GGC TGT GCA AAA GTG GAG GCA TTT TTC ACG GTC CGT ACG TCA CAA GAA CCG ACA CGT TTT CAC CTC CGT AAA AAG Cys Gin Ala Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe Phe>
1235 2330 2340 2350
ATA ATA GAA GGT GCC CAG GAA AAG ACG TAT TAT CTT CCA CGG GTC CTT TTC TGC He He Glu Gly Ala Gin Glu Lys Thr 1240 INFORMATION FOR SEQUENCE ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 760 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: Linear 1245 (ii) MOLECULE TYPE: protein
(iii) FEATURE:
(A) NAME/KEY: protein
(B) LOCATION: 1...760
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:6:
1250
10 20 30 40 50 60
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
70 80 90 100 110 120
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
1255 130 140 150 160 170 180
YRSPFIASVS DQHGWYITE NKNKTWIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
190 200 210 220 230 240
SKKGFTIPSY MISYAGMVFC EAKINDESYQ SIMYIWWG YRIYDWLSP SHGIELSVGE
250 260 270 280 290 300
1260 KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
310 320 330 340 350 360
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
370 380 390 400 410 420
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HWSLWYVP
1265 430 440 450 460 470 480
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
490 500 510 520 530 540
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
550 560 570 580 590 600 1270 RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
610 620 630 640 650 660
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCWRQLT
670 680 690 700 710 720
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
1275 730 740 750 760
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFIIEGAQEK
INFORMATION FOR SEQ ID No. 7:
(i) SEQUENCE CHARACTERISTICS: 1280 (A) LENGTH: 2568 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
1285 (iii) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1...2568
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:7:
1290
10 20 30 40
ATG GAG AGG GGG CTG CCG CTC CTC TGC GCC GTG CTC GCC CTC TAC CTC TCC CCC GAC GGC GAG GAG ACG CGG CAC GAG CGG GAG Met Glu Arg Gly Leu Pro Leu Leu Cys Ala Val Leu Ala Leu>
1295 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
50 60 70 80 GTC CTC GCC CCG GCC GGC GCT TTT CGC AAC GAT AAA TGT GGC CAG GAG CGG GGC CGG CCG CGA AAA GCG TTG CTA TTT ACA CCG 1300 Val Leu Ala Pro Ala Gly Ala Phe Arg Asn Asp Lys Cys Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
90 100 110 120 GAT ACT ATA AAA ATT GAA AGC CCC GGG TAC CTT ACA TCT CCT 1305 CTA TGA TAT TTT TAA CTT TCG GGG CCC ATG GAA TGT AGA GGA Asp Thr He Lys He Glu Ser Pro Gly Tyr Leu Thr Ser Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
130 140 150 160
1310 GGT TAT CCT CAT TCT TAT CAC CCA AGT GAA AAA TGC GAA TGG
CCA ATA GGA GTA AGA ATA GTG GGT TCA CTT TTT ACG CTT ACC Gly Tyr Pro His Ser Tyr His Pro Ser Glu Lys Cys Glu Trp> TRANSLATION OF PGNP-1CODING [A] a
1315 170 180 190 200 210
CTG ATT CAG GCT CCG GAC CCA TAC CAG AGA ATT ATG ATC AAC GAC TAA GTC CGA GGC CTG GGT ATG GTC TCT TAA TAC TAG TTG Leu He Gin Ala Pro Asp Pro Tyr Gin Arg He Met He Asn> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1320
220 230 240 250
TTC AAC CCT CAC TTC GAT TTG GAG GAC AGA GAC TGC AAG TAT AAG TTG GGA GTG AAG CTA AAC CTC CTG TCT CTG ACG TTC ATA Phe Asn Pro His Phe Asp Leu Glu Asp Arg Asp Cys Lys Tyr>
1325 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
260 270 280 290
GAC TAC GTG GAA GTC TTC GAT GGA GAA AAT GAA AAT GGA CAT CTG ATG CAC CTT CAG AAG CTA CCT CTT TTA CTT TTA CCT GTA 1330 Asp Tyr Val Glu Val Phe Asp Gly Glu Asn Glu Asn Gly His> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
300 310 320 330
TTT AGG GGA AAG TTC TGT GGA AAG ATA GCC CCT CCT CCT GTT 1335 AAA TCC CCT TTC AAG ACA CCT TTC TAT CGG GGA GGA GGA CAA
Phe Arg Gly Lys Phe Cys Gly Lys He Ala Pro Pro Pro Val> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
340 350 360 370
1340 GTG TCT TCA GGG CCA TTT CTT TTT ATC AAA TTT GTC TCT GAC
CAC AGA AGT CCC GGT AAA GAA AAA TAG TTT AAA CAG AGA CTG Val Ser Ser Gly Pro Phe Leu Phe He Lys Phe Val Ser Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1345 380 390 400 410 420 TAC GAA ACA CAT GGT GCA GGA TTT TCC ATA CGT TAT GAA ATT ATG CTT TGT GTA CCA CGT CCT AAA AGG TAT GCA ATA CTT TAA Tyr Glu Thr His Gly Ala Gly Phe Ser He Arg Tyr Glu He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1350
430 440 450 460
TTC AAG AGA GGT CCT GAA TGT TCC CAG AAC TAC ACA ACA CCT AAG TTC TCT CCA GGA CTT ACA AGG GTC TTG ATG TGT TGT GGA Phe Lys Arg Gly Pro Glu Cys Ser Gin Asn Tyr Thr Thr Pro>
1355 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
470 480 490 500 AGT GGA GTG ATA AAG TCC CCC GGA TTC CCT GAA AAA TAT CCC TCA CCT CAC TAT TTC AGG GGG CCT AAG GGA CTT TTT ATA GGG 1360 Ser Gly Val He Lys Ser Pro Gly Phe Pro Glu Lys Tyr Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
510 520 530 540 AAC AGC CTT GAA TGC ACT TAT ATT GTC TTT GCG CCA AAG ATG 1365 TTG TCG GAA CTT ACG TGA ATA TAA CAG AAA CGC GGT TTC TAC Asn Ser Leu Glu Cys Thr Tyr He Val Phe Ala Pro Lys Met> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
550 560 570 580 1370 TCA GAG ATT ATC CTG GAA TTT GAA AGC TTT GAC CTG GAG CCT AGT CTC TAA TAG GAC CTT AAA CTT TCG AAA CTG GAC CTC GGA Ser Glu He He Leu Glu Phe Glu Ser Phe Asp Leu Glu Pro> a a TRANSLATION OF PGNP-1CODING [A] a a a 1375 590 600 610 620 630
GAC TCA AAT CCT CCA GGG GGG ATG TTC TGT CGC TAC GAC CGG CTG AGT TTA GGA GGT CCC CCC TAC AAG ACA GCG ATG CTG GCC Asp Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr Asp Arg> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1380
640 650 660 670
CTA GAA ATC TGG GAT GGA TTC CCT GAT GTT GGC CCT CAC ATT GAT CTT TAG ACC CTA CCT AAG GGA CTA CAA CCG GGA GTG TAA Leu Glu He Trp Asp Gly Phe Pro Asp Val Gly Pro His He>
1385 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
680 690 700 710 GGG CGT TAC TGT GGA CAG AAA ACA CCA GGT CGA ATC CGA TCC CCC GCA ATG ACA CCT GTC TTT TGT GGT CCA GCT TAG GCT AGG 1390 Gly Arg Tyr Cys Gly Gin Lys Thr Pro Gly Arg He Arg Ser> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
720 730 740 750
TCA TCG GGC ATT CTC TCC ATG GTT TTT TAC ACC GAC AGC GCG 1395 AGT AGC CCG TAA GAG AGG TAC CAA AAA ATG TGG CTG TCG CGC
Ser Ser Gly He Leu Ser Met Val Phe Tyr Thr Asp Ser Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
760 770 780 790
1400 ATA GCA AAA GAA GGT TTC TCA GCA AAC TAC AGT GTC TTG CAG
TAT CGT TTT CTT CCA AAG AGT CGT TTG ATG TCA CAG AAC GTC He Ala Lys Glu Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1405 800 810 820 830 840 AGC AGT GTC TCA GAA GAT TTC AAA TGT ATG GAA GCT CTG GGC TCG TCA CAG AGT CTT CTA AAG TTT ACA TAC CTT CGA GAC CCG Ser Ser Val Ser Glu Asp Phe Lys Cys Met Glu Ala Leu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1410
850 860 870 880
ATG GAA TCA GGA GAA ATT CAT TCT GAC CAG ATC ACA GCT TCT TAC CTT AGT CCT CTT TAA GTA AGA CTG GTC TAG TGT CGA AGA Met Glu Ser Gly Glu He His Ser Asp Gin He Thr Ala Ser>
1415 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
890 900 910 920 TCC CAG TAT AGC ACC AAC TGG TCT GCA GAG CGC TCC CGC CTG AGG GTC ATA TCG TGG TTG ACC AGA CGT CTC GCG AGG GCG GAC 1420 Ser Gin Tyr Ser Thr Asn Trp Ser Ala Glu Arg Ser Arg Leu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
930 940 950 960 AAC TAC CCT GAG AAT GGG TGG ACT CCC GGA GAG GAT TCC TAC 1425 TTG ATG GGA CTC TTA CCC ACC TGA GGG CCT CTC CTA AGG ATG Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp Ser Tyr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
970 980 990 1000 1430 CGA GAG TGG ATA CAG GTA GAC TTG GGC CTT CTG CGC TTT GTC GCT CTC ACC TAT GTC CAT CTG AAC CCG GAA GAC GCG AAA CAG Arg Glu Trp He Gin Val Asp Leu Gly Leu Leu Arg Phe Val> a a TRANSLATION OF PGNP-1CODING [A] a a a > 1435 1010 1020 1030 1040 1050
ACG GCT GTC GGG ACA CAG GGC GCC ATT TCA AAA GAA ACC AAG TGC CGA CAG CCC TGT GTC CCG CGG TAA AGT TTT CTT TGG TTC Thr Ala Val Gly Thr Gin Gly Ala He Ser Lys Glu Thr Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1440
1060 1070 1080 1090
AAG AAA TAT TAT GTC AAG ACT TAC AAG ATC GAC GTT AGC TCC TTC TTT ATA ATA CAG TTC TGA ATG TTC TAG CTG CAA TCG AGG Lys Lys Tyr Tyr Val Lys Thr Tyr Lys He Asp Val Ser Ser>
1445 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1100 1110 1120 1130
AAC GGG GAA GAC TGG ATC ACC ATA AAA GAA GGA AAC AAA CCT TTG CCC CTT CTG ACC TAG TGG TAT TTT CTT CCT TTG TTT GGA 1450 Asn Gly Glu Asp Trp He Thr He Lys Glu Gly Asn Lys Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1140 1150 1160 1170
GTT CTC TTT CAG GGA AAC ACC AAC CCT ACA GAT GTT GTG GTT 1455 CAA GAG AAA GTC CCT TTG TGG TTG GGA TGT CTA CAA CAC CAA
Val Leu Phe Gin Gly Asn Thr Asn Pro Thr Asp Val Val Val> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1180 1190 1200 1210
1460 GCA GTA TTC CCC AAA CCA CTG ATA ACT CGA TTT GTC CGA ATC
CGT CAT AAG GGG TTT GGT GAC TAT TGA GCT AAA CAG GCT TAG Ala Val Phe Pro Lys Pro Leu He Thr Arg Phe Val Arg He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1465 1220 1230 1240 1250 1260
AAG CCT GCA ACT TGG GAA ACT GGC ATA TCT ATG AGA TTT GAA TTC GGA CGT TGA ACC CTT TGA CCG TAT AGA TAC TCT AAA CTT Lys Pro Ala Thr Trp Glu Thr Gly He Ser Met Arg Phe Glu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1470
1270 1280 1290 1300
GTA TAT GGT TGC AAG ATA ACA GAT TAT CCT TGC TCT GGA ATG CAT ATA CCA ACG TTC TAT TGT CTA ATA GGA ACG AGA CCT TAC Val Tyr Gly Cys Lys He Thr Asp Tyr Pro Cys Ser Gly Met>
1475 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1310 1320 1330 1340
TTG GGT ATG GTG TCT GGA CTT ATT TCT GAC TCC CAG ATC ACA AAC CCA TAC CAC AGA CCT GAA TAA AGA CTG AGG GTC TAG TGT 1480 Leu Gly Met Val Ser Gly Leu He Ser Asp Ser Gin He Thr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1350 1360 1370 1380
TCA TCC AAC CAA GGG GAC AGA AAC TGG ATG CCT GAA AAC ATC 1485 AGT AGG TTG GTT CCC CTG TCT TTG ACC TAC GGA CTT TTG TAG
Ser Ser Asn Gin Gly Asp Arg Asn Trp Met Pro Glu Asn He> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1390 1400 1410 1420
1490 CGC CTG GTA ACC AGT CGC TCT GGC TGG GCA CTT CCA CCC GCA
GCG GAC CAT TGG TCA GCG AGA CCG ACC CGT GAA GGT GGG CGT Arg Leu Val Thr Ser Arg Ser Gly Trp Ala Leu Pro Pro Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1495 1430 1440 1450 1460 1470 CCT CAT TCC TAC ATC AAT GAG TGG CTC CAA ATA GAC CTG GGG GGA GTA AGG ATG TAG TTA CTC ACC GAG GTT TAT CTG GAC CCC Pro His Ser Tyr He Asn Glu Trp Leu Gin He Asp Leu Gly> a a TRANSLATION OF PGNP-1CODING [A] a a a >
1500
1480 1490 1500 1510 GAG GAG AAG ATC GTG AGG GGC ATC ATC ATT CAG GGT GGG AAG CTC CTC TTC TAG CAC TCC CCG TAG TAG TAA GTC CCA CCC TTC Glu Glu Lys He Val Arg Gly He He He Gin Gly Gly Lys> 1505 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1520 1530 1540 1550 CAC CGA GAG AAC AAG GTG TTC ATG AGG AAG TTC AAG ATC GGG GTG GCT CTC TTG TTC CAC AAG TAC TCC TTC AAG TTC TAG CCC 1510 His Arg Glu Asn Lys Val Phe Met Arg Lys Phe Lys He Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1560 1570 1580 1590 TAC AGC AAC AAC GGC TCG GAC TGG AAG ATG ATT ATG GAT GAC 1515 ATG TCG TTG TTG CCG AGC CTG ACC TTC TAC TAA TAC CTA CTG Tyr Ser Asn Asn Gly Ser Asp Trp Lys Met He Met Asp Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1600 1610 1620 1630 1520 AGC AAA CGC AAG GCG AAG TCT TTT GAG GGC AAC AAC AAC TAT TCG TTT GCG TTC CGC TTC AGA AAA CTC CCG TTG TTG TTG ATA Ser Lys Arg Lys Ala Lys Ser Phe Glu Gly Asn Asn Asn Tyr> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1525 1640 1650 1660 1670 1680 GAT ACA CCT GAG CTG CGG ACT TTT CCA GCT CTC TCC ACG CGA CTA TGT GGA CTC GAC GCC TGA AAA GGT CGA GAG AGG TGC GCT Asp Thr Pro Glu Leu Arg Thr Phe Pro Ala Leu Ser Thr Arg> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1530
1690 1700 1710 1720
TTC ATC AGG ATC TAC CCC GAG AGA GCC ACT CAT GGC GGA CTG AAG TAG TCC TAG ATG GGG CTC TCT CGG TGA GTA CCG CCT GAC Phe He Arg He Tyr Pro Glu Arg Ala Thr His Gly Gly Leu>
1535 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1730 1740 1750 1760 GGG CTC AGA ATG GAG CTG CTG GGC TGT GAA GTG GAA GCC CCT CCC GAG TCT TAC CTC GAC GAC CCG ACA CTT CAC CTT CGG GGA 1540 Gly Leu Arg Met Glu Leu Leu Gly Cys Glu Val Glu Ala Pro> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1770 1780 1790 1800 ACA GCT GGA CCG ACC ACT CCC AAC GGG AAC TTG GTG GAT GAA 1545 TGT CGA CCT GGC TGG TGA GGG TTG CCC TTG AAC CAC CTA CTT Thr Ala Gly Pro Thr Thr Pro Asn Gly Asn Leu Val Asp Glu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1810 1820 1830 1840 1550 TGT GAT GAC GAC CAG GCC AAC TGC CAC AGT GGA ACA GGT GAT ACA CTA CTG CTG GTC CGG TTG ACG GTG TCA CCT TGT CCA CTA Cys Asp Asp Asp Gin Ala Asn Cys His Ser Gly Thr Gly Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1555 1850 1860 1870 1880 1890
GAC TTC CAG CTC ACA GGT GGC ACC ACT GTG CTG GCC ACA GAA CTG AAG GTC GAG TGT CCA CCG TGG TGA CAC GAC CGG TGT CTT Asp Phe Gin Leu Thr Gly Gly Thr Thr Val Leu Ala Thr Glu> a a TRANSLATION OF PGNP-1CODING [A] a a a >
1560
1900 1910 1920 1930 AAG CCC GCG GTC ATA GAC AGC ACC ATA CAA TCA GAG TTT CCA TTC GGG CGC CAG TAT CTG TCG TGG TAT GTT AGT CTC AAA GGT Lys Pro Ala Val He Asp Ser Thr He Gin Ser Glu Phe Pro> 1565 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1940 1950 1960 1970 ACA TAT GGT TTT AAC TGT GAA TTT GGC TGG GGC TCT CAC AAG TGT ATA CCA AAA TTG ACA CTT AAA CCG ACC CCG AGA GTG TTC 1570 Thr Tyr Gly Phe Asn Cys Glu Phe Gly Trp Gly Ser His Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1980 1990 2000 2010
ACC TTC TGC CAC TGG GAA CAT GAC AAT CAC GTG CAG CTC AAG 1575 TGG AAG ACG GTG ACC CTT GTA CTG TTA GTG CAC GTC GAG TTC
Thr Phe Cys His Trp Glu His Asp Asn His Val Gin Leu Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2020 2030 2040 2050 1580 TGG AGT GTG TTG ACC AGC AAG ACG GGA CCC ATT CAG GAT CAC ACC TCA CAC AAC TGG TCG TTC TGC CCT GGG TAA GTC CTA GTG Trp Ser Val Leu Thr Ser Lys Thr Gly Pro He Gin Asp His> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1585 2060 2070 2080 2090 2100 ACA GCA GGA GAT GGC AAC TTC ATC TAT TCC CAA GCT GAC GAA TGT CGT CCT CTA CCG TTG AAG TAG ATA AGG GTT CGA CTG CTT Thr Ala Gly Asp Gly Asn Phe He Tyr Ser Gin Ala Asp Glu> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1590
2110 2120 2130 2140
AAT CAG AAG GGC AAA GTG GCT CGC CTG GTG AGC CCT GTG GTT TTA GTC TTC CCG TTT CAC CGA GCG GAC CAC TCG GGA CAC CAA Asn Gin Lys Gly Lys Val Ala Arg Leu Val Ser Pro Val Val>
1595 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2150 2160 2170 2180 TAT TCC CAG AAC TCT GCC CAC TGC ATG ACC TTC TGG TAT CAC ATA AGG GTC TTG AGA CGG GTG ACG TAC TGG AAG ACC ATA GTG 1600 Tyr Ser Gin Asn Ser Ala His Cys Met Thr Phe Trp Tyr His> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2190 2200 2210 2220 ATG TCT GGG TCC CAC ATC GGC ACA CTC AGG GTC AAA CTG CGC 1605 TAC AGA CCC AGG GTG TAG CCG TGT GAG TCC CAG TTT GAC GCG Met Ser Gly Ser His He Gly Thr Leu Arg Val Lys Leu Arg> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2230 2240 2250 2260 1610 TAC CAG AAG CCA GAA GAG TAC GAT CAG CTG GTC TGG ATG GCC ATG GTC TTC GGT CTT CTC ATG CTA GTC GAC CAG ACC TAC CGG Tyr Gin Lys Pro Glu Glu Tyr Asp Gin Leu Val Trp Met Ala> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1615 2270 2280 2290 2300 2310
CTT GGA CAC CAA GGT GAC CAC TGG AAG GAA GGG CGT GTC TTG GAA CCT GTG GTT CCA CTG GTG ACC TTC CTT CCC GCA CAG AAC Leu Gly His Gin Gly Asp His Trp Lys Glu Gly Arg Val Leu> a a TRANSLATION OF PGNP- 1CODING [A] a a a >
1620
2320 2330 2340 2350 CTC CAC AAG TCT CTG AAA CTT TAT CAG GTG ATT TTC GAG GGC GAG GTG TTC AGA GAC TTT GAA ATA GTC CAC TAA AAG CTC CCG Leu His Lys Ser Leu Lys Leu Tyr Gin Val He Phe Glu Gly> 1625 a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2360 2370 2380 2390
GAA ATC GGA AAA GGA AAC CTT GGT GGG ATT GCT GTG GAT GAC CTT TAG CCT TTT CCT TTG GAA CCA CCC TAA CGA CAC CTA CTG 1630 Glu He Gly Lys Gly Asn Leu Gly Gly He Ala Val Asp Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2400 2410 2420 2430
ATT AGT ATT AAT AAC CAC ATT TCA CAA GAA GAT TGT GCA AAA 1635 TAA TCA TAA TTA TTG GTG TAA AGT GTT CTT CTA ACA CGT TTT
He Ser He Asn Asn His He Ser Gin Glu Asp Cys Ala Lys> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
2440 2450 2460 2470
1640 CCA GCA GAC CTG GAT AAA AAG AAC CCA GAA ATT AAA ATT GAT
GGT CGT CTG GAC CTA TTT TTC TTG GGT CTT TAA TTT TAA CTA Pro Ala Asp Leu Asp Lys Lys Asn Pro Glu He Lys He Asp> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1645 2480 2490 2500 2510 2520
GAA ACA GGG AGC ACG CCA GGA TAC GAA GGT GAA GGA GAA GGT CTT TGT CCC TCG TGC GGT CCT ATG CTT CCA CTT CCT CTT CCA Glu Thr Gly Ser Thr Pro Gly Tyr Glu Gly Glu Gly Glu Gly> a a TRANSLATION OF PGNP-1CODING [A] _a a a >
1650
2530 2540 2550 2560
GAC AAG AAC ATC TCC AGG AAG CCA GGC AAT GTG TTG AAG ACC CTG TTC TTG TAG AGG TCC TTC GGT CCG TTA CAC AAC TTC TGG Asp Lys Asn He Ser Arg Lys Pro Gly Asn Val Leu Lys Thr>
1655 a a TRANSLATION OF PGNP-1CODING [A] a a a >
TTA GAC AAT CTG
1660 Leu Asp
SEQUENCE DESCRIPTION: SEQ ID NO:8:
SEQUENCE ID NO:8: (i) SEQUENCE CHARACTERISTICS: 1665 (A) LENGTH: 856 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: Linear (ii) MOLECULE TYPE: protein (iii) FEATURE:
1670 (A) NAME/KEY: protein
(B) LOCATION: 1...856
(iv) SEQUENCE DESCRIPTION: SEQ ID NO:8:
10 20 30 40 50
1675 MERGLPLLCA VLALVLAPAG AFRNDKCGDT IKIESPGYLT SPGYPHSYHP
60 70 80 90 100
SEKCEWLIQA PDPYQRIMIN FNPHFDLEDR DCKYDYVEVF DGENENGHFR
1680 110 120 130 140 150
GKFCGKIAPP PWSSGPFLF IKFVSDYETH GAGFSIRYEI FKRGPECSQN
160 170 180 190 200
YTTPSGVIKS PGFPEKYPNS LECTYIVFAP KMSEIILEFE SFDLEPDSNP
1685
210 220 230 240 250
PGGMFCRYDR LEIWDGFPDV GPHIGRYCGQ KTPGRIRSSS GILSMVFYTD
260 270 280 290 300
1690 SAIAKEGFSA NYSVLQSSVS EDFKCMEALG MESGEIHSDQ ITASSQYSTN
310 320 330 340 350
WSAERSRLNY PENGWTPGED SYREWIQVDL GLLRFVTAVG TQGAISKETK
1695 360 370 380 390 400
KKYYVKTYKI DVSSNGEDWI TIKEGNKPVL FQGNTNPTDV WAVFPKPLI
410 420 430 440 450
TRFVRIKPAT WETGISMRFE VYGCKITDYP CSGMLGMVSG LISDSQITSS
1700
460 470 480 490 500
NQGDRNWMPE NIRLVTSRSG WALPPAPHSY INEWLQIDLG EEKIVRGIII
510 520 530 540 550
1705 QGGKHRENKV FMRKFKIGYS NNGSDWKMIM DDSKRKAKSF EGNNNYDTPE
560 570 580 590 600
LRTFPALSTR FIRIYPERAT HGGLGLRMEL LGCEVEAPTA GPTTPNGNLV
1710 610 620 630 640 650
DECDDDQANC HSGTGDDFQL TGGTTVLATE KPAVIDSTIQ SEFPTYGFNC
660 670 680 690 700
EFGWGSHKTF CHWEHDNHVQ LKWSVLTSKT GPIQDHTAGD GNFIYSQADE
1715
710 720 730 740 750
NQKGKVARLV SPWYSQNSA HCMTFWYHMS GSHIGTLRVK LRYQKPEEYD
760 770 780 790 800
1720 QLVWMALGHQ GDHWKEGRVL LHKSLKLYQV IFEGEIGKGN LGGIAVDDIS
810 820 830 840 850
INNHISQEDC AKPADLDKKN PEIKIDETGS TPGYEGEGEG DKNISRKPGN
1725 VLKTLD

Claims

WHAT IS CLAIMED IS:
1. A method for determining whether a compound is capable of binding to a receptor protein complex comprising a Vascular Endothelial Growth Factor Receptor-2 (VEGFR- 2) receptor protein and a Neuroplin-l (NP-1) receptor protein, the method comprising introducing a sample comprising the compound to the receptor protein complex and allowing the compound to bind to the complex.
2. A method for determining whether a test compound produces a signal upon binding to a receptor protein complex comprising a VEGFR-2 receptor protein and an NP-1 receptor protein, the method comprising:
(a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, wherein the cells naturally express both receptor proteins and/or characterized in that the cells have been transfected with a DNA sequence coding for VEGFR-2 and/or a DNA sequence coding for NP-1 such that the cells express both receptor proteins;
(b) exposing (i) a first set of the cells to a composition containing a test compound and (ii) a second set of the cells to a composition lacking the test compound;
(c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b) with both the first and the second set of cells; and
(d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells.
3. A method for determining whether a test compound blocks a signal produced by binding of VEGF,65, or another heparin-binding or NP-1 binding VEGF family member, to a receptor protein complex comprising a VEGFR-2 receptor protein and a Neuroplin-l (NP-1) receptor protein, the method comprising:
(a) providing cells expressing a VEGFR-2 receptor protein and a NP-1 receptor protein, characterized in that the cells naturally express both of the receptor proteins and/or characterized in that the cells have been transfected with a DNA sequence coding for a VEGFR-2 receptor protein and/or a DNA sequence coding for a NP-1 receptor protein such that the cells express both receptor proteins;
(b) exposing (i) a first set of the cells to VEGF,65 (or another heparin-binding VEGF family member) and a composition comprising a test compound and (ii) a second set of the cells to VEGF,65 (or another heparin-binding VEGF family member) and the composition without the test compound;
(c) quantitatively assessing a signal derived from activation of VEGFR-2 from step (b) for the first and second set of cells; and
(d) comparing the amount of signal from step (c) from the first set of cells to the amount of signal from step (c) for the second set of cells.
4. The method of any of Claims 1 through 3 characterized in that the VEGFR-2 receptor protein is a vertebrate protein derived from a species selected from the group consisting of human, mouse, rat and quail.
5. The method of any of Claims 1 through 4 characterized in that the NP-1 receptor protein is a vertebrate protein derived from a species selected from the group consisting of human, mouse, rat and chicken.
6. The method of any of Claims 1 through 3 characterized in that both the VEGFR-2 receptor protein and the NP-1 receptor protein are derived from humans.
7. The method of Claim 6 characterized in that the VEGFR-2 receptor protein has the amino acid sequence of SEQ ID NO. 2 and the NP-1 receptor protein has the amino acid sequence of SEQ ID NO. 4.
8. The method of any of Claims 1 through 6 characterized in that: (a) the VEGFR-2 receptor protein is a soluble VEGFR-2 receptor or (b) the NP-1 receptor protein is a soluble receptor or (c) both the VEGFR-2 receptor protein and the NP-1 receptor protein are soluble proteins.
9. The method of Claim 8 characterized in that the soluble VEGFR-2 receptor protein has the amino acid sequence of SEQ ID NO. 6 and the soluble NP-1 receptor protein has the amino acid sequence of SEQ ID NO. 8.
10. A complex formed by the interaction of a recombinant VEGFR-2 receptor protein and a recombinant NP-1 receptor protein.
11. The complex of Claim 10 characterized in that the VEGFR-2 receptor protein is a vertebrate protein derived from a species selected from the group consisting of human, mouse, rat and quail, preferably human; and further characterized in that the NP-1 receptor protein is a vertebrate protein derived from a species selected from the group consisting of human, mouse, rat and chicken, preferably human.
EP00975419A 1999-10-28 2000-10-26 Identification of novel pro-and anti-angiogenic agents Withdrawn EP1281088A2 (en)

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US20030113324A1 (en) * 2001-10-01 2003-06-19 Kari Alitalo Neuropilin/VEGF-C/VEGFR-3 materials and methods
US8846386B2 (en) * 2007-12-18 2014-09-30 University Of Kentucky Research Foundation sVEGFR-2 and its role in lymphangiogenesis modulation

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WO2001031346A3 (en) 2001-10-18
WO2001031346A8 (en) 2001-05-31

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