EP0620826A1 - Tie, a novel endothelial cell receptor tyrosine kinase - Google Patents

Abstract

The invention describes the cloning, sequencing and expression of a new receptor function tyrosine kinase called TIE. The TIE precursor includes 1138 amino acid residues, of which approximately 1117 contain mature TIE. The extracellular domain of TIE contains distinct extension sequences of amino acid sequence having characteristics of the families of imunoglobulin, epidermal growth factor and fibronectin type III repeat protein. In another embodiment, variants of the TIE are created by splicing in which one of the domains of epidermal growth factor homology is missing. A 117 kD glycoprotein phosphorylated with a specific tyrosine is detected by TIE antisera derived from cultured cells expressing the TIE gene. TIE mRNA is expressed in cultured endothelial cells as well as in a small number of tumor cell lines. In situ hybridization of embryonic tissue from human fetuses and mice produces specific expression in endothelial cells of blood vessels. The TIE DNAs and polypeptides described in the present invention may be useful in the diagnosis and treatment of certain diseases in which endothelial cells and their TIE receptor play a role, such as neoplastic diseases involving tumor angiogenesis, scarring wounds, thromboembolic diseases, atherosclerosis and inflammatory diseases.

Description

TIE, A NOVEL ENDOTHELIAL CELL RECEPTOR TYROSINE KINASE

FIELD OF THE INVENTION

The present invention relates generally to the field of genetic engineering and more particularly to genes for receptor tyrosine kinases, their insertion into recombinant DNA vectors, and the production of the resulting proteins in recipient strains of micro-organisms and recipient eukaryotic cells. More specifically, the present invention is directed to tie, a novel receptor tyrosine kinase, to nucleotide sequences encoding tie, and to methods for the generation of DNAs encoding tie and their gene products. Tie DNAs and polypeptides of the invention may be useful in the diagnosis and treatment of certain diseases involving endothelial cells and associated tie receptors, such as neoplastic diseases involving tumor angiogenesis, wound healing, thromboembolic diseases, atherosclerosis and inflammatory diseases.

BACKGROUND OF THE INVENTION

The cellular behavior responsible for the development, maintenance, and repair of differentiated cells and tissues is regulated, in large part, by intercellular signals conveyed via growth factors and similar ligands and their receptors. The receptors are located on the cell surface of responding cells and they bind peptides or polypeptides known as growth factors as well as other hormone-like ligands. The results of this interaction are rapid biochemical changes in the responding cells, as well as a rapid and a long-term readjustment of cellular gene expression. Several receptors associated with various cell surfaces may bind specific growth factors.

Tyrosine phosphorylation is one of the key modes of signal transduction across the plasma membrane. Several currently known protein tyrosine kinase genes encode transmembrane receptors for polypeptide growth factors and hormones such as epidermal growth factor (EGF) , insulin, insulin-like growth factor-I (IGF-I) , platelet derived growth factors (PDGF-A and -B) , and fibroblast growth factors (FGFs) . Heldin et al . , Cell Regulation , l : 555-566 (1990); Ullrich, et al . , Cell , 61 : 243-54 (1990). Growth factor receptors of endothelial cells are of particular interest due to the possible involvement of growth factors, such as FGFs, in several important physiological and pathological processes, such as vasculogenesis, angiogenesis, atherosclerosis, and inflammatory diseases. Folkman, et al . Science, 235 : 442-447 (1987) . Also, the receptors of several hematopoietic growth factors are tyrosine kinases; these include c-fms, which is the colony stimulating factor 1 receptor, Sherr, et al._r Cell , 41 : 665-676 (1985) , and c-kit, a primitive hematopoietic growth factor receptor reported in Huang, et al . , Cell , 63 : 225-33 (1990) .

On the basis of structural similarities, the receptor tyrosine kinases may be divided into evolutionary subfamilies. Ullrich, et al . Cell, 61 : 243-54 (1990) . Such subfamilies include, EGF receptor-like kinase (subclass I) and insulin receptor-like (subclass II) kinase , each of which contains repeated homologous cysteine-rich sequences in their extracellular domains. A single cysteine-rich region is also found in the extracellular domains of the eph-like kinases. Hirai, et al., Science, 238 : 1717-1720 (1987); Lindberg, et al . Mol . Cell . B±ol . , 10 : 6316-24 (1990) ; Lhotak, et. al . , Mol . Cell . Biol . , 11 : 2496-2502 (1991). PDGF receptors as well as c-fms and c-kit receptor tyrosine kinases may be grouped into subclass III; while the FGF receptors form subclass IV. Typical for the members of both of these subclasses are extracellular folding units stabilized by intrachain disulfide bonds. These so-called immunoglobulin (Ig)-like folds are found in the proteins of the immunoglobulin superfamily which contains a wide variety of other cell surface receptors having either cell-bound or soluble ligands. Williams, et al . , Ann . Rev. Immunol . , 6 : 381-405 (1988) . Receptor tyrosine kinases differ in their specificity and affinity. In general, receptor tyrosine kinases are glycoproteins, which consist of (1) an extracellular domain capable of binding the specific growth factor(s) ; (2) a transmembrane domain which usually is an alpha-helical portion of the protein; (3) a juxtame brane domain where the receptor may be regulated by, e . g . , protein phosphorylation; (4) a tyrosine kinase domain which is the enzymatic component of the receptor; and (5) a carboxyterminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.

Processes such as alternative splicing and alternative polyadenylation have recently been reported to be capable of producing several distinct polypeptides from the same gene. These polypeptides may or may not contain the various domains listed above. As a consequence, some extracellular domains may be expressed as separate, secreted proteins and some forms of the receptors may lack the tyrosine kinase domain and contain only the extracellular domain inserted in the plasma membrane via the transmembrane domain plus a short carboxyl terminal tail.

The present invention provides a novel endothelial cell receptor tyrosine kinase which was originally identified as an unknown tyrosine kinase-homologous PCR-cDNA fragment from human leukemia cells by Partanen, et al . , Proc . Natl . Acad . Scl . USA, 87: 8913-8917 (1990). This gene and its encoded protein are called tie which is an abbreviation for the "tyrosine kinase containing immunoglobulin- and EGF-like repeats".

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a DNA or RNA segment of defined structure encoding the tie receptor tyrosine kinase. A DNA or RNA according to the invention may be produced synthetically or isolated from natural sources and may be used in the production of desired recombinant DNA vectors or may be used to recover related genes from other sources. It is a further object of the present invention to provide a recombinant-DNA vector containing a heterologous segment encoding the tie receptor tyrosine kinase or a related protein which is capable of being inserted into a microorganism or eukaryotic cell for expression of the encoded protein. The present invention also provides eukaryotic cells capable of producing useful quantities of the tie receptor tyrosine kinase and proteins of similar function from multiple species. In another aspect of the invention, peptides which may be produced synthetically in a laboratory or by a microorganism which mimic the activity of the natural tie receptor tyrosine kinase protein and which may be used to produce the tie receptor tyrosine kinase or a portion thereof in eukaryotic cells in a reproducible and standardized manner are disclosed. Particularly preferred are peptides selected from the group consisting of: (a) a first sequence:

MetValTrpArgValProProPheLeuLeuProIleLeuPheLeuAlaSerHisValGly AlaAlaValAspLeuThrLeuLeuAlaAsnLeuArgLeuThrAspProGlnArgPhePhe LeuThrCysValSerGlyGluAlaGlyAlaGlyArgGlySerAspAlaTrpGlyProPro LeuLeuLeuGluLysAspAspArglleValArgThrProProGlyProProLeuArgLeu AlaArgAsnGlySerHisGlnValThrLeuArgGlyPheSerLysProSerAspLeuVal GlyValPheSerCysValGlyGlyAlaGlyAlaArgArgThrArgVallleTyrValHis AsnSerProGlyAlaHisLeuLeuProAspLysValThrHisThrValAsnLysGlyAsp ThrAlaValLeuSerAlaArgValHisLysGluLysGlnThrAspVallleTrpLysSer AsnGlySerTyrPheTyrThrLeuAspTrpHisGluAlaGlnAspGlyArgPheLeuLeu GlnLeuProAsnValGlnProProSerSerGlylleTyrSerAlaThrTyrLeuGluAla SerProLeuGlySerAlaPhePheArgLeuIleValArgGlyCysGlyAlaGlyArgTrp GlyProGlyCysThrL sGluCysProGlyCysLeuHisGlyGlyValCysHisAspHis AspGlyGluCysValCysProProGlyPheThrGlyThrArgCysGluGlnAlaCysArg GluGlyArgPheGlyGlnSerCysGlnGluGlnCysProGlylleSerGlyCysArgGly LeuThrPheCysLeuProAspProTyrGlyCysSerCysGlySerGlyTrpArgGlySer 301 GlnCysGlnGluAlaCysAlaProGlyHisPheGlyAlaAspCysArgLeuGlnCysGln

321 CysGlnAsnGlyGlyThrCysAspArgPheSerGlyCysValCysProSerGlyTrpHis

341 GlyValHisCysGluLysSerAspArglleProGlnlleLeuAsnMetAlaSerGluLeu

361 GluPheAsnLeuGluThrMetProArglleAsnCysAlaAlaAlaGlyAsnProPhePro

381 ValArgGlySerlleGluLeuArgLysProAspGlyThrValLeuLeuSerThrLysAla

401 IleValGluProGluLysThrThrAlaGluPheGluValProArgLeuValLeuAlaAsp

421 SerGlyPheTrpGluCysArgValSerThrSerGlyGlyGlnAspSerArgArgPheLys

441 ValAsnValLysValProProValProLeuAlaAlaProArgLeuLeuThrLysGlnSer

461 ArgGlnLeuValValSerProLeuValSerPheSerGlyAspGlyProIleSerThrVal

481 ArgLeuHisTyrArgProGlnAspSerThrMetAspTrpSerThrlleValValAspPro

501 SerGluAsnValThrLeuMetAsnLeuArgProLysThrGlyTyrSerValArgValGln

521 LeuSerArgProGlyGluGlyGlyGluGlyAlaTrpGlyProProThrLeuMetThrThr

541 AspCysProGluProLeuLeuGlnProTrpLeuGluGlyTrpHisValGluGlyThrAsp

561 ArgLeuArgValSerTrpSerLeuProLeuValProGlyProLeuValGlyAspGlyPhe

581 LeuLeuArgLeuTrpAspGlyThrArgGlyGlnGluArgArgGluAsnValSerSerPro

601 GlnAlaArgThrAlaLeuLeuThrGlyLeuThrProGlyThrHisTyrGlnLeuAspVal

621 GlnLeuTyrHisCysThrLeuLeuGlyProAlaSerProProAlaHisValLeuLeuPro

641 ProSerGlyProProAlaProArgHisLeuHisAlaGlnAlaLeuSerAspSerGluIle

661 GlnLeuThrTrpLysHisProGluAlaLeuProGlyProIleSerLysTyrValValGlu

681 ValGlnValAlaGlyGlyAlaGlyAspProLeuTrpIleAspValAspArgProGluGlu

701 ThrSerThrIleileArgGlyLeuAsnAlaSerThrArgTyrLeuPheArgMetArgAla

721 SerlleGlnGlyLeuGlyAspTrpSerAsnThrValGluGluSerThrLeuGlyAsnGly

741 LeuGlnAlaGluGlyProValGlnGluSerArgAlaAlaGluGluGlyLeuAspGlnGln

761 LeuIleLeuAlaValValGlySerValSerAlaThrCysLeuThrlleLeuAlaAlaLeu

781 LeuThrLeuValCysIleArgArgSerCysLeuHisArgArgArgThrPheThrTyrGln

801 SerGlySerGlyGluGluThrlleLeuGlnPheSerSerGlyThrLeuThrLeuThrArg

821 ArgProLysLeuGlnProGluProLeuSerTyrProValLeuGluTrpGluAspIleThr

841 PheGluAspLeuIleGlyGluGlyAsnPheGlyGlnVallleArgAlaMetlleLysLys 861 AspGl LeuLysMetAsnAlaAlalleLysMetLeuLysGluTyrAlaSerGluAsnAsp

881 HisArgAspPheAlaGlyGluLeuGluValLeuCysLysLeuGlyHisHisProAsnlle

901 IleAsnLeuLeuGlyAlaCysLysAsnArgGlyTyrLeuTyrlleAlalleGluTyrAla

921 ProTyrGlyAsnLeuLeuAspPheLeuArgLysSerArgValLeuGluThrAspProAla

941 PheAlaArgGluHisGlyThrAlaSerThrLeuSerSerArgGlnLeuLeuArgPheAla

961 SerAspAlaAlaAsnGlyMetGlnTyrLeuSerGluLysGlnPhelleHisArgAspLeu

981 AlaAlaArgAsnValLeuValGlyGluAsnLeuAlaSerLysIleAlaAspPheGlyLeu

1001 SerArgGlyGluGluValTyrValLysLysThrMetGlyArgLeuProValArgTrpMet

1021 AlalleGluSerLeuAsnTyrSerValTyrThrThrLysSerAspValTrpSerPheGly

1041 ValLeuLeuTrpGluIleValSerLeuGlyGlyThrProTyrCysGlyMetThrCysAla

1061 GluLeuTyrGluLysLeuProGlnAlaAspArgMetGluGlnProArgAsnCysAspAsp

1081 GluValTyrGluLeuMetArgGlnCysTrpArgAspArgProTyrGluArgProProPhe

1101 AlaGlnlleAlaLeuGlnLeuGlyArgMetLeuGluAlaArgLysAlaTyrValAsnMet

1121 SerLeuPheGluAsnPheThrTyrAlaGlylleAspAlaThrAlaGluGluAla; (SEQ ID NO 1) and

(b) a second sequence in which amino acids 214 to 257 of the first formula are absent in the second sequence.

DNA and RNA molecules, recombinant DNA vectors, and modified microorganisms or eukaryotic cells comprising a nucleotide which encodes any of the peptides indicated above are also contemplated in the present invention. In particular, sequences comprising all or part of the following two DNA sequences, their complements, or corresponding RNA sequences are preferred:

1 cgctcgtcct ggctggcctg ggtcggcctc tggagtatgg tctggcgggt

51- gccccctttc ttgctcccca tcctcttctt ggcttctcat gtgggcgcgg

101 cggtggacct gacgctgctg gccaacctgc ggctcacgga cccccagcgc 151 ttcttcctga cttgcgtgtc tggggaggcc ggggcgggga ggggctcgga

201 cgcctggggc ccgcccctgc tgctggagaa ggacgaccgt atcgtgcgca

251 ccccgcccgg gccacccctg cgcctggcgc gcaacggttc gcaccaggtc

301 acgcttcgcg gcttctccaa gccctcggac ctcgtgggcg tcttctcctg

351 cgtgggcggt gctggggcgc ggcgcacgcg cgtcatctac gtgcacaaca

401 gccctggagc ccacctgctt ccagacaagg tcacacacac tgtgaacaaa

451 ggtgacaccg ctgtactttc tgcacgtgtg cacaaggaga agcagacaga

501 cgtgatctgg aagagcaacg gatcctactt ctacaccctg gactggcatg

551 aagcccagga tgggcggttc ctgctgcagc tcccaaatgt gcagccacca

601 tcgagcggca tctacagtgc cacttacctg gaagccagcc ccctgggcag

651 cgccttcttt cggctcatcg tgcggggttg tggggctggg cgctgggggc

701 caggctgtac caaggagtgc ccaggttgcc tacatggagg tgtctgccac

751 gaccatgacg gcgaatgtgt atgcccccct ggcttcactg gcacccgctg

801 tgaacaggcc tgcagagagg gccgttttgg gcagagctgc caggagcagt

851 gcccaggcat atcaggctgc cggggcctca ccttctgcct cccagacccc

901 tatggctgct cttgtggatc tggctggaga ggaagccagt gccaagaagc

951 ttgtgcccct ggtcattttg gggctgattg ccgactccag tgccagtgtc

1001 agaatggtgg cacttgtgac cggttcagtg gttgtgtctg cccctctggg

1051 tggcatggag tgcactgtga gaagtcagac cggatccccc agatcctcaa

1101 catggcctca gaactggagt tcaacttaga gacgatgccc cggatcaact

1151 gtgcagctgc agggaacccc ttccccgtgc ggggcagcat agagctacgc

1201 aagccagacg gcactgtgct cctgtccacc aaggccattg tggagccaga 1251 gaagaccaca gctgagttcg aggtgccccg cttggttctt gcggacagtg

1301 ggttctggga gtgccgtgtg tccacatctg gcggccaaga cagccggcgc 1351 ttcaaggtca atgtgaaagt gccccccgtg cccctggctg cacctcggct 1401 cctgaccaag cagagccgcc agcttgtggt ctccccgctg gtctcgttct 1451 ctggggatgg acccatctcc actgtccgcc tgcactaccg gccccaggac 1501 agtaccatgg actggtcgac cattgtggtg gaccccagtg agaacgtgac 1551 gttaatgaac ctgaggccaa agacaggata cagtgttcgt gtgcagctga

1601 gccggccagg ggaaggagga gagggggcct gggggcctcc caccctcatg

1651 accacagact gtcctgagcc tttgttgcag ccgtggttgg agggctggca

1701 tgtggaaggc actgaccggc tgcgagtgag ctggtccttg cccttggtgc

1751 ccgggccact ggtgggcgac ggtttcctgc tgcgcctgtg ggacgggaca

1801 cgggggcagg agcggcggga gaacgtctca tccccccagg cccgcactgc

1851 cctcctgacg ggactcacgc ctggcaccca ctaccagctg gatgtgcagc

1901 tctaccactg caccctcctg ggcccggcct cgccccctgc acacgtgctt

1951 ctgcccccca gtgggcctcc agccccccga cacctccacg cccaggccct

2001 ctcagactcc gagatccagc tgacatggaa gcacccggag gctctgcctg

2051 ggccaatatc caagtacgtt gtggaggtgc aggtggctgg gggtgcagga

2101 gacccactgt ggatagacgt ggacaggcct gaggagacaa gcaccatcat

2151 ccgtggcctc aacgccagca cgcgctacct cttccgcatg cgggccagca

2201 ttcaggggct cggggactgg agcaacacag tagaagagtc caccctgggc

2251 aacgggctgc aggctgaggg cccagtccaa gagagccggg cagctgaaga

2301 gggcctggat cagcagctga tcctggcggt ggtgggctcc gtgtctgcca

2351 cctgcctcac catcctggcc gcccttttaa ccctggtgtg catccgcaga

2401 agctgcctgc atcggagacg caccttcacc taccagtcag gctcgggcga

2451 ggagaccatc ctgcagttca gctcagggac cttgacactt acccggcggc

2501 caaaactgca gcccgagccc ctgagctacc cagtgctaga gtgggaggac

2551 atcacctttg aggacctcat cggggagggg aacttcggcc aggtcatccg

2601 ggccatgatc aagaaggacg ggctgaagat gaacgcagcc atcaaaatgc

2651 tgaaagagta tgcctctgaa aatgaccatc gtgactttgc gggagaactg

2701 gaagttctgt gcaaattggg gcatcacccc aacatcatca acctcctggg

2751 ggcctgtaag aaccgaggtt acttgtatat cgctattgaa tatgccccct

2801 acgggaacct gctagatttt ctgcggaaaa gccgggtcct agagactgac

2851 ccagcttttg ctcgagagca tgggacagcc tctaccctta gctcccggca

2901 gctgctgcgt ttcgccagtg atgcggccaa tggcatgcag tacctgagtg 2951 agaagcagtt catccacagg gacctggctg cccggaatgt gctggtcgga

3001 gagaacctag cctccaagat tgcagacttc ggcctttctc ggggagagga

3051 ggtttatgtg aagaagacga tggggcgtct ccctgtgcgc tggatggcca

3101 ttgagtccct gaactacagt gtctatacca ccaagagtga tgtctggtcc

3151 tttggagtcc ttctttggga gatagtgagc cttggaggta caccctactg

3201 tggcatgacc tgtgccgagc tctatgaaaa gctgccccag gctgaccgca

3251 tggagcagcc tcgaaactgt gacgatgaag tgtacgagct gatgcgtcag

3301 tgctggcggg accgtcccta tgagcgaccc ccctttgccc agattgcgct

3351 acagctaggc cgcatgctgg aagccaggaa ggcctatgtg aacatgtcgc

3401 tgtttgagaa cttcacttac gcgggcattg atgccacagc tgaggaggcc

3451 tgagctgcca tccagccaga acgtggctct gctggccgga gcaaactctg

3501 ctgtctaacc tgtgaccagt ctgaccctta cagcctctga cttaagctgc

3551 ctcaaggaat ttttttaact taagggagaa aaaaagggat ctggggatgg

3601 ggtgggctta ggggaactgg gttcccatgc tttgtaggtg tctcatagct

3651 atcctgggca tccttctttc tagttcagct gccccacagg tgtgtttccc

3701 atcccactgc tcccccaaca caaaccccca ctccagctcc ttcgcttaag

3751 ccagcactca caccactaac atgccctgtt cagctactcc cactcccggc

3801 ctgtcattca gaaaaaaata aatgttctaa taagctccaa aaaaa (SEQ ID 2) ; or

a second sequence, wherein the nucleotides corresponding to positions 676 to 807 in the first sequence are absent from the second sequence.

DNA and RNA molecules containing segments of the larger sequence are also provided for use in carrying out preferred aspects of the invention relating to the production of such peptides by the techniques of genetic engineering and the production of oligonucleotide probes. Since the DNA sequence encoding the tie protein has been fully identified, it is possible to produce an entire gene by, for example, polymerase chain reaction or by synthetic chemistry using commercially available equipment, after which the gene can be inserted into any of the many available DNA vectors using known techniques of recombinant DNA technology. Furthermore, automated equipment is also available which makes direct synthesis of any of the peptides disclosed herein readily available. Thus, the present invention may be carried out using reagents, plasmids, and microorganism which are readily available to the skilled artisan.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures 1 to 13 are provided to illustrate the invention but are not considered to be limiting thereof unless so specified. Figure 1. Nucleic and deduced amino acid sequence of the tie cDNA. The 3845 bp nucleotide sequence compiled from two overlapping cDNA clones isolated from HEL library contains an open reading frame of 1138 amino acids (marked in the single-letter code) . The tie precursor begins from nucleotide number 37 and the mature tie protein from amino acid 22 (nucleotide number 100) . The hydrophobic signal sequence and the putative transmembrane domain are underlined (thick lines) as are the sites for potential N-linked glycosylation (thin lines) . The mature tie protein Cysteine residues found in the extracellular domain have been boxed, the tyrosine kinase domain is shown by horizontal arrows and the kinase insert with italics. The three cysteine-rich segments having homology to EGF-like domains are also boxed (EGFH I-III) . Their alignment is shown in Fig. 2. The first of the EGF repeats missing in clone 3a is indicated by vertical arrows. The sequence has been deposited to GenBank/EMBL (Accession no. X60957) . A is alanine_Λ C is cysteine, D is aspartate, E is glutamate, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, \L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine.

Figure 2. A. Alignment of the EGF-like domains of tie. Comparison is made with human EGF sequence (amino acid residues 1-44) and homologous sequence^'s in the growth factor CRIPTO (67-108), laminin A chain (1092-1138), Drosophila melanogaster Notch (897-945) and Caenorhabditis elegans Lin-12 (204-246) developmental control proteins, human blood coagulation factor IXa (83-130) and mouse urokinase type plasminogen activator (18-65) . The asterisks point out conserved residues and the homologous cysteine residues are boxed. The consensus residues for s-hydroxylation present in the repeats of Notch and factor IXa are printed in boldface. B. Comparison of the three fibronectin type III repeats of the tie protein and the first three FNIII repeats of the human LAR receptor phosphotyrosine phosphatase. The cysteine residues as well as some other consensus residues typical for immunoglobulin domains are shown above the second FNIII repeat of the tie protein.

Figure 3. Expression of tie cDNA in COS cells. COS cells were transfected with SV40-based expression vectors for tie (SV14-1, SV14-2) and FGFR-4 (C, Partanen, J. , T. P. Makela, E. Eerola, J. Korhonen, H. Hirvonen, L. Claesson-Welsh, and K. Alitalo, EMBO J . 10 : 1347-1354, 1991), labelled with ³⁵S-methionine, lysed and immunoprecipitated as described in materials and methods of example 3. Autoradiograms of the SDS-PAGE analysis of the precipitated proteins are shown. A. Identification of tie polypeptides expressed in the COS cells. HI, immune serum against s-gal-tie fusion protein; HO, preim une serum. The immune serum was blocked with the antigen where indicated (+) . B. Effect of tunicamycin on the molecular weight of the tie protein. MI, immune serum.against a carboxyl terminal tie peptide; MO preimmune serum. Where indicated (+) , the transfected cell cultures were labelled in the presence of tunicamycin. Mobilities of the molecular weight markers are shown on the left.

Figure 4. Immunoblot analysis of cell lines expressing the tie protein. Cell lysates of NIH3T3 cells transfected (LTR14-2) or not transfected (NE01) with a tie expression vector as well as porcine aortic endothelial cells (PAE) were analyzed by immunoblotting with antiserum against a carboxyterminal tie peptide. The samples in the two right most lanes (aPY, IP) were immunoprecipitate with ahti-phosphotyrosine antibodies prior to immunoblotting.

Figure 5. Chromosomal mapping of the tie locus. Radiolabeled JTK14 DNA was hybridized to normal human male peripheral lymphocyte metaphase preparations; slides were washed, developed after exposure and chromosomes were G-banded to distinguish individual chromosomes. Grain localization is illustrated on the schematic chromosome 1 where each dot represents 3 grains. Some nonspecific background signal was detected on the other chromosomes; 12,6% (40/317) on other chromosomes of group A, 8,5% (27/317) on chromosomes of group B, 29.6% (94/317) on C-group chromosomes and 14.8% (47/317) on the other chromosome groups.

^' Figure 6. tie mRNA expression in leukemia cell lines. Poly (A)+ RNA from the indicated cell lines was analyzed by Northern blotting and hybridization with the tie cDNA probe. Hybridization with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used as an internal control for the loading of even amounts of RNA to the analysis.

Figure 7. tie mRNA expression in endothelial cell lines. Northern blot analysis of tie mRNA expression in PAE and EA hy926 endothelial cell lines. A lane containing poly(A)+ RNA from Dami cells was included as a positive control.

Figure 8. Location of tie mRNA in endothelium of the kidney vessels by in situ hybridization. The dark field image showing the hybridization signal is on the top (A) . A corresponding phase-contrast micrograph is shown below (B) .

Figure 9. Comparison of the structure of the tie protein with some other receptor tyrosine kinases containing immunoglobulin and fibronectin type III repeats.

The open circles represent immunoglobulin loops, the open boxes fibronectin type III repeats and the filled ovals EGF homology domains. The shaded box represents the cysteine rich region of the eph-like kinases. The cytoplasmic tyrosine kinase domains are drawn as black boxes. Figure 10. Schematic structure of the human tie receptor tyrosine kinase and comparison of its deduced amino acid sequence with two mouse tie cDNA clones (1C1D and D10E5) .

The tie receptor consists of two immunoglobulin-like loops (Ig) , three (or two) epidermal growth factor domains (EGF) followed by three fibronectin III like domains, a transmembrane region (TM) and two cytoplasmic tyrosine kinase domains (TK1 and TK2) . Amino acid homology between mouse and human tie amino acid sequences is 96% and 95% for the segments 1C1D and D10E5, respectively. Amino acid residue symbols are as in Fig. 1.

Figure 11. Expression of tie mRNA in human tissues. Total RNA isolated from 17-19 week fetal tissues was analyzed by Northern blotting (A) . Hybridization of polyadenylated RNA from human adult tissues is shown in B. The s-actin and GAPDH probes were used as internal controls for the amount of RNA loaded.

Figure 12. In situ hybridization analysis of tie mRNA expression in 12 day p.c. mouse embryo.

Shown are light-field (A) and dark-field (B,C) photomicrographs of a sagittal section hybridized with 1C1D antisense (A,B) and sense probes (C) . Expression of tie mRNA is restricted to the endothelium of blood vessels. Used abbreviations: br (brain) , mg ( eninges) , lg (lung) , mb (mandible) , ht (heart) , vn (ventricle) , at (atrium) , sc (spinal cord) , pv (prevertebra) , and cv (posterior cardinal vein) .

Figure 13. Comparison of tie mRNA (A) and factor VIII (B) expression in a 8 day p.c. mouse placenta.

Factor VIII is seen as the dark deposit surrounding the blood lacunae in (A) and the tie signal in a similar but separate section (B) is seen as white grains. As can be seen from the figure, both signals are localized to endothelial cells of blood lacunae which form the labyrinth. DETAILED DESCRIPTION

In the description which follows, a number of terms used in reco binant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided.

Gene: A DNA sequence containing a template for an RNA polymerase. The RNA transcribed from a gene may or may not code for a protein. RNA that codes for a protein is termed messenger RNA (mRNA) and, in eukaryotes, is transcribed by RNA polymerase II. However, it is also known to construct a gene containing an RNA polymerase II template wherein a RNA sequence is transcribed which has a sequence complementary to that of a specific mRNA but is not normally translated. Such a gene construct is herein termed an "antisense RNA gene" and such a RNA transcript is termed an "antisense RNA." Antisense RNAs are not normally translatable due to the presence of translational stop codons in the antisense RNA sequence. A "complementary DNA" or "cDNA" gene includes recombinant genes synthesized by reverse transcription of mRNA lacking intervening sequences (introns) .

Cloning vehicle: A plasmid or phage DNA or other DNA sequence which is able to replicate autonomously in a host cell, and which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning. The cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word "vector" is sometimes used for "cloning vehicle."

Expression vector: A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences. Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites. The present invention pertains both to expression of recombinant tie protein, and to the functional derivatives of this protein.

Functional Derivative: A "functional derivative" of tie protein is a protein which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of non-recombinant tie protein. A functional derivative of tie protein may or may not contain post-translational modifications such as covalentiy linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term "functional derivative" is intended to include the "fragments," "variants," "analogues," or "chemical derivatives" of a molecule. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980) . Procedure for coupling such moieties to a molecule are well known in the art.

Fragment: A "fragment" of a molecule such as tie protein is meant to refer to any variant of the molecule, such as the peptide core, or a variant of the peptide core. Variant. A "variant" of a molecule such as tie protein is meant to refer to a molecule substantially similar in structure and biological activity to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.

Analog: An "analog" of tie protein or genetic sequences is meant to refer to a protein or genetic sequence substantially similar in function to the tie protein or genetic sequence herein.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to "tie", a novel receptor tyrosine kinase, tie-encoding nucleic acid molecules (e . g. , cDNAs, genomic DNAs, RNAs, anti-sense RNAs, etc.), production of tie peptides or tie protein from a tie gene and its product, recombinant tie expression vectors, tie analogs and derivatives, and diagnostic and/or therapeutic uses of tie and related proteins, tie -encoding nucleic acid molecules, tie ligands, tie antagonists and anti-tie antibodies.

EXAMPLE 1

Production of Recombinant Tie Biologically-active tie may be produced by the cloning and expression of a tie-encoding nucleotide or its functional equivalent in a suitable host cell. Production of tie using recombinant DNA technology may be divided into a step-wise process for the purpose of description, which process includes: (1) isolating or generating the coding sequence (gene) for the desired tie; (2) constructing an expression vector capable of directing the synthesis of the desired tie;

(3) transfecting or transforming appropriate host cells capable of replicating and expressing the tie gene and/or processing the gene product to produce the desired tie; and

(4) identifying and purifying the desired tie product.

A. isolation of the tie gene The nucleotide coding sequence of tie, or functional equivalents thereof, may be used to construct recombinant expression vectors which direct the expression of the desired tie product. The nucleotide coding sequence for tie is depicted in SEQ ID NO. 1. The nucleotide sequence depicted therein, or fragments or functional equivalents thereof, may be used to generate the recombinant molecules which direct the expression of the recombinant tie product in appropriate host cells. Tie-encoding nucleotide sequences may be obtained from a variety of cell sources which produce products with tie-like activities and/or which express tie-encoding mRNA. The Applicants have identified a number of suitable human cell sources for tie including endothelial cells, leukemia cells, and rhabdomyosarcoma and fibrosarcoma cells.

The tie coding sequence may be obtained by cDNA cloning from RNA isolated and purified from such cell sources or by genomic cloning. For example, the tie sequence may be amplified by polymerase chain reaction from cDNA or genomic DNA material using techniques well-known in the art. Either cDNA or genomic libraries of clones may be prepared using techniques well-known in the art and may be screened for particular tie DNAs with nucleotide probes which are substantially complementary to any portion of the tie gene. Full length clones, i.e., those containing the entire coding region of the desired tie gene, may be selected for use in constructing expression vectors. Alteratively, tie-encoding DNAs may be synthesized, in whole or in part, by chemical synthesis using standard techniques.

Due to the inherent degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the method of the invention. Such alterations of tie nucleotide sequences include deletions, additions, or substitutions of different nucleotides resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product may contain deletions, additions, or substitutions of amino acid residues within the sequence which result in "silent" changes thus producing a bioactive tie product. Such amino acid deletions, additions, or substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the a phipathic nature of the amino acids involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids indude lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values indude the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

B. construction of tie expression vectors

Using the above information, a variety of recombinant DNA vectors capable of expressing the tie receptor tyrosine kinase in reasonable quantities are provided. Additional recombinant DNA vectors of related structure which code for synthetic proteins having the key structural features identified herein as well as for proteins of the same family from other sources may be produced from the tie receptor tyrosine kinase cDNA using standard techniques of recombinant DNA technology. A transformant expressing the tie receptor tyrosine kinase has been produced as an example of this technology (see EXAMPLES 3 and 4) . The newly discovered sequence and structure information may be used, through transfection of eukaryotic cells, to prepare the tie receptor tyrosine kinase and its various domains for biological purposes.

C. Identification of Transfectants or Transformants Expressing tie Gene Products

The host cells which contain recombinant coding sequences and which express the biologically active, mature product may be identified by at least four general approaches: (a) DNA-DNA, DNA-RNA or RNA-antisense RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expression of tie mRNA transcripts in the host cell; and (d) detection of the mature gene product as measured by immunoassay and, ultimately, by its biological activities.

In the first approach, the presence of tie coding sequences inserted into expression vectors may be detected by DNA-DNA hybridization using probes comprising nucleotide sequences that are homologous to the tie coding sequence.

In the second approach, the recombinant expression vector/host system may be identified and selected based upon the presence or absence of certain "marker" gene functions (e . g. , thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.). For example, if the tie coding sequence is inserted within a marker gene sequence of the vector, recombinants containing that coding sequence may be identified by the absence of the marker gene function. Alternatively, a marker gene may be placed in tandem with the tie sequence under the control of the same or different promoter used to control the expression of the tie coding sequence. Expression of the marker in response to induction or selection indicates expression of the tie coding sequence.

In the third approach, transcriptional activity of the tie coding region may be assessed by hybridization assays. For example, polyadenylated RNA may be isolated and analyzed by Northern blotting using a probe homologous to the tie coding sequence or particular portions thereof. Alternatively, the total nucleic acid of the host cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of tie may be assessed immunologically, for example, by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like. The ultimate test of the success of the expression system, however, involves the detection of the biologically-active tie gene product. A cell-free media obtained from the cultured transfectant host cell may be assayed for tie activity when the gene product is secreted. When the gene product is not secreted, cell lysates may be assayed for such activity. In either case, assays which measure ligand binding to tie or other bioactivities of tie may be used.

D. tie Derivatives, Analogs and Peptides

The production and use of derivatives, analogs, and peptides related to tie are also within the scope of the invention. Such derivatives, analogs, or peptides may have enhanced or diminished biological activities in comparison to native tie. tie-related derivatives, analogs, and peptides of the invention may be produced by a variety of means known in the art. Procedures and manipulations at the genetic and protein levels are within the scope of the invention. Peptide synthesis, which is standard in the art, may be used to obtain tie peptides. At the protein level, numerous chemical modifications may be used to produce tie like derivatives, analogs, or peptides by techniques known in the art, including but not limited to, specific chemical cleavage by endopeptidases (e . g. cyanogen bromides, trypsin, chymotrypsin, V8 protease, and the like) or exopeptidases, acetylation, formulation, oxidation, etc.

E. Anti-tie Antibodies

Also within the scope of the invention is the production of polyclonal and monoclonal antibodies which recognize tie or related proteins. Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of tie. For the production of antibodies, various host animals may be immunized by injection with tie, or a synthetic tie peptide, including but not limited to, rabbits, mice, and rats. Various adjuvants may be used to increase the immunological response, depending upon the host species, including but not limited, to Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum .

A monoclonal antibody directed against an epitope of tie may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein, Nature, 256 : 495-497 (1975) , and the more recent human B-cell hybridoma technique of Kosbor et al . , Immunology Today, 4 : 12 (1983) and the EBV-hybridoma technique of Cole et al . , Monoclonal Antibodies and Cancer Therapy, 77-96 (Alan R. Liss, Inc. 1985) .

Antibody fragments which contain the idiotype of the molecule may be generated by known techniques. For example, such fragments include, but are not limited to, the F(ab')2 fragment which may be produced by pepsin digestion of the antibody molecule; the Fab' fragments which may be generated by reducing the disulfide bridges of the F(ab')2 fragment; and the two Fab fragments which may be generated by treating the antibody molecule with papain and a reducing agent.

Antibodies to tie may find use in the qualitative and quantitative detection of mature tie and its precursor and subcomponent forms, in the affinity purification of tie polypeptides, and in the elucidation of tie biosynthesis, metabolism and function. Detection of tie tyrosine kinase activity may be used as an enzymatic means of generating and amplifying a tie-specific signal in such assays. Antibodies to tie may also be useful as diagnostic and therapeutic agents.

F. Uses of tie, tie-encoding Nucleic Acid Molecules and Anti-tie Antibodies

Compositions of the present invention may be applied to a wide variety of uses, including diagnostic and/or therapeutic uses of tie, tie analogs and derivatives, tie-encoding nucleic acid molecules, antisense nucleic acid molecules and anti-tie antibodies.

Tie-encoding nucleic acid molecules or fragments thereof may be used as probes to detect and quantify mRNAs encoding tie. Assays which utilize nucleic acid probes to detect sequences comprising all or part of a known gene sequence are well-known in the art. Tie mRNA levels may indicate emerging and/or exiting neoplasias as well as the onset and/or progression of other human diseases. Therefore, assays which detect and quantify tie mRNA may be of considerable diagnostic value.

Anti-sense tie RNA molecules may be useful therapeutically to inhibit the translation of tie-encoding mRNAs where the therapeutic objective involves the elimination of the presence of tie or to downregulate its levels. Tie anti-sense RNA, for example, may be useful as a tie- antagonizing agent in the treatment of diseases for which tie is involved as a causative agent, for example due to its overexpression.

Additionally, tie anti-sense RNAs may be useful in elucidating tie functional mechanisms. Tie-encoding nucleic acid molecules may be used for the production of recombinant tie proteins and related molecules as separately discussed in this application.

Anti-tie antibodies may be used to diagnose and quantify tie in various contexts. For example, antibodies against various domains of tie may be used as a basis for tie immunoassays or immunohistochemical assessment of tie. Tyrosine kinase activity of tie may be useful in these assays as an enzymatic amplification reaction for the generation of a tie signal. Anti-tie antibodies may also be useful in studying the amount of tie on cell surfaces.

Antibodies may be produced which function as tie ligand agonists or antagonists whereby the regulation of tie activity becomes possible. Since tie apparently is located on endothelial surfaces facing the vascular lumen, introduction of tie extracellular domain, its fragments or analogs, ligands or anti-tie extracellular domain antibodies into the bloodstream may allow the manipulation of tie activity and function in vivo with consequences on endothelial cell behavior and disease onset/progression.

The introduction and expression of genes in endothelial cells in vivo is envisioned and will allow further manipulation of tie activity via expression vectors producing tie or its various functional derivatives in endothelial cells. For example, when sufficiently overexpressed, receptor tyrosine kinases in which the tyrosine kinase domain has been specifically inactivated by in vitro mutagenesis function as dominant inhibitors of receptor function. Cloning of the tie promoter and regulatory sequences may allow targeting of gene expression mainly to endothelial cells in vivo .

G. Molecular Biology of Tie

Containing EGF-like, immunoglobulin-like, fibronectin-like and tyrosine kinase domains, tie belongs to four different gene superfamilies. A combination of motifs from all immunoglobulin, fibronectin and EGF-homology superfamilies in the extracellular domain is a unique feature among known receptor tyrosine kinases.

The EGF-like domain is a commonly found structural motif in cell surface and extracellular proteins involved in protein-protein interactions. Davis, The New Biologist, 2 : 410-419, (1990). Many transmembrane receptors for either soluble or cell bound ligands contain EGF repeats. Furthermore, two of the six EGF repeats of thrombomodulin, an endothelial cell surface glycoprotein, have been reported to be responsible for thrombin binding, Stearns, et al . , J. Biol . Chem . , 264 : 3352-3356, (1989), and the EGF domain of the lymph node homing receptor has been implicated in the adhesion of lymphocytes to high endothelial venules. Siegelman, et al . , Cell , 61 : 611-22, (1990). Also, some homeotic genes, such as Notch, delta, and crumbs of Drosophila melanogaster (Wharton, et al., Cell , 43 : 567-581, 1985, Vissin, et al., EMBO J . , 6 : 3431-3440, 1987, Tepass, et al., Cell, 61: 787-799, 1990) and lin!2 and grlp-1 of Caenorhabditis elegans (Yochem, et al., Nature, 335 : 547-550, 1988, Yochem and Greenwald, Cell, 58 : 553-563 1989) encode large transmembrane proteins containing several EGF-like repeats. These proteins participate in several cell-fate decisions, requiring cell-cell communication. Genetic evidence further suggests, that the various EGF motifs function in different protein-protein interactions. Kelley, et al . , Cell, 51 : 539-548, (1987). Multiple EGF repeats are found also in extracellular matrix proteins mediating cell adhesion, such as laminin and tenascin. In addition, EGF repeats are a common motif in secreted proteins involved in blood clotting, including coagulation factors VII, IX, X, proteins C and S as well as tissue- and urokinase-type plasminogen activators. Furie and Furie, Cell, 53 : 505-518, (1988). The EGF like domain of urokinase-type plasminogen activator has been reported to be responsible for its receptor binding. Appella, et al . , J. Biol . Chem . , 262 : 4437-4440, (1987).

The EGF-like repeats of tie contain eight cysteine residues instead of the usual six. Although eight cysteines are also found in the EGF repeats of laminin, the tie repeats are clearly most related to each other. None of the repeats of tie contains the consensus sequence required for asparagine/aspartate s-hydroxylation and calcium binding. The finding of a tie cDNA clone which encoded a protein lacking the first of the EGF-like repeats further suggests that these domains are located in separate exons and that the repeat structure was presumably created by exon duplication in the course of the molecular evolution of the tie receptor tyrosine kinase. In addition, the observation of several tie mRNA forms in EAhy926 cells supports the notion that various forms of the tie receptor are produced, presumably due to differential splicing.

The immunoglobulin and fibfonectin superfamilies also comprise glycoproteins implicated in extracellular protein-protein interactions with either soluble or cell bound molecules. Williams and Barclay, Ann . Rev. Immunol . , 6: 381-405, (1988) . Many receptor tyrosine kinases such as PDGF, CSF-1 receptors, c-kit proto-oncogene as well as the FGF receptors contain Ig-like loops. Ullrich and Schlessinger, Cell, 61 : 243-54, (1990). In many cases both immunoglobulin and fibronectin type III domains are found in the same protein. This type of multidomain structure has recently been reported to be present in some receptor tyrosine kinases. O'Bryan, et al . , Mol . Cell . Biol . , 11 : 5016-5031, (1991); Rescigno, et al . , Oncogene, 6 : 1909-1913, (1991). As both immunoglobulin and FNIII repeats have been suggested to have a common evolutionary origin (Bazan, Proc . Natl . Acad . Sci . USA, 87 : 6934-6938, 1990), it is interesting to note that repeat regions of the tie protein possess features of both of these classes. The presence of EGF, immunoglobulin, and fibronectin-like structural motifs in the extracellular domain of the tie protein suggests that the tie receptor might interact with several different extracellular molecules.

The regional localization of the tie gene at Ip33-p34 indicates that the tie locus is telomeric of the jun locus since the PB5-5 hybrid which is negative for tie is positive for jun . Haluska, et al . , Proc . Natl . Acad . Sci . USA, 85 : 2215-2218, (1988). The chromosomal region Ip32-p34 is involved in deletions in neuroblastoma, malignant lymphoma, glioma and other malignancies. Trent, et al . , Cytogenet . Cell Genet . , 51 : 533-562, (1989).

Our earlier and present experiments indicate that the mRNA for the tie receptor is expressed only in few tumor cell lines in culture. In contrast, expression was evident in Northern blotting of all mouse and human fetal tissues studied. This pattern of expression is compatible with the possibility that the signal obtained from tissues is derived from endothelial cells, as suggested by the finding of tie mRNA in the EA.hy926 and PAEC endothelial cell lines as well as in primary cultured human endothelial cells. Furthermore, in situ hybridization analyses of tie expression in human as well as in mouse tissues indicate that tie mRNA is present in endothelial cells. The above-stated findings on tie mRNA expression suggest that the tie expression product is characteristic of the bipotential hematopoietic cell lineage retaining erythroid and megakaryoblastic differentiation capacities as well as for the endothelial cell lineage. Several differentiation antigens shared between megakaryoblastic and endothelial cells have been shown to exist, one example being the platelet glycoprotein Ilia Blood, 72 : 1478-1486, (1988); Kieffer, et al.. Blood, 72 : 1209-1215, (1988); Berridge, et al . , Blood, 66: 76-85, (1985) . The observed expression pattern of tie mRNA is rather intriguing as EGF motifs are a common theme of proteins controlling hemostasis as well as proteins mediating associations with the endothelium.

EXAMPLE 1 Isolation and characterization of cDNA clones encoding tie An oligo-dT primed human HEL cell cDNA library in bacteriophage Igtll (A kind gift from Dr. Mortimer Poncz, Childrens Hospital of Philadelphia, PA; (Poncz, et al., Blood, 69 : 219-223, 1987)) and a random primed human endothelial cell cDNA library (Clontech Cat. #107Ob) were screened with the JTK14 cDNA fragment PCR-amplified from the reverse-transcribed polyadenylated RNA of K562 leukemia cells. Partanen, et al . , Proc . Natl . Acad . Sci . U S A, 87 : 8913-8917, (1990) . Positive plaques were identified and purified as described in Sambrook, et al . , Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, 1989. cDNA inserts of bacteriophage lambda were isolated as -Bco-RI-fragments and subcloned into GEM3Z (+) plasmid (Promega) . The entire tie protein coding region was isolated from both libraries. Two overlapping clones isolated from a HEL-library (HE11-1, nucleotides 62 to 3845 in Fig.l, and 12a, nucleotides 1 to 2446) were sequenced using the dideoxy chain termination method with oligonucleotide primers designed according to the sequences obtained. All portions of the cDNAs were sequenced on both strands. Sequence analyses were performed using the GCG package programs (Devereux, et al., Nucleic Acids Res . , 12 : 387-395, 1984) and the Prosite program for Apple Macintosh.

A 200 bp long tie cDNA fragment isolated by a PCR cloning method from K562 cell cDNA was used as a molecular probe to screen an oligo dT-primed human erythroleukemia cell cDNA library and a random-primed human endothelial cell cDNA library. Nucleotide sequence analysis of clones HE11-1 and 12a isolated from the HEL library revealed an open reading frame of 1138 amino acids (Fig. 1) . The translational initiator, methionine, marked in Figure 1 is surrounded by a typical consensus sequence (Kozak, Nucleic Acids Res . , 12 : 857-872, 1984) and followed by a hydrophobic amino acid sequence characteristic of signal sequences for translocation into the endoplasmic reticulum. Beginning with amino acid residue 214 of the reading frame there is a region of 130 amino acid residues containing 24 cysteine residues. This region may be aligned into three repeated homologous domains containing eight cysteine residues each (Fig. 2A) . Figure 2A also shows the comparison of the tie cysteine-rich domains with the epidermal growth factor (EGF) and CRIPTO growth factor proteins and the EGF-like repeats of laminin A chain, the Notch and Linl2 developmental control proteins of Drosophila melanogaster and Caenorhabditis elegans , respectively, and blood coagulation factor IXa. Significant structural similarities are present between tie and the EGF family, which allow the inclusion of the cysteine-rich repeats of tie in the EGF repeat family. However, the tie repeats are more closely related to each other than to other members of the EGF repeat family. This is particularly evident when examining the amino terminal ends of the repeats, whose three cysteine residues are not conserved in other EGF repeats (Fig. 2A) . In addition to several tie cDNA molecules encoding three EGF repeats, a cDNA clone was isolated from HEL cell cDNA library, which lacked the first of the EGF repeats (marked between the arrowheads in Fig. 1) without otherwise affecting the reading frame. The amino-terminal region of the tie extracellular domain shows weak, but significant, homology to the amino terminus of chicken N-CAM protein (Cunningham, et al., Science, 236: 799-806, 1987). As with N-CAM, a pair of cysteine residues surrounded by consensus motifs characteristic for the proteins of the immunoglobulin superfamily (Williams and Barclay, Ann. Rev. Immunol . , 6: 381-405, 1988) is found in this region (Igl in Fig.l). In addition, two pairs of cysteine residues are located carboxyl terminal of the three EGF repeats. The amino acid sequence around the first cysteine pair shows additional homology to immunoglobulin domains (Ig2 in Fig.l). The extracellular region following the Ig2 domain (including one of the cysteine pairs) may be aligned into three repeats that are homologous to fibronectin type III (FNIII) repeats. The three repeats of the tie protein and their comparison with the FNIII repeats present in the human LAR phosphotyrosine phosphatase (Streuli, et al._r J. Exp . Med . , 168 : 1553-1562, 1988) are shown in Fig. 2B. Interestingly, the second of these three repeats (FN2) contains a pair of cysteine residues as well as some other features of an immunoglobulin domain (Fig. 2B) and thus represents an intermediate of a FNIII repeat and an immunoglobulin domain.

Five consensus sites for potential N-linked glycosylation (NXS/T, X = any amino acid) may be distinguished in the extracellular domain. None of these appears in the EGF repeats. Amino acids 761-787 form a hydrophobic region of the sequence, which is likely to function as the transmembrane domain of the receptor, followed by several basic residues on the putative cytoplasmic side of the polypeptide. The juxtamembrane domain is 50 residues long before the beginning of a tyrosine kinase sequence homology at amino acid 837. With the interruption of homology in the kinase insert sequence of 14 aa (indicated by italics in the Fig. 1) , this homology is first lost at the beginning of the 31 amino acid carboxyl terminal tail of the receptor. A search for related tyrosine kinase domains in the amino acid sequence database (Swissprot and NBRF) identified the FGFR-1, ret, c-fms, PDGFR and c-kit receptor tyrosine kinases as the closest homologs of tie (about 40% amino acid sequence identity in the tyrosine kinase domain) .

EXAMPLE 2

Preparation of Antisera A tie cDNA fragment encoding 196 carboxyl terminal amino acids was inserted into pEX2 bacterial expression vector (Stanley and Luzio, EMBO J . 3 : 1429-1434, 1984) using an internal Xhol site. The resulting s-galactosidase fusion protein was produced in bacteria and partially purified by preparative SDS-polyacrylamide gel electrophoresis. Polypeptide bands were cut out from the gel, minced, mixed with Freund's adjuvant, and used for immunization of rabbits. Antisera were used after the third booster immunization. A peptide corresponding to 15 amino acids from the carboxyl terminus of the predicted tie protein was synthesized and cross-linked by glutaraldehyde to keyhole limpet hemocyanin (KLH, Calbiochem) . The immunizations were performed as in Example 1. Briefly, 7.5 mg carrier protein was dissolved in 0.5 ml of 0.1 M phosphate, pH 8.0, mixed with 7.5 mg of peptide and 5 ml of 20 mM glutaraldehyde was added. After mixing the solution, it was left for 15 min. at room temperature, after which 2.5 ml of glutaraldehyde was again added and the 15 min. incubation was repeated. Then, 0.1 ml of 1 M glycine, pH 6.0 was added to block unreacted glutaraldehyde and the stirring resumed for an additional 10 min. The product was dialyzed exhaustively against phosphate-buffered saline. For immunization, 1.25 mg of synthetic peptide-KLH conjugate in 0.5 ml PBS pH 7.5 was mixed with 0.5 ml complete Freund's adjuvant. The emulsion was delivered by subcutaneous injections, 0.1 ml in each of ten sites, into 3 month old New Zealand white rabbits. After biweekly intervals, the immunization was repeated with an identical quantity of immunogen. Serum was prepared from blood collected from auricular vein one week after the second and subsequent booster injections. EXAMPLE 3 Expression of tie in COS cells

The full-length tie protein coding sequence (combined from two overlapping clones, HE11-1 and 12a) was inserted into the -EcoRI site of an SV poly-mammalian expression vector (Stacey and Schnieke, Nucleic Acids Res . , 18 : 1829, 1990; construct SV14-2) . The SV14-1 vector lacks the first seven amino acids from its signal sequence, but it is initiated from an ATG codon present in the SV-poly vector. The expression vectors (SV14-2, SV14-1) were introduced into COS-1 cells by the DEAE-dextran transfection method (McCutchan and Pagano, <J. Natl Cancer Inst. , 41 : 351-357, 1968). Two days after transfection the cells were labelled for 4 hours with ³⁵S-methionine in the presence or absence of 10 g/ml tunicamycin. The cells were washed with PBS and scraped into immunoprecipitation buffer (lOmM Tris pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40, 0.1% SDS, 0.1 TlU/ml Aprotinin) . The lysates were sonicated, centrifuged for 15' at 10 000 g, and incubated overnight on ice with 3 ml of the antisera. Protein A sepharose (Pharmacia) was added and the incubation was continued for 30' with rotation. The precipitates were washed four times with the immunoprecipitation buffer, once with PBS, and once with aqua before analysis in SDS-PAGE.

The structural predictions of the tie cDNA sequence were tested by cloning the full-length tie protein coding region into the -EcoRI site of the pSVpoly expression vector (constructs pSV14-2 and pSV14-l) , and these expression vectors were then transfected into COS cells. The proteins produced by these two constructs differ in their signal sequence as noted above, but the predicted mature protein products are identical. After two days, the cells were metabolically labelled and immunoprecipitated with antibodies generated against as s-galactosidase-tie fusion protein containing 195 carboxyl terminal amino acid residues of the predicted tie protein (antiserum HI) , or against a 15 amino acid peptide corresponding to the tie carboxyl terminus (antiserum MI) . Fig. 3 shows analysis of the immunoprecipitated radioactive polypeptides by SDS-polyacrylamide gel electrophoresis. As can be seen from Fig. 3A, the HI immune serum precipitated some weakly labeled polypeptides from untransfected COS cells. These polypeptides were probably not related to tie because the COS cells do not express its mRNA.

Cells transfected with the pSV14 expression vector show an additional specific polypeptide of 117 kD (marked tie in figure 3) . This tie polypeptide was not precipitated with the preimmune serum or the antiserum blocked with the immunogen. The 117 kD polypeptide was recognized also by the MI antiserum against a carboxyl terminal peptide (Fig. 3B) . Immunoprecipitation of tie polypeptides from transfected COS cells metabolically labeled in the presence of tunicamycin to prevent N-linked glycosylation of proteins gave a specific polypeptide of approximately 105 kD apparent molecular weight (marked tie* in Fig. 3B) .

EXAMPLE 4

Expression of tie in NIH3T3 cells The full-length tie cDNA was subcloned under the control of Moloney murine leukemia virus long terminal repeat promoter. This expression vector was used to co-transfect NIH3T3 cells with the pSVneol marker plasmid and G418 resistant clones were analyzed for tie expression. Cells on one confluent plate were lysed in 2.5% SDS, 125mM Tris, pH 6.5 for immunoblot analysis. Cell lysates were electrophoresed on SDS-page and electroblotted on nitrocellulose membrane. The membrane was incubated with the anti-peptide antiserum against the tie carboxyterminus and bound antibodies were visualized using horseradish peroxidase conjugated swine anti-rabbit antiserum (Dako) and ECL reagents (Amersham) . Tyrosine phosphorylated proteins were immunoprecipitated as described (Frackelton, et al., 1991, In T. Hunterand B. M. Sefton (ed.), Protein phosphorylation part B, Meth. Enzymol. 201:79-91). Briefly, cells on one confluent were lysed in extraction buffer (1% Triton X-100, lOmM Tris pH 7.6, 5mM EDTA, 50mM NaCl, 100 M Sodium orthovanadate, ImM PMSF) and the lysates were incubated with rotation for 2 hours on ice with agarose conjugated anti-phosphotyrosine antibodies (1G2-A, Oncogene Science) . The immunoprecipitates were washed four times with extraction buffer, and tyrosine phosphorylated proteins were eluted with ImM phenyl phosphate. Eluted proteins were analyzed with immunoblotting as described above.

The 117 kD tie protein was detected by immunoblotting with the antiserum raised against the peptide corresponding the tie carboxyterminus (Fig. 4) . In addition, endogenous tie protein of a similar molecular weight was detected in PAE (porcine aortic endothelial) cells. The tie protein was also detected in anti-phosphotyrosine immunoprecipitates of the tie -transfected cells.

EXAMPLE 5

Chromosomal mapping of the tie locus Metaphase spreads from normal human male peripheral blood buffy coat leukocytes were prepared and hybridized essentially as described in Harper and Saunders, Chromosoma, 83 : 431-439, (1981) . For in situ hybridization, about 1 mg of HE11-1 cDNA insert was labeled by nick translation using four ³H-labeled NTPs to a specific activity of about 4-8xl07cpm/mg. After hybridization, slides were washed in 50% formamide, 2 x SSC at 39°C, and exposed to Kodak NTB2 nuclear track emulsion for 12 days at 4°C. The slides were developed with Kodak Dektol developer and Kodafix solution, and chromosomes were first G-banded with Wright-Giemsa stain (Cannizarro and Emanuel, Cytogenet . Cell Genet . , 38 : 308-309) , and if necessary, rebanded by the trypsin-Giemsa (GTG) technique.

In situ hybridization of radiolabeled tie probe to normal human metaphase chromosomes localized tie sequences to chromosome 1. A total of 317 chromosomally-localized grains were scored on 145 metaphases. Thirty-four percent (109/317) of the grains were on chromosome 1 with 69% (75/109) of chromosome 1 grains localized to Ip33-p34. Grain localization on chromosome 1 is illustrated schematically in Fig. 5, where each dot represents 3 grains. This narrows the localization to lp33 - p34, with the highest concentration of grains close to the border between bands lp33 and p34. Chromosomal localization using a panel of somatic mouse-human hybrid cell lines also placed the tie locus to human chromosome 1.

EXAMPLE 6

Expression of the tie mRNA in leukemia cell lines and endothelial cells

The leukemia cell lines used in this study have been reported in several previous publications; K562 (Lozzio and Lozzio, Blood, 45 : 321-334, 1975), HL-60 (Collins, et al. , Nature, 270 : 347-349, 1977) , HEL (Martin and Papayannopuolou, Science, 216 : 1233-1235, 1982), Dami (Greenberg, et al. , Blood, 72 : 1968-1977, 1988), MOLT-4 (Minowada, et al., J . Natl . Cancer Inst . , 49 : 891-895, 1972), Jurkat (Schwenk and Schneider, Blut, 31 : 299-306, 1975), U937 Sundstrom and Nilsson, Int . J. Cancer, 17 : 565-577, 1976), KG-1 (Koeffler and Golde, Science, 200 : 1153-1154, 1978), JOK-1 (Andersson et al., 1982, In R. F. Revoltella (ed.), Expression of differentiated functions in cancer cells, p.239-245, Raven Press, New York), ML-2 (Gahmberg et al., 1985, In L. C. Andersson, et al . (ed.), Gene expression during normal and malignant differentiation. p. 107-123, Academic Press, London) and RC-2A (Bradley, et al . , Br . J . Haemat . , 51 : 595, 1982) . The leukemia cells were grown in RPMI containing 10% FCS and antibiotics. Dami cells were cultivated in Iscoves modified DMEM with 10% horse serum. A permanent hybrid cell line (EA.hy926) obtained by fusing first-passage human umbilical vein endothelial cells with the A549 lung carcinoma cells (Edgell, et al., Proc . Natl . Acad . Sci . USA, 50 : 3734-3737) was cultured in DMEM-HAT medium containing 10% FCS and antibiotics. The PAE cells (a kind gift from Dr. Lena Claesson-Welsh, Ludwig Institute for Cancer Research, Uppsala, Sweden) were grown in Ham's F12 medium containing 10% FCS Poly(A)+ RNA was extracted from the cell lines as described in Sambrook, et al . , Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, 1989. Five grams of the Poly(A)+ RNA samples were electrophoresed in agarose gels containing formaldehyde and blotted using standard conditions (Sambrook, et al. , supra) . The insert of the HE11-1 cDΝA clone was labelled by the random priming method and hybridized to the blots. Hybridization was carried out in 50% formamide, 5 x Denhardt's solution (lOOx Denhardt's solution comprises 2% each of Ficoll, polyvinylpyrrolidone and bovine serum albumin), 5 x SSPE (3M ΝaCl, 200mM ΝaH2P04 . H20, 20 mM EDTA, pH 7.0), 0.1% SDS (sodium dodecyl sulphate), and 0.1 mg/ l of sonicated salmon sperm DNA at 42°C for 18-24 h. The filters were washed at 65°C in IxSSC (150 mM NaCl, 15mM sodium citrate, pH 7.0), 0,1% SDS and exposed to Kodak XAR-5 film.

Figure 6 shows the results of analysis of tie mRNA expression in ten leukemia cell lines. Only the HEL erythroleukemia cells, KG-1 myeloid leukemia cells and Dami megakaryoblastic leukemia cells expressed a 4.4 kb tie mRNA, as detected with the 3.8 kb tie cDNA probe. The Jurkat and MOLT-4 T-cell leukemias, as well as HL-60 promyelocytic leukemia, U937 and RC-2A onocytic leukemias, JOK-1 hairy cell leukemia and ML-2 myeloid leukemia cells were negative for the tie mRNA. The tie mRNA was also induced after TPA treatment of the K562 cells, when the cells undergo megakaryoblastoid differentiation. Interestingly, porcine aortic endothelial cells (PAE) as well as a hybrid human endothelial cell line, EA.hy926, which has been reported to express several endothelial markers in vitro (Edgell, et al. , Proc. Natl . Acad . Sci . USA, 50 : 3734-3737, 1983, Emeis and Edgell, Blood, 71 : 1669-1675, 1988), expressed tie mRNA abundantly (Fig. 7) . The EA.hy926 cell line was created by the fusion of human umbilical vein endothelial cells with A549 lung carcinoma cell line. The A549 cells were negative for tie mRNA expression. In addition to the 4.4 kb mRNA, the EA.hy926 cells expressed tie mRNA species of 3.9, 4.2 and 4.7 kb. The results of Northern blot analyses of the tie mRNA expression in cell lines are summarized in table 1. EXAMPLE 7

Expression of tie in blood vessels Selected fragments of cloned human tie cDNA outside of the tyrosine kinase domain exhibiting a low degree of homology to other receptor tyrosine kinases were used as in situ hybridization probes to detect the tie mRNA. Specifically, we used a Smal fragment (nucleotides 268-1767) of the full-length cDNA clone corresponding to the extracellular domain of the tie clone further digested to smaller fragments with PstI and Sail. The probe was labeled with ³⁵S-deoxy(thio)ATP for the in situ hybridizations (Feinberg and Vogelstein, Anal . Biochem . , 132 : 6-13, 1983). Fragments of 100-790-bp generated by -Bgrll of bacteriophage Lambda DNA were labeled similarly and used as a negative control probe. All specimens from fetal abortuses were obtained with permission of the joint ethical committee of the University Central Hospital and University of Turku (Turku, Finland) . In situ hybridizations were carried out as described previously (Sandberg and Vuorio, -J. Cell . Biol . , 104: 1077-1084). In brief, tissue samples of 15-19-week human fetuses obtained from therapeutic abortions were fixed with formaline and embedded in paraffin for sectioning. The sections were pretreated with proteinase K and HC1 and acetylated. The hybridizations were carried out at 42°C for 24 h using ³⁵S-deoxy(thio)ATP-labeled probes, followed by washing, autoradiography at +4°C for 5-25 days, and staining of the sections with hematoxylin.

Tie mRNA expression in tissues was studied by mRNA in situ hybridization of 15-19 week old human fetal tissues. In agreement with the tie expression in endothelial cell lines, tie mRNA was seen to be located in the walls of medium and large vessels of the kidney (Fig. 8) . Labelled Lambda DNA used as a negative control provided no detectable hybridization signal over the background. EXAMPLE 8

Analysis of tie mRNA in mouse embryos Approximately 106 plaques from two lgtlO libraries (a kind gift of Dr. Brigitte Galliot, Zentru fur Molekularbiologie Heidelberg, Germany) prepared from 10- and 11-day post coitu (p.c.) mouse embryonic mRNA were screened with a S-mal fragment (nucleotides 155 - 1765 ; this cDNA encodes the first immunoglobulin domain and EGF-like domains I - Ill of the extracellular part of the tie receptor) and 3.8 kb .EcoRl fragments of human tie receptor cDNA (Partanen et al. , Mol . Cell Biol . , in press). These cDNA segments have only little homology with other known genes. The probe was labelled with [a-³⁵P]dCTP by the random priming method. The nitrocellulose replicas of each phage-infected plate were hybridized in 50 % deionized formamide, 5 x Denhardt's solution, 5xSSPE, 0.1 % SDS and 100 mg/ml ssDNA. Seven positive clones were purified out of which four were subcloned into pGEM 3Zf(+) (Promega) and sequenced. DNA sequencing was performed by the dideoxy chain termination method of Sanger, .et al . , Proc . Natl . Acad . Sci . USA 74 : 5463-5467, (1977) , using a modified T7 DNA polymerase (Sequenase TM, U.S. Biochemical) . Sequences were generated from both ends of subcloned restriction fragments using universal pGEM sequencing primers. The internal sequence of large fragments as well as the complementary strands of all fragments were determined using oligonucleotide primers synthesized according to sequence information on preceding sequences. Two nonoverlapping inserts of the mouse tie cDNA plasmid clones, designated D10E5 and 1C1D (Fig. 10) were used as hybridization probes. Mouse embryos of 8 - 14 -days p.c. were derived from matings of CBA and NMR mice. Embryonic age was calculated from the day on which the copulation plug was detected as day 0 (estimated copulation time 2 a.m.). Pregnant mice were killed by cervical dislocation, the embryos were removed and transferred immediately via phosphate buffered saline (PBS) into 4 % paraformaldehyde in PBS, pH 7.2. The embryos were fixed for 18 h at 4°C, dehydrated, embedded in wax (Fisher Scientific Co) and cut into 5-6 mm sections. Isolated mouse organs were treated similarly. Total RNA was isolated from adult mouse organs and developing embryos according to Chirgwin, et al . , Biochemistry, 18 : 5294-5299. Poly(A)+ RNA (5 g) and total RNA (20 g) were electrophoresed in 0.8 % agarose gels containing formaldehyde and blotted into Hybond-N (Amersha ) filters using standard conditions. After transfer, the filters were exposed to ultraviolet radiation for 4 minutes, hybridized and washed in stringent conditions (Sambrook, et al . , Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, 1989) . In situ hybridization of sections was performed according to Wilkinson, et al . , Development 99 : 493-500 (1987) with the following modifications: 1) instead of toluene, xylene was used before embedding in paraffin wax, 2) 5 - 6 mm sections were cut, placed on a layer of diethyl pyrocarbonate-treated (DEPC) water on the surface of glass slides pretreated with 2% 3-triethoxysilylpropylamine (TESPA) (Sigma) , 3) alkaline hydrolysis of the probes was omitted, 4) the hybridization mixture contained 60 % deionized formamide, 5) the high stringency wash was for 80 minutes at 65°C in a solution containing 50 mM DTT and 1 x SSC, 6) the sections were covered with NTB-2 emulsion (Kodak) and stored at 4°C. After an exposure time of about 14 days the slides were developed for 2.5 min. in a Kodak D-19 developer and fixed for 5 min. with Unifix (Kodak). The sections were stained with 0.02 % toluidine blue in water. Control hybridizations with sense strand and RNAse A-treated sections did not give a specific signal above background. I munoperoxidase staining was done using human monoclonal anti-factor VIII antibodies and standard techniques.

Total RNA and polyadenylated RNA were isolated from various human fetal and adult tissues as well as mouse tissues and subjected to Northern blotting and hybridization with the tie cDNA probes. Figure 11A shows that all fetal human tissues tested contain a 4.4 kb tie mRNA. In polyadenylated RNA from human adult tissues the tie signal is most prominent in the highly vascularized lung, placenta and heart, but a weaker signal can also be recognized in other tissues as well, particularly in long exposure of the autoradiogram (Fig. 11B) The expression of tie begins very early; from 9 to 10 days of gestation tie is expressed weakly, then the number of tie transcripts increases (maximum at 14 days gestation) . The newborn and postnatal mice have lower amounts of tie mRNA. Sagittal sections of 12 day p.c. mouse embryos were hybridized with the antisense and sense RNA transcribed from the insert of the 1C1D plasmid. Figure 12A shows the brightfield image of a representative section probed with antisense RNA. Figure 12A illustrates that the autoradiographic grains decorate the linings of major blood vessels. These signals were, however, better visualized in the darkfield microscopy of the same section (Fig. 12B) . This result demonstrates that the tie mRNA is ubiquitously expressed in all vessels. The cells responsible for tie expression were endothelial cells as shown by Factor VIII immunostaining, which is specific to endothelial cells. The sense probe did not give signals above background, as can be seen from Fig. 12C. The tie hybridization signal in a 8-day p.c. mouse placenta (Fig. 13A) was very similar and practically superi posable with the pattern of factor VIII staining of adjacent sections (Fig. 13B) .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Partanen, Juha

Armstrong, Elina Makela, Tomi Korhonen, Jaana Alitalo, Kari (ii) TITLE OF INVENTION: Tie, A Novel Endothelial Cell Recptor Tyrosine Kinase (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Helsinki University Holding Ltd.

(B) STREET: Teollisuuskatu 23

(C) CITY: 00510 Helsinki

(E) COUNTRY: Finland

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(A) MEDIUM TYPE: Floppy disk

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(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Oy Kolster Ab

(B) STREET: Stora Robertsgatan 23

(C) CITY: 00120 Helsinki

(E) COUNTRY: Finland

(F) ZIP: 00120

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1138 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Val Trp Arg Val Pro Pro Phe Leu Leu Pro lie Leu Phe Leu Ala 1 5 10 15

Ser His Val Gly Ala Ala Val Asp Leu Thr Leu Leu Ala Asn Leu Arg 20 25 30

Leu Thr Asp Pro Gin Arg Phe Phe Leu Thr Cys Val Ser Gly Glu Ala 35 40 45

Gly Ala Gly Arg Gly Ser Asp Ala Trp Gly Pro Pro Leu Leu Leu Glu 50 55 60

Lys Asp Asp Arg lie Val Arg Thr Pro Pro Gly Pro Pro Leu Arg Leu 65 70 75 80

Ala Arg Asn Gly Ser His Gin Val Thr Leu Arg Gly Phe Ser Lys Pro 85 90 95

Ser Asp Leu Val Gly Val Phe Ser Cys Val Gly Gly Ala Gly Ala Arg 100 105 110

Arg Thr Arg Val lie Tyr Val His Asn Ser Pro Gly Ala His Leu Leu 115 120 125

Pro Asp Lys Val Thr His Thr Val Asn Lys Gly Asp Thr Ala Val Leu 130 135 140

Ser Ala Arg Val His Lys Glu Lys Gin Thr Asp Val lie Trp Lys Ser 145 150 155 160

Asn Gly Ser Tyr Phe Tyr Thr Leu Asp Trp His Glu Ala Gin Asp Gly 165 170 175

Arg Phe Leu Leu Gin Leu Pro Asn Val Gin Pro Pro Ser Ser Gly lie 180 185 190

Tyr Ser Ala Thr Tyr Leu Glu Ala Ser Pro Leu Gly Ser Ala Phe Phe 195 200 205

Arg Leu lie Val Arg Gly Cys Gly Ala Gly Arg Trp Gly Pro Gly Cys 210 215 220

Thr Lys Glu Cys Pro Gly Cys Leu His Gly Gly Val Cys His Asp His 225 230 235 240

Asp Gly Glu Cys Val Cys Pro Pro Gly Phe Thr Gly Thr Arg Cys Glu 245 250 255

Gin Ala Cys Arg Glu Gly Arg Phe Gly Gin Ser Cys Gin Glu Gin Cys 260 265 270

Pro Gly lie Ser Gly Cys Arg Gly Leu Thr Phe Cys Leu Pro Asp Pro 275 280 285

Tyr Gly Cys Ser Cys Gly Ser Gly Trp Arg Gly Ser Gin Cys Gin Glu 290 295 300

Ala Cys Ala Pro Gly His Phe Gly Ala Asp Cys Arg Leu Gin Cys Gin 305 310 315 320

Cys Gin Asn Gly Gly Thr Cys Asp Arg Phe Ser Gly Cys Val Cys Pro 325 330 335

Ser Gly Trp His Gly Val His Cys Glu Lys Ser Asp Arg lie Pro Gin 340 345 350

lie Leu Asn Met Ala Ser Glu Leu Glu Phe Asn Leu Glu Thr Met Pro 355 360 365

Arg lie Asn Cys Ala Ala Ala Gly Asn Pro Phe Pro Val Arg Gly Ser 370 375 380

lie Glu Leu Arg Lys Pro Asp Gly Thr Val Leu Leu Ser Thr Lys Ala 385 390 395 400

lie Val Glu Pro Glu Lys Thr Thr Ala Glu Phe Glu Val Pro Arg Leu 405 410 415

Val Leu Ala Asp Ser Gly Phe Trp Glu Cys Arg Val Ser Thr Ser Gly 420 425 430

Gly Gin Asp Ser Arg Arg Phe Lys Val Asn Val Lys Val Pro Pro Val, 435 440 445

Pro Leu Ala Ala Pro Arg Leu Leu Thr Lys Gin Ser Arg Gin Leu Val 450 455 460

Val Ser Pro Leu Val Ser Phe Ser Gly Asp Gly Pro lie Ser Thr Val 465 470 475 480

Arg Leu His Tyr Arg Pro Gin Asp Ser Thr Met Asp Trp Ser Thr lie 485 490 495

Val Val Asp Pro Ser Glu Asn Val Thr Leu Met Asn Leu Arg Pro Lys 500 505 510

Thr Gly Tyr Ser Val Arg Val Gin Leu Ser Arg Pro Gly Glu Gly Gly 515 520 525

Glu Gly Ala Trp Gly Pro Pro Thr Leu Met Thr Thr Asp Cys Pro Glu 530 535 540

Pro Leu Leu Gin Pro Trp Leu Glu Gly Trp His Val Glu Gly Thr Asp 545 550 555 560

Arg Leu Arg Val Ser Trp Ser Leu Pro Leu Val Pro Gly Pro Leu Val 565 570 575

Gly Asp Gly Phe Leu Leu Arg Leu Trp Asp-Gly Thr Arg Gly Gin Glu 580 585 590

Arg Arg Glu Asn Val Ser Ser Pro Gin Ala Arg Thr Ala Leu Leu Thr 595 600 605

Gly Leu Thr Pro Gly Thr His Tyr Gin Leu Asp Val Gin Leu Tyr His 610 615 620

Cys Thr Leu Leu Gly Pro Ala Ser Pro Pro Ala His Val Leu Leu Pro 625 630 635 640

Pro Ser Gly Pro Pro Ala Pro Arg His Leu His Ala Gin Ala Leu Ser 645 650 655

Asp Ser Glu lie Gin Leu Thr Trp Lys His Pro Glu Ala Leu Pro Gly 660 665 670

Pro lie Ser Lys Tyr Val Val Glu Val Gin Val Ala Gly Gly Ala Gly 675 680 685

Asp Pro Leu Trp lie Asp Val Asp Arg Pro Glu Glu Thr Ser Thr lie 690 695 700

lie Arg Gly Leu Asn Ala Ser Thr Arg Tyr Leu Phe Arg Met Arg Ala 705 710 715 720

Ser lie Gin Gly Leu Gly Asp Trp Ser Asn Thr Val Glu Glu Ser Thr 725 730 735

Leu Gly Asn Gly Leu Gin Ala Glu Gly Pro Val Gin Glu Ser Arg Ala 740 745 750

Ala Glu Glu Gly Leu Asp Gin Gin Leu lie Leu Ala Val Val Gly Ser 755 760 765

Val Ser Ala Thr Cys Leu Thr lie Leu Ala Ala Leu Leu Thr Leu Val 770 775 780

Cys lie Arg Arg Ser Cys Leu His Arg Arg Arg Thr Phe Thr Tyr Gin 785 790 795 800

Ser Gly Ser Gly Glu Glu Thr lie Leu Gin Phe Ser Ser Gly Thr Leu 805 810 815

Thr Leu Thr Arg Arg Pro Lys Leu Gin Pro Glu Pro Leu Ser Tyr Pro 820 825 830

Val Leu Glu Trp Glu Asp lie Thr Phe Glu Asp Leu lie Gly Glu Gly 835 840 845

Asn Phe Gly Gin Val lie Arg Ala Met lie Lys Lys Asp Gly Leu Lys 850 855 860

Met Asn Ala Ala lie Lys Met Leu Lys Glu Tyr Ala Ser Glu Asn Asp 865 870 875 880

His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly His 885 890 895

His Pro Asn lie lie Asn Leu Leu Gly Ala Cys Lys Asn Arg Gly Tyr 900 905 910

Leu Tyr lie Ala lie Glu Tyr Ala Pro Tyr Gly Asn Leu Leu Asp Phe 915 920 925

Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala Phe Ala Arg Glu 930 935 940

His Gly Thr Ala Ser Thr Leu Ser Ser Arg Gin Leu Leu Arg Phe Ala 945 950 955 960

Ser Asp Ala Ala Asn Gly Met Gin Tyr Leu Ser Glu Lys Gin Phe lie 965 970 975

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gly Glu Asn Leu Ala 980 985 990

Ser Lys lie Ala Asp Phe Gly Leu Ser Arg Gly Glu Glu Val Tyr Val 995 1000 1005

Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala lie Glu Ser 1010 1015 1020

Leu Asn Tyr Ser Val Tyr Thr Thr Lys Ser Asp Val Trp Ser- Phe Gly 1025 1030 1035 1040

Val Leu Leu Trp Glu lie Val Ser Leu Gly Gly Thr Pro Tyr Cys Gly 1045 1050 1055

Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gin Ala Asp Arg Met 1060 1065 1070

Glu Gin Pro Arg Asn Cys Asp Asp Glu Val Tyr Glu Leu Met Arg Gin 1075 1080 1085

Cys Trp Arg Asp Arg Pro Tyr Glu Arg Pro Pro Phe Ala Gin lie Ala 1090 1095 1100

Leu Gin Leu Gly Arg Met Leu Glu Ala Arg Lys Ala Tyr Val Asn Met 1105 1110 1115 1120

Ser Leu Phe Glu Asn Phe Thr Tyr Ala Gly lie Asp Ala Thr Ala Glu 1125 1130 1135

Glu Ala

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1094 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Val Trp Arg Val Pro Pro Phe Leu Leu Pro lie Leu Phe Leu Ala

1 5 10 15

Ser His Val Gly Ala Ala Val Asp Leu Thr Leu Leu Ala Asn Leu Arg 20 25 30

Leu Thr Asp Pro Gin Arg Phe Phe Leu Thr Cys Val Ser Gly Glu Ala 35 40 45

Gly Ala Gly Arg Gly Ser Asp Ala Trp Gly Pro Pro Leu Leu Leu Glu 50 55 60

Lys Asp Asp Arg lie Val Arg Thr Pro Pro Gly Pro Pro Leu Arg Leu 65 70 75 80

Ala Arg Asn Gly Ser His Gin Val Thr Leu Arg Gly Phe Ser Lys Pro 85 90 95

Ser Asp Leu Val Gly Val Phe Ser Cys Val Gly Gly Ala Gly Ala Arg 100 105 110

Arg Thr Arg Val lie Tyr Val His Asn Ser Pro Gly Ala His Leu Leu 115 120 125

Pro Asp Lys Val Thr His Thr Val Asn Lys Gly Asp Thr Ala Val Leu 130 135 140

Ser Ala Arg Val His Lys Glu Lys Gin Thr Asp Val lie Trp Lys Ser 145 150 155 160

Asn Gly Ser Tyr Phe Tyr Thr Leu Asp Trp His Glu Ala Gin Asp Gly 165 170 175

Arg Phe Leu Leu Gin Leu Pro Asn Val Gin Pro Pro Ser Ser Gly He 180 185 190

Tyr Ser Ala Thr Tyr Leu Glu Ala Ser Pro Leu Gly Ser Ala Phe Phe 195 200 205

Arg Leu He Val Arg Ala Cys Arg Glu Gly Arg Phe Gly Gin Ser Cys 210 215 220

Gin Glu Gin Cys Pro Gly He Ser Gly Cys Arg Gly Leu Thr Phe Cys 225 230 235 240

Leu Pro Asp Pro Tyr Gly Cys Ser Cys Gly Ser Gly Trp Arg Gly Ser 245 250 255

Gin Cys Gin Glu Ala Cys Ala Pro Gly His Phe Gly Ala Asp Cys Arg 260 265 270

Leu Gin Cys Gin Cys Gin Asn Gly Gly Thr Cys Asp Arg Phe Ser Gly 275 280 285

Cys Val Cys Pro Ser Gly Trp His Gly Val His Cys Glu Lys Ser Asp 290 295 300

Arg He Pro Gin He Leu Asn Met Ala Ser Glu Leu Glu Phe Asn Leu 305 310 315 320

Glu Thr Met Pro Arg He Asn Cys Ala Ala Ala Gly Asn Pro Phe Pro 325 330 335

Val Arg Gly Ser He Glu Leu Arg Lys Pro Asp Gly Thr Val Leu Leu 340 345 350

Ser Thr Lys Ala He Val Glu Pro Glu Lys Thr Thr Ala Glu Phe Glu 355 360 365

Val Pro Arg Leu Val Leu Ala Asp Ser Gly Phe Trp Glu Cys Arg Val 370 375 380

Ser Thr Ser Gly Gly Gin Asp Ser Arg Arg Phe Lys Val Asn Val Lys 385 390 395 400

Val Pro Pro Val Pro Leu Ala Ala Pro Arg Leu Leu Thr Lys Gin Ser 405 410 415

Arg Gin Leu Val Val Ser Pro Leu Val Ser Phe Ser Gly Asp Gly Pro 420 425 430

He Ser Thr Val Arg Leu His Tyr Arg Pro Gin Asp Ser Thr Met Asp 435 440 445

Trp Ser Thr He Val Val Asp Pro Ser Glu Asn Val Thr Leu Met Asn 450 455 460

Leu Arg Pro Lys Thr Gly Tyr Ser Val Arg Val Gin Leu Ser Arg Pro 465 470 475 480

Gly Glu Gly Gly Glu Gly Ala Trp Gly Pro Pro Thr Leu Met Thr Thr 485 490 495

Asp Cys Pro Glu Pro Leu Leu Gin Pro Trp Leu Glu Gly Trp His Val 500 505 510

Glu Gly Thr Asp Arg Leu Arg Val Ser Trp Ser Leu Pro Leu Val Pro 515 520 525

Gly Pro Leu Val Gly Asp Gly Phe Leu Leu Arg Leu Trp Asp Gly Thr 530 535 540

Arg Gly Gin Glu Arg Arg Glu Asn Val Ser Ser Pro Gin Ala Arg Thr 545 550 555 560

Ala Leu Leu Thr Gly Leu Thr Pro Gly Thr His Tyr Gin Leu Asp Val 565 570 575

Gin Leu Tyr His Cys Thr Leu Leu Gly Pro Ala Ser Pro Pro Ala His 580 585 590

Val Leu Leu Pro Pro Ser Gly Pro Pro Ala Pro Arg His Leu His Ala 595 600 605

Gin Ala Leu Ser Asp Ser Glu He Gin Leu Thr Trp Lys His Pro Glu 610 615 620

Ala Leu Pro Gly Pro He Ser Lys Tyr Val Val Glu Val Gin Val Ala 625 630 635 640

Gly Gly Ala Gly Asp Pro Leu Trp He Asp Val Asp Arg Pro Glu Glu 645 650 655

Thr Ser Thr He He Arg Gly Leu Asn Ala Ser Thr Arg Tyr Leu Phe 660 665 670

Arg Met Arg Ala Ser He Gin Gly Leu Gly Asp Trp Ser Asn Thr Val 675 680 685

Glu Glu Ser Thr Leu Gly Asn Gly Leu Gin Ala Glu Gly Pro Val Gin 690 695 700

Glu Ser Arg Ala Ala Glu Glu Gly Leu Asp Gin Gin Leu He Leu Ala 705 710 715 720

Val Val Gly Ser Val Ser Ala Thr Cys Leu Thr He Leu Ala Ala Leu 725 730 735

Leu Thr Leu Val Cys He Arg Arg Ser Cys Leu His Arg Arg Arg Thr 740 745 750

Phe Thr Tyr Gin Ser Gly Ser Gly Glu Glu Thr He Leu Gin Phe Ser 755 760 765

Ser Gly Thr Leu Thr Leu Thr Arg Arg Pro Lys Leu Gin Pro Glu Pro 770 775 780

Leu Ser Tyr Pro Val Leu Glu Trp Glu Asp He Thr Phe Glu Asp Leu 785 790 795 800

He Gly Glu Gly Asn Phe Gly Gin Val He Arg Ala Met He Lys Lys 805 810 815

Asp Gly Leu Lys Met Asn Ala Ala He Lys Met Leu Lys Glu Tyr Ala 820 825 830

Ser Glu Asn Asp His Arg Asp Phe Ala Gly Glu Leu Glu Val Leu Cys 835 840 845

Lys Leu Gly His His Pro Asn He He Asn Leu Leu Gly Ala Cys Lys 850 855 860

Asn Arg Gly Tyr Leu Tyr He Ala He Glu Tyr Ala Pro Tyr Gly Asn 865 870 875 880

Leu Leu Asp Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro Ala 885 890 895

Phe Ala Arg Glu His Gly Thr Ala Ser Thr Leu Ser Ser Arg Gin Leu 900 905 910

Leu Arg Phe Ala Ser Asp Ala Ala Asn Gly Met Gin Tyr Leu Ser Glu 915 920 925

Lys Gin Phe He His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Gly 930 935 940

Glu Asn Leu Ala Ser Lys He Ala Asp Phe Gly Leu Ser Arg Gly Glu 945 950 955 960

Glu Val Tyr Val Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met 965 970 975

Ala He Glu Ser Leu Asn Tyr Ser Val Tyr Thr Thr Lys Ser Asp Val 980 985 990

Trp Ser Phe Gly Val Leu Leu Trp Glu He Val Ser Leu Gly Gly Thr 995 1000 1005

Pro Tyr Cys Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro Gin 1010 1015 1020

Ala Asp Arg Met Glu Gin Pro Arg Asn Cys Asp Asp Glu Val Tyr Glu 1025 1030 1035 1040

Leu Met Arg Gin Cys Trp Arg Asp Arg Pro Tyr Glu Arg Pro Pro Phe 1045 1050 1055

Ala Gin He Ala Leu Gin Leu Gly Arg Met Leu Glu Ala Arg Lys Ala 1060 1065 1070

Tyr Val Asn Met Ser Leu Phe Glu Asn Phe Thr Tyr Ala Gly He Asp 1075 1080 1085

Ala Thr Ala Glu Glu Ala 1090

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3845 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CGCTCGTCCT GGCTGGCCTG GGTCGGCCTC TGGAGTATGG TCTGGCGGGT GCCCCCTTTC 60

TTGCTCCCCA TCCTCTTCTT GGCTTCTCAT GTGGGCGCGG CGGTGGACCT GACGCTGCTG 120

GCCAACCTGC GGCTCACGGA CCCCCAGCGC TTCTTCCTGA CTTGCGTGTC TGGGGAGGCC 180

GGGGCGGGGA GGGGCTCGGA CGCCTGGGGC CCGCCCCTGC TGCTGGAGAA GGACGACCGT 240

ATCGTGCGCA CCCCGCCCGG GCCACCCCTG CGCCTGGCGC GCAACGGTTC GCACCAGGTC 300

ACGCTTCGCG GCTTCTCCAA GCCCTCGGAC CTCGTGGGCG TCTTCTCCTG CGTGGGCGGT 360

GCTGGGGCGC GGCGCACGCG CGTCATCTAC GTGCACAACA GCCCTGGAGC CCACCTGCTT 420

CCAGACAAGG TCACACACAC TGTGAACAAA GGTGACACCG CTGTACTTTC TGCACGTGTG 480

CACAAGGAGA AGCAGACAGA CGTGATCTGG AAGAGCAACG GATCCTACTT CTACACCCTG 540

GACTGGCATG AAGCCCAGGA TGGGCGGTTC CTGCTGCAGC TCCCAAATGT GCAGCCACCA 600

TCGAGCGGCA TCTACAGTGC CACTTACCTG GAAGCCAGCC CCCTGGGCAG CGCCTTCTTT 660

CGGCTCATCG TGCGGGGTTG TGGGGCTGGG CGCTGGGGGC CAGGCTGTAC CAAGGAGTGC 720

CCAGGTTGCC TACATGGAGG TGTCTGCCAC GACCATGACG GCGAATGTGT ATGCCCCCCT 780

GGCTTCACTG GCACCCGCTG TGAACAGGCC TGCAGAGAGG GCCGTTTTGG GCAGAGCTGC 840

CAGGAGCAGT GCCCAGGCAT ATCAGGCTGC CGGGGCCTCA CCTTCTGCCT CCCAGACCCC 900

TATGGCTGCT CTTGTGGATC TGGCTGGAGA GGAAGCCAGT GCCAAGAAGC TTGTGCCCCT 960

GGTCATTTTG GGGCTGATTG CCGACTCCAG TGCCAGTGTC AGAATGGTGG CACTTGTGAC 1020 CGGTTCAGTG GTTGTGTCTG CCCCTCTGGG TGGCATGGAG TGCACTGTGA GAAGTCAGAC 1080

CGGATCCCCC AGATCCTCAA CATGGCCTCA GAACTGGAGT TCAACTTAGA GACGATGCCC 1140

CGGATCAACT GTGCAGCTGC AGGGAACCCC TTCCCCGTGC GGGGCAGCAT AGAGCTACGC 1200

AAGCCAGACG GCACTGTGCT CCTGTCCACC AAGGCCATTG TGGAGCCAGA GAAGACCACA 1260

GCTGAGTTCG AGGTGCCCCG CTTGGTTCTT GCGGACAGTG GGTTCTGGGA GTGCCGTGTG 1320

TCCACATCTG GCGGCCAAGA CAGCCGGCGC TTCAAGGTCA ATGTGAAAGT GCCCCCCGTG 1380

CCCCTGGCTG CACCTCGGCT CCTGACCAAG CAGAGCCGCC AGCTTGTGGT CTCCCCGCTG 1440

GTCTCGTTCT CTGGGGATGG ACCCATCTCC ACTGTCCGCC TGCACTACCG GCCCCAGGAC 1500

AGTACCATGG ACTGGTCGAC CATTGTGGTG GACCCCAGTG AGAACGTGAC GTTAATGAAC 1560

CTGAGGCCAA AGACAGGATA CAGTGTTCGT GTGCAGCTGA GCCGGCCAGG GGAAGGAGGA 1620

GAGGGGGCCT GGGGGCCTCC CACCCTCATG ACCACAGACT GTCCTGAGCC TTTGTTGCAG 1680

CCGTGGTTGG AGGGCTGGCA TGTGGAAGGC ACTGACCGGC TGCGAGTGAG CTGGTCCTTG 1740

CCCTTGGTGC CCGGGCCACT GGTGGGCGAC GGTTTCCTGC TGCGCCTGTG GGACGGGACA 1800

CGGGGGCAGG AGCGGCGGGA GAACGTCTCA TCCCCCCAGG CCCGCACTGC CCTCCTGACG 1860

GGACTCACGC CTGGCACCCA CTACCAGCTG GATGTGCAGC TCTACCACTG CACCCTCCTG 1920

GGCCCGGCCT CGCCCCCTGC ACACGTGCTT CTGCCCCCCA GTGGGCCTCC AGCCCCCCGA 1980

CACCTCCACG CCCAGGCCCT CTCAGACTCC GAGATCCAGC TGACATGGAA GCACCCGGAG 2040

GCTCTGCCTG GGCCAATATC CAAGTACGTT GTGGAGGTGC AGGTGGCTGG GGGTGCAGGA 2100

GACCCACTGT GGATAGACGT GGACAGGCCT GAGGAGACAA GCACCATCAT CCGTGGCCTC 2160

AACGCCAGCA CGCGCTACCT CTTCCGCATG CGGGCCAGCA TTCAGGGGCT CGGGGACTGG 2220

AGCAACACAG TAGAAGAGTC CACCCTGGGC AACGGGCTGC AGGCTGAGGG CCCAGTCCAA 2280

GAGAGCCGGG CAGCTGAAGA GGGCCTGGAT CAGCAGCTGA TCCTGGCGGT GGTGGGCTCC 2340

GTGTCTGCCA CCTGCCTCAC CATCCTGGCC GCCCTTTTAA CCCTGGTGTG CATCCGCAGA 2400 AGCTGCCTGC ATCGGAGACG CACCTTCACC TACCAGTCAG GCTCGGGCGA GGAGACCATC 2460

CTGCAGTTCA GCTCAGGGAC CTTGACACTT ACCCGGCGGC CAAAACTGCA GCCCGAGCCC 2520

CTGAGCTACC CAGTGCTAGA GTGGGAGGAC ATCACCTTTG AGGACCTCAT CGGGGAGGGG 2580

AACTTCGGCC AGGTCATCCG GGCCATGATC AAGAAGGACG GGCTGAAGAT GAACGCAGCC 2640

ATCAAAATGC TGAAAGAGTA TGCCTCTGAA AATGACCATC GTGACTTTGC GGGAGAACTG 2700

GAAGTTCTGT GCAAATTGGG GCATCACCCC AACATCATCA ACCTCCTGGG GGCCTGTAAG 2760

AACCGAGGTT ACTTGTATAT CGCTATTGAA TATGCCCCCT ACGGGAACCT GCTAGATTTT 2820

CTGCGGAAAA GCCGGGTCCT AGAGACTGAC CCAGCTTTTG CTCGAGAGCA TGGGACAGCC 2880

TCTACCCTTA GCTCCCGGCA GCTGCTGCGT TTCGCCAGTG ATGCGGCCAA TGGCATGCAG 2940

TACCTGAGTG AGAAGCAGTT CATCCACAGG GACCTGGCTG CCCGGAATGT GCTGGTCGGA 3000

GAGAACCTAG CCTCCAAGAT TGCAGACTTC GGCCTTTCTC GGGGAGAGGA GGTTTATGTG 3060

AAGAAGACGA TGGGGCGTCT CCCTGTGCGC TGGATGGCCA TTGAGTCCCT GAACTACAGT 3120

GTCTATACCA CCAAGAGTGA TGTCTGGTCC TTTGGAGTCC TTCTTTGGGA GATAGTGAGC 3180

CTTGGAGGTA CACCCTACTG TGGCATGACC TGTGCCGAGC TCTATGAAAA GCTGCCCCAG 3240

GCTGACCGCA TGGAGCAGCC TCGAAACTGT GACGATGAAG TGTACGAGCT GATGCGTCAG 3300

TGCTGGCGGG ACCGTCCCTA TGAGCGACCC CCCTTTGCCC AGATTGCGCT ACAGCTAGGC 3360

CGCATGCTGG AAGCCAGGAA GGCCTATGTG AACATGTCGC TGTTTGAGAA CTTCACTTAC 3420

GCGGGCATTG ATGCCACAGC TGAGGAGGCC TGAGCTGCCA TCCAGCCAGA ACGTGGCTCT 3480

GCTGGCCGGA GCAAACTCTG CTGTCTAACC TGTGACCAGT CTGACCCTTA CAGCCTCTGA 3540

CTTAAGCTGC CTCAAGGAAT TTTTTTAACT TAAGGGAGAA AAAAAGGGAT CTGGGGATGG 3600

GGTGGGCTTA GGGGAACTGG GTTCCCATGC TTTGTAGGTG TCTCATAGCT ATCCTGGGCA 3660

TCCTTCTTTC TAGTTCAGCT GCCCCACAGG TGTGTTTCCC ATCCCACTGC TCCCCCAACA 3720

CAAACCCCCA CTCCAGCTCC TTCGCTTAAG CCAGCACTCA CACCACTAAC ATGCCCTGTT 3780 CAGCTACTCC CACTCCCGGC CTGTCATTCA GAAAAAAATA AATGTTCTAA TAAGCTCCAA 3840

AAAAA 3845

(2) INFORMATION FOR SEQ ID NO: :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3713 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CGCTCGTCCT GGCTGGCCTG GGTCGGCCTC TGGAGTATGG TCTGGCGGGT GCCCCCTTTC 60

TTGCTCCCCA TCCTCTTCTT GGCTTCTCAT GTGGGCGCGG CGGTGGACCT GACGCTGCTG 120

GCCAACCTGC GGCTCACGGA CCCCCAGCGC TTCTTCCTGA CTTGCGTGTC TGGGGAGGCC 180

GGGGCGGGGA GGGGCTCGGA CGCCTGGGGC CCGCCCCTGC TGCTGGAGAA GGACGACCGT 240

ATCGTGCGCA CCCCGCCCGG GCCACCCCTG CGCCTGGCGC GCAACGGTTC GCACCAGGTC 300

ACGCTTCGCG GCTTCTCCAA GCCCTCGGAC CTCGTGGGCG TCTTCTCCTG CGTGGGCGGT 360

GCTGGGGCGC GGCGCACGCG CGTCATCTAC GTGCACAACA GCCCTGGAGC CCACCTGCTT 420

CCAGACAAGG TCACACACAC TGTGAACAAA GGTGACACCG CTGTACTTTC TGCACGTGTG 480

CACAAGGAGA AGCAGACAGA CGTGATCTGG AAGAGCAACG GATCCTACTT CTACACCCTG 540

GACTGGCATG AAGCCCAGGA TGGGCGGTTC CTGCTGCAGC TCCCAAATGT GCAGCCACCA 600

TCGAGCGGCA TCTACAGTGC CACTTACCTG GAAGCCAGCC CCCTGGGCAG CGCCTTCTTT 660

CGGCTCATCG TGCGGGCCTG CAGAGAGGGC CGTTTTGGGC AGAGCTGCCA GGAGCAGTGC 720

CCAGGCATAT CAGGCTGCCG GGGCCTCACC TTCTGCCTCC CAGACCCCTA TGGCTGCTCT 780 TGTGGATCTG GCTGGAGAGG AAGCCAGTGC CAAGAAGCTT GTGCCCCTGG TCATTTTGGG 840

GCTGATTGCC GACTCCAGTG CCAGTGTCAG AATGGTGGCA CTTGTGACCG GTTCAGTGGT 900

TGTGTCTGCC CCTCTGGGTG GCATGGAGTG CACTGTGAGA AGTCAGACCG GATCCCCCAG 960

ATCCTCAACA TGGCCTCAGA ACTGGAGTTC AACTTAGAGA CGATGCCCCG GATCAACTGT 1020

GCAGCTGCAG GGAACCCCTT CCCCGTGCGG GGCAGCATAG AGCTACGCAA GCCAGACGGC 1080

ACTGTGCTCC TGTCCACCAA GGCCATTGTG GAGCCAGAGA AGACCACAGC TGAGTTCGAG 1140

GTGCCCCGCT TGGTTCTTGC GGACAGTGGG TTCTGGGAGT GCCGTGTGTC CACATCTGGC 1200

GGCCAAGACA GCCGGCGCTT CAAGGTCAAT GTGAAAGTGC CCCCCGTGCC CCTGGCTGCA 1260

CCTCGGCTCC TGACCAAGCA GAGCCGCCAG CTTGTGGTCT CCCCGCTGGT CTCGTTCTCT 1320

GGGGATGGAC CCATCTCCAC TGTCCGCCTG CACTACCGGC CCCAGGACAG TACCATGGAC 1380

TGGTCGACCA TTGTGGTGGA CCCCAGTGAG AACGTGACGT TAATGAACCT GAGGCCAAAG 1440

ACAGGATACA GTGTTCGTGT GCAGCTGAGC CGGCCAGGGG AAGGAGGAGA GGGGGCCTGG 1500

GGGCCTCCCA CCCTCATGAC CACAGACTGT CCTGAGCCTT TGTTGCAGCC GTGGTTGGAG 1560

GGCTGGCATG TGGAAGGCAC TGACCGGCTG CGAGTGAGCT GGTCCTTGCC CTTGGTGCCC 1620

GGGCCACTGG TGGGCGACGG TTTCCTGCTG CGCCTGTGGG ACGGGACACG GGGGCAGGAG 1680

CGGCGGGAGA ACGTCTCATC CCCCCAGGCC CGCACTGCCC TCCTGACGGG ACTCACGCCT 1740

GGCACCCACT ACCAGCTGGA TGTGCAGCTC TACCACTGCA CCCTCCTGGG CCCGGCCTCG 1800

CCCCCTGCAC ACGTGCTTCT GCCCCCCAGT GGGCCTCCAG CCCCCCGACA CCTCCACGCC 1860

CAGGCCCTCT CAGACTCCGA GATCCAGCTG ACATGGAAGC ACCCGGAGGC TCTGCCTGGG 1920

CCAATATCCA AGTACGTTGT GGAGGTGCAG GTGGCTGGGG GTGCAGGAGA CCCACTGTGG 1980

ATAGACGTGG ACAGGCCTGA GGAGACAAGC ACCATCATCC GTGGCCTCAA CGCCAGCACG 2040

CGCTACCTCT TCCGCATGCG GGCCAGCATT CAGGGGCTCG GGGACTGGAG CAACACAGTA 2100

GAAGAGTCCA CCCTGGGCAA CGGGCTGCAG GCTGAGGGCC CAGTCCAAGA GAGCCGGGCA 2160 GCTGAAGAGG GCCTGGATCA GCAGCTGATC CTGGCGGTGG TGGGCTCCGT GTCTGCCACC 2220

TGCCTCACCA TCCTGGCCGC CCTTTTAACC CTGGTGTGCA TCCGCAGAAG CTGCCTGCAT 2280

CGGAGACGCA CCTTCACCTA CCAGTCAGGC TCGGGCGAGG AGACCATCCT GCAGTTCAGC 2340

TCAGGGACCT TGACACTTAC CCGGCGGCCA AAACTGCAGC CCGAGCCCCT GAGCTACCCA 2400

GTGCTAGAGT GGGAGGACAT CACCTTTGAG GACCTCATCG GGGAGGGGAA CTTCGGCCAG 2460

GTCATCCGGG CCATGATCAA GAAGGACGGG CTGAAGATGA ACGCAGCCAT CAAAATGCTG 2520

AAAGAGTATG CCTCTGAAAA TGACCATCGT GACTTTGCGG GAGAACTGGA AGTTCTGTGC 2580

AAATTGGGGC ATCACCCCAA CATCATCAAC CTCCTGGGGG CCTGTAAGAA CCGAGGTTAC 2640

TTGTATATCG CTATTGAATA TGCCCCCTAC GGGAACCTGC TAGATTTTCT GCGGAAAAGC 2700

CGGGTCCTAG AGACTGACCC AGCTTTTGCT CGAGAGCATG GGACAGCCTC TACCCTTAGC 2760

TCCCGGCAGC TGCTGCGTTT CGCCAGTGAT GCGGCCAATG GCATGCAGTA CCTGAGTGAG 2820

AAGCAGTTCA TCCACAGGGA CCTGGCTGCC CGGAATGTGC TGGTCGGAGA GAACCTAGCC 2880

TCCAAGATTG CAGACTTCGG CCTTTCTCGG GGAGAGGAGG TTTATGTGAA GAAGACGATG 2940

GGGCGTCTCC CTGTGCGCTG GATGGCCATT GAGTCCCTGA ACTACAGTGT CTATACCACC 3000

AAGAGTGATG TCTGGTCCTT TGGAGTCCTT CTTTGGGAGA TAGTGAGCCT TGGAGGTACA 3060

CCCTACTGTG GCATGACCTG TGCCGAGCTC TATGAAAAGC TGCCCCAGGC TGACCGCATG 3120

GAGCAGCCTC GAAACTGTGA CGATGAAGTG TACGAGCTGA TGCGTCAGTG CTGGCGGGAC 3180

CGTCCCTATG AGCGACCCCC CTTTGCCCAG ATTGCGCTAC AGCTAGGCCG CATGCTGGAA 3240

GCCAGGAAGG CCTATGTGAA CATGTCGCTG TTTGAGAACT TCACTTACGC GGGCATTGAT 3300

GCCACAGCTG AGGAGGCCTG AGCTGCCATC CAGCCAGAAC GTGGCTCTGC TGGCCGGAGC 3360

AAACTCTGCT GTCTAACCTG TGACCAGTCT GACCCTTACA GCCTCTGACT TAAGCTGCCT 3420

CAAGGAATTT TTTTAACTTA AGGGAGAAAA AAAGGGATCT GGGGATGGGG TGGGCTTAGG 3480

GGAACTGGGT TCCCATGCTT TGTAGGTGTC TCATAGCTAT CCTGGGCATC CTTCTTTCTA 3540 GTTCAGCTGC CCCACAGGTG TGTTTCCCAT CCCACTGCTC CCCCAACACA AACCCCCACT 3600

CCAGCTCCTT CGCTTAAGCC AGCACTCACA CCACTAACAT GCCCTGTTCA GCTACTCCCA 3660

CTCCCGGCCT GTCATTCAGA AAAAAATAAA TGTTCTAATA AGCTCCAAAA AAA 3713

Claims

What is claimed is:

1. An isolated nucleotide sequence comprising a sequence encoding the protein sequence of tie receptor tyrosine kinase or a functional derivative or a fragment thereof.

2. An isolated nucleotide sequence according to claim 1 encoding a tie precursor comprising the nucleotide sequence substantially as depicted in SEQ ID NO: 1 from about nucleotide number 1 to about nucleotide number 3845.

3. An isolated nucleotide sequence according to claim 1 encoding a tie precursor comprising the nucleotide coding sequence substantially as depicted in SEQ ID NO: 1 from about nucleotide number 37 to about nucleotide number 3845.

4. An isolated nucleotide sequence according to claim 1 encoding a mature tie comprising the nucleotide coding sequence substantially as depicted in SEQ ID NO: 1 from about nucleotide number 100 to about nuoleotide number 3845.

5. A recombinant-DNA-molecule which comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences according to any of the claims 1 to 4.

6. The recombinant-DNA-molecule according to claim 5, wherein said nucleotide sequence is operably linked to a suitable expression control sequence.

7. The recombinant-DNA-molecule according to claim 6, wherein said expression control sequence renders the said recombinant-DNA-molecule capable of expressing the said nucleotide sequence.

8. The recombinant-DNA-molecule according to claim 6, wherein said expression control sequence renders the said recombinant DNA molecule capable of expressing antisense RNA to the said nucleotide sequence.

9. A host cell transformed with the recombinant-DNA-molecule of claim 5.

10. The host cell according to claim 9, wherein said cell is a eukaryotic cell.

11. The host cell according to claim 10, wherein said cell is a mammalian cell.

12. A substantially pure tie protein or a functional derivative or a fragment thereof.

13. The substantially pure tie protein according to claim 12 _r wherein the tie protein is a tie precursor comprising the amino acid sequence substantially as depicted in SEQ ID NO: 1 from about amino acid residue number 1 to about amino acid residue number 1138.

14. The substantially pure tie protein according to claim 12, wherein the tie protein is a mature tie comprising the amino acid sequence substantially as depicted in SEQ ID NO: 1 from about amino acid residue number 22 to about amino acid residue number 1138.

15. The substantially pure tie protein according to claim 12, wherein said protein is human tie.

16. The substantially pure tie protein according to claim 12, wherein said protein is a recombinantly-produced tie.

17. The substantially pure tie protein according to claim 16, wherein said tie is produced in a mammalian cell culture.

18. A process for producing recombinant tie protein, which process comprises:

1) isolating nucleotide sequence encoding the said tie protein,

2) constructing an expression vector by introducing the nucleotide sequence into an appropriate cloning vector,

3) transforming appropriate host cells with said expression vector,

4) culturing said host cells, and

5) isolating the tie product.

19. The process for producing the recombinant tie according to claim 18, wherein said tie is human tie.

20. The process for producing the recombinant tie according to claim 18, wherein said host cells are mammalian cells.

21. The process according to claim 18, wherein the nucleotide seguence encoding tie comprises the nucleotide sequence substantially as depicted in SEQ ID NO: 1 from about nucleotide number l to about nucleotide number 3845.

22. The process according to claim 18, wherein the nucleotide seguence encoding tie comprises the nucleotide coding sequence substantially as depicted in SEQ ID NO: 1 from about- nucleotide number 37 to about nucleotide number 3845. .

23. The process according to claim 18, wherein the nucleotide seguence encoding tie comprises the nucleotide coding sequence substantially as depicted in SEQ ID NO: 1 from about nucleotide number 100 to about nucleotide number 3845.