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The Specificity of Receptor Binding by Vascular Endothelial Growth Factor-D Is Different in Mouse and Man

2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês

10.1074/jbc.m100097200

ISSN

1083-351X

Autores

Megan E. Baldwin, Bruno Catimel, Edouard C. Nice, Sally Roufail, Nathan E. Hall, Kaye L. Stenvers, Marika J. Karkkainen, Kari Alitalo, Steven A. Stacker, Marc G. Achen,

Tópico(s)

Angiogenesis and VEGF in Cancer

Resumo

Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where theVEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man. Human vascular endothelial growth factor-D (VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed on vascular and lymphatic endothelial cells. As VEGFR-2 signals for angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it was proposed that VEGF-D stimulates growth of blood vessels and lymphatic vessels into regions of embryos and tumors. Here we report the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3 cross-linking. Mutation of amino acids in mouse VEGF-D to those in the human homologue indicated that residues important for the VEGFR-2 interaction are clustered at, or are near, the predicted receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3 in mouse embryos was detected in the developing skin where theVEGF-D gene was expressed in a layer of cells beneath the developing epidermis and VEGFR-3 was localized on a network of vessels immediately beneath the VEGF-D-positive cells. This suggests that VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin by a paracrine mechanism. Our study of receptor specificity suggests that VEGF-D may have different biological functions in mouse and man. vascular endothelial growth factor VEGF receptor VEGF homology domain erythropoietin receptor interleukin-3 embryonic day human mouse Blood vessel development in embryos and tumors depends on members of the vascular endothelial growth factor (VEGF)1 family of proteins (for review see (1Achen M.G. Stacker S.A. Int. J. Exp. Path. 1998; 79: 255-265Crossref PubMed Scopus (107) Google Scholar)). VEGF (also known as VEGF-A and vascular permeability factor) is essential for vascular development during embryogenesis (2Carmeliet P. Ferreira V. Breier G. Pollofeyt S. Keickens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declerq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3506) Google Scholar, 3Ferrara N. Carver-Moore K. Chen H. Dowd M. Lu L. O'Shea K.S. Powel-Braxton L. Hillan K.J. Moore M.W. 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Cancer Res. 1996; 56: 1615-1620PubMed Google Scholar), other ligands that activate this receptor, such as VEGF-D and VEGF-C (14Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1177) Google Scholar, 15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar), could also stimulate angiogenesis. VEGF-C binds both VEGFR-2 and VEGFR-3 (Flt-4) (14Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1177) Google Scholar), is angiogenic (16Witzenbichler B. Asahara T. Murohara T. Silver M. Spyridopoulos I. Magner M. Principe N. Kearney M. Hu J.-S. Isner J.M. Am. J. Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 17Cao Y. Linden P. 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Immunol. 1999; 237: 85-96Crossref PubMed Scopus (67) Google Scholar), a receptor expressed on venous endothelial cells during early embryogenesis that subsequently becomes restricted to the endothelium of lymphatic vessels (21Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W. Fang G.H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1201) Google Scholar, 22Lymboussaki A. Partanen T.A. Olofsson B. Thomas-Crusells J. Fletcher C.D.M. de Waal R.M.W. Kaipainen A. Alitalo K. Am. J. Pathol. 1998; 153: 395-403Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). VEGFR-3 signaling is thought to induce lymphangiogenesis (20Taipale J. Makinen T. Arighi E. Kukk E. Karkkainen M. Alitalo K. Curr. Top. Microbiol. Immunol. 1999; 237: 85-96Crossref PubMed Scopus (67) Google Scholar) and hyperplasia of lymphatic vessels 2Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Petrova, T. V., Kubo, H., Thurston, G., McDonald, D. M., Achen, M. G., Stacker, S. A., and Alitalo, K. (2001)EMBO J. 20, 1223–1231., although this receptor can be up-regulated on tumor blood vessels (23Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar) and is involved in signaling for tumor angiogenesis (24Kubo H. Fujiwara T. Jussila L. Hashi H. Ogawa M. Shimizu K. Awane M. Sakai Y. Takabayashi A. Alitalo K. Yamaoka Y. Nishikawa S.-I. Blood. 2000; 96: 546-553Crossref PubMed Google Scholar). The most recently discovered mammalian member of the VEGF family, VEGF-D (also known as c-fos-induced growth factor) (25Orlandini M. Marconcini L. Ferruzzi R. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11675-11680Crossref PubMed Scopus (268) Google Scholar), is angiogenic (26Marconcini L. Marchio S. Morbidelli L. Cartocci E. Albini A. Ziche M. Bussolino F. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9671-9676Crossref PubMed Scopus (233) Google Scholar), mitogenic for endothelial cells in vitro(15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar), is expressed at many sites in the developing embryo (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 28Avantaggiato V. Orlandini M. Acampora D. Oliviero S. Simeone A. Mech. Dev. 1998; 73: 221-224Crossref PubMed Scopus (53) Google Scholar), and is localized in human tumors (29Achen M.G. Williams R.A. Minekus M.P. Thornton G.E. Stenvers K. Rogers P.A.W. Lederman F. Roufail S. Stacker S.A. J. Pathol. 2000; 193: 147-154Crossref Scopus (133) Google Scholar). VEGF-D also induced lymphangiogenesis and metastatic spread via the lymphatics in a mouse tumor model (30Stacker S.A. Caesar C. Baldwin M.E. Thornton G.E. Williams R.A. Prevo R. Jackson D.G. Nishikawa S.-I. Kubo H. Achen M.G. Nat. Med. 2001; 7: 186-191Crossref PubMed Scopus (1076) Google Scholar). VEGF-D is initially synthesized as a precursor protein containing N- and C-terminal propeptides in addition to the VEGF homology domain (VHD), the region of the protein that shares homology with all VEGF family members and contains receptor-binding epitopes (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar, 27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The N- and C-terminal propeptides are proteolytically cleaved from the VHD during biosynthesis to generate a mature, secreted form consisting of dimers of the VHD (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The mature form of human VEGF-D binds both VEGFR-2 and VEGFR-3 with much higher affinity than does unprocessed VEGF-D (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Therefore proteolytic processing is important for activating human VEGF-D. As human VEGF-D activates VEGFR-2 and VEGFR-3 (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar), it has been proposed that VEGF-D can stimulate the growth of blood vessels and lymphatic vessels into regions of developing embryos and tumors. Previous studies of receptor binding by VEGF-D were carried out using the human protein (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar, 27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Here we characterize the receptor-binding specificity of mouse VEGF-D. Unlike human VEGF-D, that binds both VEGFR-2 and VEGFR-3, mouse VEGF-D is specific for VEGFR-3 in the mouse; it does not bind mouse VEGFR-2. This finding suggests that the biological functions of VEGF-D in mouse and man may differ. Mouse VEGF-D consists of the VHD, amino acid residues 97–206 tagged at the N terminus with the FLAG octapeptide (Scientific Imaging Systems) (31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar). Mouse VEGF-D-Cterm-FLAG is identical to mouse VEGF-D except that it is tagged at the C terminus with FLAG instead of at the N terminus. Human VEGF-D consists of amino acid residues 93–201, the region exactly homologous to residues 98–206 of mouse VEGF-D, and is tagged at the N terminus with FLAG (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar). Mutants of mouse VEGF-D were generated using polymerase chain reaction with oligonucleotides encoding the mutated residues essentially as described previously (32Li X.-M. Shapiro L.J. Nucleic Acids Res. 1993; 21: 3745-3748Crossref PubMed Scopus (24) Google Scholar). Expression plasmids derived from pEFBOSSFLAG (C. McFarlane, Walter and Eliza Hall Institute, Melbourne, Australia) encoding human, mouse, and mutant VEGF-D were transiently transfected into 293EBNA cells using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) and the proteins purified by affinity chromatography with M2 (anti-FLAG) monoclonal antibody as described elsewhere (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Expression and purification of the extracellular domain of mouse VEGFR-2 (VEGFR-2-FLAG) has been described previously (33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Chimeric proteins consisting of the extracellular domain of human VEGFR-2 (the first four Ig-like domains), human VEGFR-3 (the first three Ig-like domains), or mouse VEGFR-3 (the first three Ig-like domains) and the Fc portion of IgG (hVEGFR-2-Ig, hVEGFR-3-Ig, and mVEGFR-3-Ig, respectively) were expressed by transient transfection of 293EBNA cells with expression plasmids for these Ig fusion proteins (kind gifts from K. Pajusola and Y. Gunji, Helsinki, Finland). Ig fusion proteins were purified by affinity chromatography with protein A-Sepharose (Amersham Pharmacia Biotech) according to the manufacturer's instructions. SDS-polyacrylamide gel electrophoresis and Western blotting with M2 monoclonal antibody were carried out as described previously (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). The extinction coefficient for mouse and human VEGF-D was estimated based on amino acid composition using the ProtParam tool program at the ExPASy website 3Contact corresponding author for Website address.). Quantitation of mouse and human VEGF-D was by spectrophotometry at 280 nm. The relative abundance of mouse, human, and mutants of VEGF-D was confirmed by SDS-polyacrylamide gel electrophoresis followed by silver staining of serial dilutions of these proteins. Bioassays for monitoring the binding and cross-linking of VEGFR-2 and VEGFR-3 involved the use of cell lines expressing chimeric receptors consisting of the extracellular, ligand-binding domain of mouse VEGFR-2 or human VEGFR-3 and the transmembrane and cytoplasmic domains of the erythropoietin receptor (EpoR). In addition, cells expressing chimeric receptors consisting of the extracellular domain of the mouse endothelial cell receptor Tie2 and the transmembrane and cytoplasmic domains of EpoR were used as a non-responding cell line. The chimeric receptors had been transfected into the Ba/F3 cell line, a line of pre-B cells, which survives and proliferates in the presence of interleukin-3 (IL-3) but which dies after IL-3 deprivation. It has been shown previously that signaling from the cytoplasmic EpoR domain of chimeric receptors upon ligand binding is capable of rescuing these cells in the absence of IL-3 (34Pacifici R.E. Thomason A.R. J. Biol. Chem. 1994; 269: 1571-1574Abstract Full Text PDF PubMed Google Scholar). The expression of the VEGFR-2/EpoR, VEGFR-3/EpoR, and Tie2/EpoR chimeric receptors in Ba/F3 cells allows detection of specific ligand binding and cross-linking of the extracellular domains of these receptors, which results in signaling from the cytoplasmic domain of the EpoR and cell survival and proliferation in the absence of IL-3. The cell lines expressing the chimeric receptors are designated VEGFR-2-EpoR-Ba/F3 (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar, 33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), VEGFR-3-EpoR-Ba/F3 (31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar), and Tie2-EpoR-Ba/F3 (35Stacker S.A. Runting A.S. Caesar C. Vitali A. Lackmann M. Chang J. Ward L. Wilks A.F. Growth Factors. 2000; 18: 177-191Crossref PubMed Scopus (13) Google Scholar). Samples of purified mouse and human VEGF-D were diluted in cell culture medium (Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum, 50 mml-glutamine, 50 μg/ml gentamicin, 1.2 mg/ml G418) deficient in IL-3. Bioassay cell lines were then incubated in the media for 48 h at 37 °C, and DNA synthesis was then quantitated by the addition of 1 μCi of [3H]thymidine and further incubation for 4 h prior to harvesting using an automated cell harvester (Tomtec®). Incorporated [3H]thymidine was measured by β-counting (Canberra Packard "Top Count NXT™ "; scintillation counter, Meriden, CT). All protein preparations were analyzed for homogeneity, and buffer was exchanged into the appropriate buffers by micropreparative size exclusion high pressure liquid chromatography using a Superose 12 (3.2/30) column installed in a SMART™ system (Amersham Pharmacia Biotech) immediately prior to use (36Nice E.C. Catimel B. Bioessays. 1999; 21: 339-352Crossref PubMed Scopus (184) Google Scholar). Receptor domains were coupled to the carboxymethylated dextran layer of a CM5 sensor chip using standard amine coupling chemistry (36Nice E.C. Catimel B. Bioessays. 1999; 21: 339-352Crossref PubMed Scopus (184) Google Scholar) for analysis of ligand binding using a BIAcore 3000 optical biosensor (BIAcore, Uppsala, Sweden). Automatic targeting of immobilization levels was achieved using BIAcore 3.1 control software (37Catimel B. Domagala T. Nerrie M. Weinstock J. White S. Abud H. Heath J. Nice E. Protein Pept. Lett. 1999; 6: 319-340Google Scholar). Following immobilization, residual activated ester groups were blocked by treatment with 1 m ethanolamine hydrochloride, pH 8.5, followed by washing with 10 mm diethylamine to remove non-covalently bound material. The 10 mm diethylamine was also used to regenerate the sensor surface between analyses. Samples for assay were diluted in running buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, 0.005% Tween 20). The integrity of the surface was assessed by binding of purified mouse and human VEGF-D. The apparent binding affinities of mouse and human VEGF-D to receptor domains were determined by analysis of the initial dissociation phase to obtain the kd, which was then used to constrain a global analysis of the association region of the curves assuming a 1:1 Langmuirian model. Data were analyzed using BIAevaluation 3.0 (BIAcore, Uppsala, Sweden) as described previously (38Catimel B. Nerrie M. Lee F.T. Scott A.M. Ritter G. Welt S. Old L.J. Burgess A.W. Nice E.C. J. Chromatogr. A. 1997; 776: 15-30Crossref PubMed Scopus (79) Google Scholar). In situ hybridization was carried out as described elsewhere (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Two non-overlapping antisense RNA probes, homologous to the regions of mouse VEGF-D cDNA (GenBankTM accession number X99572) encoding from amino acid residues 1–85 (probe A) and 199–317 (probe B), were used in this study. In addition to encoding the N-terminal 85 amino acids of VEGF-D, probe A also contained 80 nucleotides of the 5′ untranslated region immediately upstream from the translation start codon. Cryostat sections of embryonic day (E) 15.5-mouse embryos were fixed in cold acetone for 10 min, and endogenous peroxidase activity was quenched by incubation in 0.2% H2O2. Sections were blocked for 15 min in phosphate-buffered saline/0.5% bovine serum albumin and incubated with goat anti-human VEGFR-3 (Flt-4) affinity-purified antiserum (R&D Systems, Minneapolis, MN) for 1.5 h at room temperature. Sections were then incubated with rabbit anti-goat Ig-horseradish peroxidase (DAKO Corp., Carpinteria, CA) for 45 min. VEGFR-3 staining was detected using 3,3′-diaminobenzidine (DAKO Corp.). As an adsorption control, the VEGFR-3 anti-serum was incubated for 1 h at room temperature with a 10-fold molar excess of a chimeric protein consisting of the extracellular domain of human VEGFR-3 fused to the Fc region of human IgG1 (R&D) before being used for immunohistochemistry. Models of human VEGF-D and human VEGFR-2, based on the crystal structure of VEGF in complex with VEGFR-1 (PDB entry 1FLT (39Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar)), were created using MODELLER (40Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10779) Google Scholar). The model complex includes residues Ile94-Pro197 of VEGF-D and Arg122-Gly220 of VEGFR-2, encompassing the second Ig-like domain that interacts with VEGF (41Fuh G. Li B. Crowley C. Cunningham B. Wells J.A. J. Biol. Chem. 1998; 273: 11197-11204Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). The 1:2 complex of VEGF-D with Ig-like domain 2 of VEGFR-2 was generated by superimposing the coordinates of the VEGF-D and the VEGFR-2 models onto the co-crystal structure. Identification of the highest quality model was achieved using a combination of the MODELLER and ProsaII scores (42Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1821) Google Scholar). We have previously shown that mature human VEGF-D, consisting of the VHD, binds, cross-links, and activates VEGFR-2 and VEGFR-3 (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar, 27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar). In preliminary studies to analyze the interaction of mouse VEGF-D with VEGFR-2, we observed a lack of binding (data not shown). This was surprising given that the VHDs of mouse and human VEGF-D are closely related in primary structure (Fig.1 A) and that human VEGF-D binds VEGFR-2 (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar). Therefore we used biosensor analysis to investigate the binding of the VHDs of mouse and human VEGF-D to immobilized receptor extracellular domains of VEGFR-2 and VEGFR-3. Mouse and human VEGF-D, each consisting of the VHD tagged at the N terminus with the FLAG octapeptide, were expressed and purified as described under "Experimental Procedures."; The identity of these proteins was confirmed by silver staining and Western blot analysis. As expected, the apparent molecular masses of the subunits of these proteins were ∼20 kDa (Fig. 1 B). Analysis of the biosensor binding curves for the interactions of mouse and human VEGF-D with mouse and human VEGFR-2 and VEGFR-3 demonstrated that mouse VEGF-D did not bind the extracellular domain of mouse VEGFR-2 but bound to mouse VEGFR-3 (Fig. 1 C); human VEGF-D bound both human VEGFR-2 and human VEGFR-3 (Fig. 1 D). These findings indicate that mouse VEGF-D is specific for VEGFR-3 in the mouse but that human VEGF-D interacts with both VEGFR-2 and VEGFR-3 in human. Interestingly, mouse VEGF-D bound human VEGFR-2 and human VEGF-D bound mouse VEGFR-2, indicating that the inability of mouse VEGF-D to bind mouse VEGFR-2 may be a consequence of differences in both growth factor and receptor in comparison to the human homologues. The kinetics of the interactions of human VEGF-D with mouse VEGFR-2 and human VEGFR-3 were similar to those documented previously (27Stacker S.A. Stenvers K. Caesar C. Vitali A. Domagala T. Nice E. Roufail S. Simpson R.J. Moritz R. Karpanen T. Alitalo K. Achen M.G. J. Biol. Chem. 1999; 274: 32127-32136Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). To further analyze the interaction of mouse VEGF-D with VEGFR-2 and VEGFR-3, we used bioassays of receptor binding and cross-linking. The assays involved the use of Ba/F3 pre-B cell lines designated VEGFR-2-EpoR-Ba/F3 (33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and VEGFR-3-EpoR-Ba/F3 (31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar), which survive and proliferate only in the presence of growth factors capable of binding and cross-linking the extracellular domains of mouse VEGFR-2 and human VEGFR-3, respectively. In previous studies, all of the activating ligands for VEGFR-2 or VEGFR-3 (i.e. VEGF, VEGF-C, VEGF-D, and viral VEGFs) stimulated the VEGFR-2-EpoR-Ba/F3 or VEGFR-3-EpoR-Ba/F3 bioassay cell lines (15Achen M.G. Jeltsch M. Kukk E. Mäkinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar, 31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar, 33Stacker S.A. Vitali A. Caesar C. Domagala T. Groenen L.C. Nice E. Achen M.G. Wilks A.F. J. Biol. Chem. 1999; 274: 34884-34892Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 43Wise L.M. Veikkola T. Mercer A.A. Savory L.J. Fleming S.B. Caesar C. Vitali A. Makinen T. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3071-3076Crossref PubMed Scopus (234) Google Scholar). Both mouse and human VEGF-D were tested in the mouse VEGFR-2 and human VEGFR-3 bioassays (Fig. 2). Mouse VEGF-D did not promote significant proliferation of the VEGFR-2-EpoR-Ba/F3 cells indicating that this ligand cannot bind and cross-link mouse VEGFR-2 at the cell surface (Fig. 2 A). This result is consistent with the biosensor data demonstrating that mouse VEGF-D does not bind immobilized mouse VEGFR-2. Surprisingly, mouse VEGF-D was more potent than human VEGF-D in the human VEGFR-3 bioassay, an ∼10-fold greater concentration of human VEGF-D was required to give comparable stimulation of the VEGFR-3 cell line (Fig. 2 B). Mouse VEGF-D-Cterm-FLAG, a derivative of mature mouse VEGF-D tagged with FLAG at the C terminus rather than at the N terminus, exhibited comparable activity to the N-terminal-tagged derivative in both bioassays (data not shown), indicating that the position of FLAG did not influence receptor interactions. To identify amino acid residues important for the interaction of VEGF-D with its receptors, a mutant of mouse VEGF-D, designated VEGF-D(SLI), was generated in which the three sequential residues, Gly155-Val156-Met157, were altered to the corresponding sequence of Ser-Leu-Ile found in human VEGF-D (see Fig. 1 A for positions of these altered residues in the primary structure). These three residues in mouse VEGF-D were chosen for alteration because a predicted three-dimensional structure of human VEGF-D, based on the crystal structure of VEGF in complex with VEGFR-1 (39Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), indicated that the corresponding residues in human VEGF-D, Ser150-Leu151-Ile152, are near or part of a putative receptor-binding surface (Fig. 2 C). Mouse VEGF-D(SLI) exhibited reduced activity compared with wild-type mouse VEGF-D in the VEGFR-3 bioassay, comparable with the activity of human VEGF-D (Fig. 2 B). However, this mutant did not exhibit significantly enhanced activity in the VEGFR-2 bioassay (Fig.2 A). As the three altered residues in mouse VEGF-D(SLI) did not confer activity in the VEGFR-2 bioassay, further mutants were generated to recapitulate human VEGF-D-like activity. Of the differences in amino acid sequence between mouse VEGF-D(SLI) and human VEGF-D, Gly200 in the mouse protein, corresponding to Ala195 in the human protein, is most closely located to the predicted receptor-binding surface (Fig. 2 C). A mutant of VEGF-D(SLI), designated VEGF-D(ASLI), in which residue Gly200 was altered to Ala, exhibited greatly enhanced activity in the VEGFR-2 bioassay in comparison with the wild-type mouse protein but ∼13-fold less activity than human VEGF-D (Fig.2 A). In contrast, mouse mutant VEGF-D(A), in which the only alteration from wild-type was Gly200 to Ala, did not exhibit activity in the bioassay (data not shown), indicating that the Gly200 to Ala alteration is required in combination with the SLI mutation to restore activity. A derivative of VEGF-D(ASLI), in which Thr101 was altered to the corresponding Ile residue at position 96 in human VEGF-D, was designated VEGF-D(AISLI) and exhibited marginally more activity than VEGF-D(ASLI) but ∼8-fold less activity than human VEGF-D. As Ile96 in human VEGF-D is distant from the predicted receptor-binding surface (Fig.2 C), the enhanced activity of mouse VEGF-D(AISLI) in comparison with the ASLI mutant indicates that the residues distant from the receptor-binding surface, which differ between mouse and human VEGF-D, can influence the VEGFR-2 receptor interaction. The finding that mouse VEGF-D binds mouse VEGFR-3 but not mouse VEGFR-2 suggests that the vessels in the mouse that respond to mouse VEGF-D are those expressing VEGFR-3. To determine whether VEGF-D and VEGFR-3 expression is coordinated during embryogenesis, we analyzed E15.5 mouse embryos for these markers. A strong signal for VEGF-D mRNA was detected by in situ hybridization in a layer of cells in the developing skin, beneath the developing epidermis (Fig.3 A, B, andD). Immunohistochemical analysis revealed a network of VEGFR-3-positive vessels immediately beneath the layer of VEGF-D-positive cells (Fig. 3 E). This finding is consistent with previous studies in which VEGFR-3 mRNA was detected on a network of vessels immediately beneath the skin of E12.5 mouse embryos (21Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W. Fang G.H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1201) Google Scholar). Although vessels in the E15.5 skin were also immunopositive for VEGFR-2, one of the ligands for this receptor, VEGF, was undetectable and the other, VEGF-C, was only very weakly positive in hair follicles (data not shown). The localization of a VEGFR-3-positive network of vessels in the developing skin immediately beneath a layer of cells expressing VEGF-D suggests that the interaction of VEGF-D with VEGFR-3 could occur in vivo and may have a role in the development of these vessels. The finding that VEGF-D is a specific ligand for VEGFR-3 in the mouse was unexpected as it is often assumed that homologous ligands exhibit similar receptor-binding characteristics in different species. This is the first report of a VEGF family member with different receptor-binding specificity in mouse and man. This difference in specificity was not observed for the closely related growth factor VEGF-C, as human VEGF-C activated human VEGFR-2 (14Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1177) Google Scholar) and mouse VEGF-C bound and cross-linked mouse VEGFR-2 4M. E. Baldwin and M. G. Achen, unpublished data.. Given that VEGFR-2 signals for angiogenesis (11Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3391) Google Scholar, 12Millauer B. Shawver L.K. Plate K.H. Risau W. Ullrich A. Nature. 1994; 367: 576-579Crossref PubMed Scopus (1166) Google Scholar, 13Millauer B. Longhi M.P. Plate K.H. Shawver L.K. Risau W. Ullrich A. Strawn L.M. Cancer Res. 1996; 56: 1615-1620PubMed Google Scholar), whereas VEGFR-3 is thought to signal for lymphangiogenesis (20Taipale J. Makinen T. Arighi E. Kukk E. Karkkainen M. Alitalo K. Curr. Top. Microbiol. Immunol. 1999; 237: 85-96Crossref PubMed Scopus (67) Google Scholar), the data reported here suggest that VEGF-D may induce both angiogenesis and lymphangiogenesis in man but only lymphangiogenesis in the mouse. However, this issue is complicated by the findings that VEGFR-3, although specific to lymphatic endothelium in normal adult tissues (21Kaipainen A. Korhonen J. Mustonen T. van Hinsbergh V.W. Fang G.H. Dumont D. Breitman M. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3566-3570Crossref PubMed Scopus (1201) Google Scholar, 22Lymboussaki A. Partanen T.A. Olofsson B. Thomas-Crusells J. Fletcher C.D.M. de Waal R.M.W. Kaipainen A. Alitalo K. Am. J. Pathol. 1998; 153: 395-403Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), is up-regulated on angiogenic blood vessels in cancer (23Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, 44Partanen T.A. Alitalo K. Miettinen M. Cancer (Phila.). 1999; 86: 2406-2412Crossref PubMed Scopus (238) Google Scholar), and signaling via this receptor appears to be required for tumor angiogenesis (24Kubo H. Fujiwara T. Jussila L. Hashi H. Ogawa M. Shimizu K. Awane M. Sakai Y. Takabayashi A. Alitalo K. Yamaoka Y. Nishikawa S.-I. Blood. 2000; 96: 546-553Crossref PubMed Google Scholar). Furthermore, VEGFR-3 can also be up-regulated on blood vessels during wound healing (45Paavonen K. Puolakkainen P. Jussila L. Jahkola T. Alitalo K. Am. J. Pathol. 2000; 156: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). The predicted structure of the VHD of human VEGF-D indicated that three of the amino acid residues that differ between human and mouse VEGF-D, which occur sequentially in the amino acid sequence (residues Ser150-Leu151-Ile152 in the human protein), were located near, or were part of, a putative receptor-binding surface. This putative receptor-binding surface in human VEGF-D is likely to interact with both VEGFR-2 and VEGFR-3 as it is known that VEGF binds both VEGFR-1 and VEGFR-2 using the same binding interface (39Wiesmann C. Fuh G. Christinger H.W. Eigenbrot C. Wells J.A. de Vos A.M. Cell. 1997; 91: 695-704Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar). Furthermore, a neutralizing antibody to human VEGF-D that blocks the interaction with VEGFR-2 also blocks binding to VEGFR-3 (31Achen M.G. Roufail S. Domagala T. Catimel B. Nice E.C. Geleick D.M. Murphy R. Scott A.M. Caesar C. Makinen T. Alitalo K. Stacker S.A. Eur. J. Biochem. 2000; 267: 2505-2515Crossref PubMed Scopus (101) Google Scholar). Mutation of these three residues in mouse VEGF-D to the homologous residues in human VEGF-D decreased the potency of binding and cross-linking of human VEGFR-3 in the Ba/F3 bioassay to that exhibited by the human protein, indicating that this region is important for the VEGFR-3 interaction. Although mouse VEGF-D binds and cross-links human VEGFR-3 more potently than human VEGF-D, the kinetics for the binding of mouse VEGF-D measured with a biosensor were not significantly different from those observed for human VEGF-D. This suggests that the greater activity of mouse VEGF-D in the bioassay may be due to more efficient dimerization of the receptor or differences in the efficiency of receptor internalization and recycling of receptors to the cell surface, phenomena that can be dramatically altered by single amino acid substitutions in growth factors (46Walker F. Nice E. Fabri L. Moy F.J. Liu J.F. Wu R. Scheraga H.A. Burgess A.W. Biochemistry. 1990; 29: 10635-10640Crossref PubMed Scopus (16) Google Scholar, 47Clackson T. Ultsch M.H. Wells J.A. de Vos A.M. J. Mol. Biol. 1998; 277: 1111-1128Crossref PubMed Scopus (254) Google Scholar). The greater activity of mouse VEGF-D in the human VEGFR-3 bioassay also indicates that mouse VEGF-D may be a better therapeutic for inducing signaling via VEGFR-3 and has a more appropriate structure on which to base design of small molecule VEGFR-3 agonists. The capacity of the mouse VEGF-D mutants generated in this study to bind and cross-link VEGFR-2 indicated that residues in human VEGF-D at, or near, the putative receptor-binding surface, including Ser150, Leu151, Ile152, and Ala195, are important for the VEGFR-2 interaction. Although the ASLI mutant of mouse VEGF-D, containing these four residues of human VEGF-D, had much greater activity than wild-type mouse VEGF-D in the VEGFR-2 bioassay, it still had ∼13-fold less activity than human VEGF-D. This indicates that residues distant from the receptor-binding interface do play a role. The locations of the residues Ser150,Leu151, Ile152, and Ala195 in human VEGF-D, which are important for the VEGFR-2 interaction, are different from those for the residues in VEGF (Arg82, Lys,84 and His86), which were shown to be critical for the binding of this receptor by alanine-scanning mutagenesis (48Keyt B.A. Nguyen H.V. Berleau L.T. Duarte C.M. Park J. Chen H. Ferrara N. J. Biol. Chem. 1996; 271: 5638-5646Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). This suggests that different sets of residues in the receptor-binding surfaces of these two growth factors are critical for the VEGFR-2 interaction. Previous studies indicated that mouse VEGF-D induced proliferation of endothelial cells in vitro and was angiogenic both in vivo and in vitro (26Marconcini L. Marchio S. Morbidelli L. Cartocci E. Albini A. Ziche M. Bussolino F. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9671-9676Crossref PubMed Scopus (233) Google Scholar). The angiogenesis activityin vivo was demonstrated in the rabbit corneal assay, andin vitro studies of angiogenesis and mitogenic activity were performed using human umbilical vein endothelial cells. As we have shown that mouse VEGF-D binds human VEGFR-2, the mitogenic effects of mouse VEGF-D on human umbilical vein endothelial cells observed previously could indeed have been mediated by VEGFR-2, although the involvement of VEGFR-3 cannot be discounted. The angiogenic activity observed in the rabbit corneal assay may have been a consequence of the capacity of mouse VEGF-D to activate rabbit VEGFR-2, although it is not known if this ligand binds VEGFR-2 in the rabbit. Our finding that VEGF-D binds VEGFR-3 but not VEGFR-2 in the mouse suggests that expression of genes for VEGFR-3 and VEGF-D should be coordinated at sites in the mouse embryo during development to establish a paracrine mode of action that is typical of VEGF family members (49Kukk E. Lymboussaki A. Taira S. Kaipainen A. Jeltsch M. Joukov V. Alitalo K. Development. 1996; 122: 3829-3837PubMed Google Scholar, 50Plate K.H. Breier G. Risau W. Brain Pathol. 1994; 4: 207-218Crossref PubMed Scopus (226) Google Scholar). Indeed, expression of VEGFR-3 and VEGF-D is coordinated in developing mouse skin, as a layer of cells positive for VEGF-D mRNA is immediately adjacent to a network of VEGFR-3-positive vessels. Therefore, it will be important to monitor the effect of VEGF-D deficiency on the development of vessels in the skin. The unexpected finding that mouse VEGF-D does not bind mouse VEGFR-2 has important implications for the interpretation of biological models and pharmacological approaches for monitoring the function and utility of VEGF-D. We thank Prof. Antony Burgess for critical reading of this manuscript and Dr. Tanya Petrova for providing the mVEGFR-3-Ig construct.

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