MXPA99007237A - Ligands for discoidin domain receptor tyrosine kinases and complexes thereof - Google Patents

Abstract

Members of the collagen family are ligands for the discoidin domain receptor tyrosine kinases, DDR1 and DDR2. Collagen directly interacts with the extracellular domains and evokes tyrosine phosphorylation of DDRs in a time and concentration dependent manner. Collagen activation of DDR1 induced phosphorylation of a docking site for the Shc phosphotyrosine binding domain. Therefore, isolated complexes are described comprising (a) a discoidin domain receptor tyrosine kinase or a part thereof, and a collagen or a part thereof;or (b) a discoidin domain receptor tyrosine kinase or a part thereof and Shc or PTB binding domain of Shc or a protein containing a PDZ domain or a PDZ domain. Compositions, methods, and uses are also described based on the interaction of DDRs with collagens.

Description

LIGANDOS PARA T1ROSINA KINASAS DE RECEPTOR DE DOMINIO DE DISCOIDIIMA AND COMPLEXES OF THE SAME FIELD OF THE INVENTION The invention relates to novel complexes, methods for activating a discoidine domain receptor tyrosine kinase-mediated signaling pathway (DDR) in a cell, and methods for identifying substances that affect the pathway. BACKGROUND OF THE INVENTION The cloning of cDNA for receptor tyrosine kinases in mammals (RTK) has recently resulted in the identification of a new sub-family of receptors that have an extracellular domain related to discoidin of iecitin, Found in the mold Dictyostelium discoideum. Two different RTKs have been identified with discoidin repeats, namely discoidin 1 domain receptor (DDR1) (also called DDR, MCK10, Cak, NEP, trkE, Ptk3 or RTK6) (1-7) and discoidin 2 domain receptor (DDR2) (also called CCK2, TKT or Tyro 10) (1, 8, 9) DDR1 is mainly expressed in epithelial cells (1), and is particularly abundant in cells neuroepithelial cells during embryonic development of mice (4) Higher levels of DDR1 have been detected in human ovarian and breast cancer samples, suggesting that over-expression of DDR1 may be involved in the development of tumors (1, 10). Extracellular DDR1 has the motif RXRR, which is a potential recognition site for the separation of endoproteases. In addition, a considerable fraction of DDR1 is processed to a subunit β associated with truncated membranes and a subunit to soluble (1). In comparison with other receptor tyrosine kinases, DDR1 has a relatively long juxtamembrane region, which is modified by alternating to give two different isoforms of DDR1 (1). The DDR1a spectrum has 139 amino acids in the juxtamembrane region, while the DDR1b isoform differs from the a isoform by incorporating a further extension of 17 amino acids in the juxtamembrane region, encoded by an extra exon (7). DDR receptors may be implicated in tumorigenesis. DDR1 RNA transcripts specific for both a and b have been detected in several human ovarian (7) and breast2 cell lines. In situ analyzes of several human primary mammary carcinomas have shown that the expression of DDR1 mRNA can be at least three times higher in tumor cells than in adjacent normal epithelia (Barker et al., Oncogene 11: 569-575, 1995) Using tests for both people, in situ hybridization on adjacent sections of human ovaries or lung carcinomas have shown that DDR1 is expressed in the tumor cells themselves while DDR2 was detected in stromal cells surrounding the tumor (Alves et al., Oncogene 10: 609-618, 1995). The specific insert for b, in DDR1b, exhibits sequence motifs suggesting that the insert may be involved in signaling downstream of the DDR1 receptor (1). Notably, the insert specific for isoform b contains an LLSNPAY sequence, which can potentially serve as a coupling site for the phosphotyrosine binding domain (PTB) of the Shc adapter protein. The Shc protein contains both the C-terminal SH2 domain and an N-terminal PTB domain of approximately 160 residues, which, unlike the SH2 domains, recognizes the phosphotyrosine (pTyr) sites with the consensual sequence: Hydrophobic -X-Asn-Pro-X-pTyr (11-14): said phosphorylated motifs can be found in the juxtamembrane region of nerve growth factor receptor (NGF) and in the terminal ends in C of the epidermal growth factor receptor (EGF), ErbB2 and ErbB3 (15-18). the interaction of the PTB domain of Shc with activated receptors can stimulate the phosphorylation of Shc to Tyr 317, within a motif (YVNV) which is recognized by the Grb2 SH2 domain (19. The association of phosphorylated Shc with Grb2 provides a mechanism by which Shc can stimulate the Ras path. In addition to the growth factor receptors , cytoplasmic proteins such as polyomavirus medium T antigen and inositol containing SHIP SH2 contain motifs of NPXY that can potentially bind to the PTB domain of Shc under phosphorylation (15,20) .The analysis of both the PTB domain of Shc as its binding sites have shown that three N-terminal residues of Asn for the phospho-Tyr (to the -3 position) is essential to bind and that Pro is favored in the -2 position (21-25). to form a ß turn that places the phosphotyrosine in a basic PTB domain bag (26) The high affinity binding of phosphopeptides of the PTB domain of Shc also requires a hydrophobic residue in the -5 position (16, 18). In addition, a resident uo hydrophobic in position -6 can make contact with the PTB domain (26). The LLSNPAY sequence within the insert specific for the isoform b of DDR1 conforms to this consensus for the binding of PTB to Shc. SUMMARY OF THE INVENTION The present inventors have shown significantly that members of the collagen family are ligands for receptor tyrosine kinases of the discoidin domain, DDR1 (also known as MCK-10, DDR, NEP, cak, trkE RTK6, and ptk3). ) and DDR2 (also known as CCK-2, tyro-10 and TKT). It was found that collagen interacts directly with the extracellular domains and evokes the tyrosine phosphorylation of DDR in a manner that depends on time and concentration. In particular, collagen groups types II, III, IV and V were shown to be good ligands for DDR1. It was shown that collagen types I and III are highly potent ligands for DDR2 while collagen types II and V, they showed moderate activity. The present inventors also showed that glycosylation of collagen is essential for the activation of DDR, in particular the activation of DDR2. The stimulation of DDR receptor tyrosine kinase activity required the natural triple helix structure of collagen. Collagen activation of DDR1 induced phosphorylation of a coupling site for the phosphotyrosine binding domain of Shc, whose presence is controlled by alternative cleavage. In particular, the present inventors showed direct evidence that the PTB domain of the Shc adapter protein binds selectively to DDR1b using the LLSNPAY motif encoded by the exon specific for b divided alternatively. It was also shown that the activation of DDR2 by collagen results in upregulation of the expression of matrix metalloproteinase-1 (MMP-1). Therefore, DDR1 and DDR2 are novel collagen receptors that can control cellular responses of the extracellular matrix. Broadly, the present invention provides an isolated complex comprising a DDR or a part thereof and a collagen or a part thereof, or a complex comprising a DDR or a part thereof and Shc, or a protein containing a PDZ domain. Peptides derived from the binding domain of a DDR that interacts with a collagen or part of a collagen or interacts with Shc or a PDZ domain. A molecule derived from the collagen binding domain that interacts with a DDR or a part thereof is also contemplated. The invention also includes antibodies specific for the complexes and peptides. The present invention also provides a method for modulating, and in particular, activating a discoidine domain receptor tyrosine kinase (DDR) -mediated signaling pathway in a cell, comprising reacting a discoidine domain receptor tyrosine kinase protein. , or an isoform or a part of the protein on the cell, with a collagen or part of a collagen, thus modulating the signaling path in the cell. In one embodiment, the protein or part of the protein comprises an oligomerized receptor or extracellular domain or an oligomerized extracellular tyrosine kinase domain of discoidin domain receptor. The route can also be activated using a complex or peptide of the invention. In one embodiment, the invention contemplates a method for modulating extracellular matrix synthesis, degradation or remodeling responses in a cells comprising reacting a discoidine domain receptor tyrosine kinase protein, or an isoform or a part of the protein having at least 20 contiguous amino acids of the protein on a cell, with a collagen or part of a collagen, thus modulating the responses of synthesis, degradation or remodeling of extracellular matrix. The responses of synthesis, degradation or extracellular matrix remodeling responses can be modulated using a complex or peptide of the invention.

Still further, the invention provides a method for evaluating a compound for its ability to modulate a signaling pathway mediated by DDR. For example, a substance that inhibits or enhances the interaction of a DDR and a collagen, or a substance that binds to DDR or part thereof or to a collagen or part of a collagen can be evaluated. In one embodiment, the invention provides a method for identifying a substance that affects a DDR receptor tyrosine kinase-mediated signaling path, comprising the steps of: (a) reacting a collagen and at least one tyrosine kinase protein of discoidin domain receptor, or an isoform or part of the protein, and a test substance, wherein the collagen and the discoid domain receptor tyrosine kinase protein is selected such that they bind to form a collagen complex Tyrosidine domain receptor tyrosine kinase protein; and (b) compare it with a control in the absence of the substance to determine the effect of the substance. In particular, a method is provided for identifying a substance that affects a DDR receptor tyrosine kinase-mediated signaling pathway in a cell, comprising (a) reacting a collagen or part thereof, and at least one protein receptor tyrosine kinase of discoidma domain. or an isoform or a part of the protein and a test substance, wherein the collagen and the discoid domain receptor tyrosine kinase protein are selected such that they bind to form a collagen-protein tyrosine kinase complex of discoidin domain receptor, under conditions that allow the formation of collagen-protein tyrosine kinase complexes of discoidin domain receptor, and (b) analyze complexes, for free substances, for non-complexed collagen, or for activation of the protein. In one embodiment of the method, the substance is a carbohydrate moiety of a collagen, or an imitation thereof, or a peptide derived from the domain of a DDR that binds to a collagen, or an imitation thereof. The invention further provides a method for treating or avoiding a condition involving a tyrosine kinase-mediated signaling pathway of discoidin domain receptor, which method comprises administering to a patient in need thereof an amount of a substance that is effective to interfere with the signaling path wherein the substance is (a) a tyrosine kinase of discoidin domain receptor or part thereof; (b) a collagen or part thereof; (c) a substance identified first by (i) reacting a collagen, and at least one discoidine domain receptor tyrosine kinase protein, or an isoform or a part of the protein, and the test substance, wherein the collagen and the tyrosine kinase protein of discoidin domain receptor are selected so that they bind to form a collagen-tyrosine kinase protein complex of discoidin domain receptor; and (i) compare with a control in the absence of the substance to determine the effect of the substance. The substance can also be an isolated complex comprising a DDR and a collagen; peptides derived from the binding domain of a DDR that interacts with a collagen or part of a collagen, or that interacts with a Shc or a PDZ domain; a molecule derived from the collagen binding domain that interacts with a DDR or a part thereof; or, antibodies specific for the complexes and peptides. The invention also relates to a pharmaceutical composition comprising a receptor tyrosine kinase protein of the isolated discoidin domain sub-family or an isoform or part of the protein, a collagen or a part of a collagen, a complex, antibody, a peptide of the invention, or a substance as described herein, in an amount effective to affect a tyrosine kinase-mediated signaling pathway of discoidin domain receptor, and a pharmaceutically acceptable carrier, diluent, or excipient. The composition may comprise an extracellular domain of a discoidine domain receptor tyrosine kinase, or the portion of the extracellular domain that binds to the carbohydrate portion of a collagen or portions thereof. In one embodiment the composition comprises a collagen or a portion thereof, preferably a carbohydrate portion of collagen. The methods and compositions of the invention can be used to alter transformation or metastasis in a mammal, to treat conditions involving structural or functional dysregulation of collagens such as Cleidocranial dysplasia and Sickler syndrome, conditions that require synthesis modulation, degradation or extracellular matrix remodeling or to treat conditions that require modulation of MMP-1 expression (e.g., for use in wound healing). Other objects, aspects and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art. the matter from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described in relation to the drawings in which: Figure 1A. is an immunoassay of immunoprecipitated spots with anti-Shc antibodies from cell lysates of embryonic 293 cells of kidney fibroblasts, human, transfected with expression plasmids encoding DDR1a (MCKIOa) or DDR1b (CKIOb) and stimulated with an antibody-tested orthovanadate for phosphotyrosine; Figure 1B is the spot immunoassay of Figure 1A again tested with antibodies against DDR1; Figure 1C is a spot immunoassay of Figure 1B retested with antibodies against Shc; Figure 1D is a spot immunoassay of the total cell lysates used in Figure 1A, analyzed by Western blot analysis with anti-phosphotyrosine antibodies; Figure 1E, is an immunoassay of lysate protein spots (of 293 cells that have been transfected with DDRIA or DDR1b and stimulated with orthovanadate) that binds to the GST fusion proteins containing the SH2 domain of Shc or the domino of PTB of Shc, Figure 2A, is a spot immunoassay of an experiment involving incubation of the GST-PTB domain fusion protein of Shc bound to glutathione beads with DDR1b lysates that overexpress 293 cells in the absence or presence of increasing concentrations of a competing peptide, ALLLSNPApYRLLLA and detect the bound protein by the immunoassay with antibodies to DDR1. Figure 2B is a graph depicting analysis of the binding of the GST-PTB domain of Shc purified for the phosphopeptide of medium T antigen by resonance of surface plasmons in the presence of increasing amounts of DDR1 phosphopeptide (ALLLSNPApYRLLLA, open circles) or NGF receptor phosphopeptides (HIIENPQpYFSD, closed circles), respectively; wherein the percentage of the PTB domain of Shc bound to the micrometer surface is plotted against the concentration of competing peptides; Figure 3A is a triptych map of two-dimensional phosphopeptides of the in vivo-labeled isoform of DDR1; Figure 3B is a triptych map of two-dimensional phosphopeptides of the labeled β isoform of DDR1; Figure 3C is a schematic representation of the phosphopeptides in Figures 3B and 3F; Figure 3D shows the results of the tryptic mapping of the in vitro-labeled protein of the DDR1a isoform; Figure 3E shows the results of the tryptic mapping of the in vitro labeled protein of the DDR1b isoform; Figure 3F shows the results of the tryptic mapping of the phosphopeptides of DDR1a and DDR1b; Figure 4A is an immunoassay of the results of the experiments in which DDR1a and DDR1b, a mixture of DDR2, and TrkA, are temporarily expressed in 293 cells stimulated with type I collagen or treated with 100 μM acetic acid and cell lysates total are graphed and tested with apTyr antibodies [Sigma # C-7661: rat tail collagen type I]; Figure 4B. is an immunoassay of spots from the results of the experiments where DDR1a. DDR1b, DDR2 and TrkA are expressed temporally in 293 cells stimulated with type I or IV collagen, and the total cell lysates are graphed and tested with apTyr antibodies [Sigma # C-3511: bovine skin collagen type I, C-7521 : collagen of human placenta type IV]; Figure 4C is a spot immunoassay of the results of the experiments where DDR1a, DDR1b and TrkA are temporarily expressed in 293 cells stimulated with type I collagens and the total cell lysates are graphed and tested with apTyr antibodies [Sigma #C -8897: rat tail collagen type I, C-7774: type I collagen from human placenta]; Figure 5A is an analysis of a-pY spots of total cell lysates of 293 cells temporarily expressing DDR1b in the presence of collagen types I and IV at different concentrations [Sigma # C-7661: rat tail type I collagen, C-0543: mouse type IV collagen]; Figure 5B is an a-pY analysis of total cell lysates of 293 cells expressing temporally DDR2 stimulated with type I or IV collagens at different concentrations; Figure 5C is an analysis of a-pY of total cell lysates of 293 cells temporarily expressing DDR2 in the presence of type I collagens at different concentrations [CBP: Collaborative Biomedical Products # 40236- collagen type I], Figure 6 is a a-PY analysis of DDR1b immunoprecipitated from lysates of human mammary carcinoma cells stimulated with Collagen I [C-7661], Collagen IV [C-0543] and orthovanadate; Figure 7A, is an analysis of total protein cell lysate spots for 293 cells of human kidney fibroblasts transfected with DDR1b and stimulated with different concentrations of Matrigel, tested with antiphosphotyrosine antibodies; Figure 7B is a spot analysis of Figure 7A separated and retested with antibodies raised against DDR1; Figure 7C is an analysis of antiphosphotyrosine stains of cell lysates of 293 cells transfected with DDR1b and treated with the following reagents: 400 μM acetic acid (a), 50 μl / ml matrigel (b), 10 μg / ml laminin type IV (c), 10 μg / ml fibronectin (d), type IV collagen, partially purified from matrigel by extraction with guanidinium hydrochloride (e) or by extraction of acetic acid and pepsin (f), 10 μg / ml of type IV mouse collagen, Sigma C-0543 (g), 10 μg / ml of human type IV collagen, Sigma c-5533 (h); Figure 7D is a stain analysis of anti-DDR1 antibodies of cell lysates of 293 cells treated with the following reagents: 400 μM acetic acid (a), 50 μl / ml matrigel (b), 10 μg / ml laminin type IV (c), 10 μg / ml fibronectin (d), collagen type IV, partially purified from matrigel by extraction with guanidinium hydrochloride (e) or by extraction of acetic acid and pepsin (f), 10 μg / ml of type IV mouse collagen, Sigma C-0543 (g), 10 μg / ml of human type IV collagen, Sigma c-5533 (h); Figure 8A is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells transfected with plasmids encoding human insulin receptors (Ins-R), DDR1a, DDR1b or DDR2 stimulated with 10 μg / ml of type I mouse collagen, 100 nM of insulin or left unstimulated. Figure 8B is a spot analysis of Figure 8A retested with a mixture of antibodies against DDR1, DDR2 and insulin receptor; Figure 9A, is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells transfected with DDR1b and stimulated with type I collagen for different periods of time; Figure 9B is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells transfected with DDR2 and stimulated with type 1 collagen for different periods of time. Figure 9C is an immunoprecipitated DDR1 spot analysis of cell lysates of human mammary carcinoma T-47D cells with type I collagen for various periods of time. Figure 9D is a spot analysis of Figure 9C re-tested with an antibody specific to C-terminus of DDR1; Figure 9E is an immunoprecipitated DDR1 spot analysis of 293 cells and subjected to an in vitro kinase reaction. Figure 9F is an immunoprecipitated DDR1 spot analysis of T-47D cells and subjected to an in vitro kinase reaction; Figure 10A, is an analysis of antiphosphotyrosine antibody stains from cellular islets of 293 cells transfected with DDR1b stimulated with 10 μg / ml of human collagen types I, III, IV or V and collagen from bovine type II; Figure 10B is the spot analysis of Figure 10a retested with the receptor specific antibodies for DDR1; Figure 10C is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells transfected with DDR2 stimulated with 10 μg / ml of human collagen types I, III, IV or V and collagen of cattle type II; Figure 10D is a spot analysis of Figure 10C retested with specific receptor antibodies for DDR2; Figure 10E, is an anti-phosphotyrosine antibody staining of DDR1b immunoprecipitated from T-47D cells and stimulated for 9 minutes with collagen types I, II, III, IV, V or gelatin or treated with 1 mM of orthovanadate; Figure 10F is the spot analysis of Figure 10E retested with specific antibodies to DDR1; Figure 10G is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells that have been transfected with DDR1b and contacted with human collagen types I, III, IV and V; Figure 10H, is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells that have been transfected with DDR2 and contacted with human collagen types I, III, IV and V; Figure 11A shows type I collagen isolated from mouse or human tissue or BSA treated with collagenase or pepsin and analyzed by SDS-PAGE and visualized by Coomassie tinsion; Figure 11B, is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells that over-express DDR2 stimulated with collagenase type I collagenase or pepsin; Figure 11C is an analysis in Figure 11B retested with specific antibody for DDR2; Figure 11D is a spectrum of mouse type I collagen (500 ng / ml in 10 mM acetic acid) fused in a previous spectropolarimeter (frames) and then by heat denaturation (diamonds); Figure 11E, is a stain analysis of antiphosphotyrosine antibodies of cell lysates of 293 cells that over-express DDR2 stimulated with aliquots of mouse type I collagen that has been incubated at various temperatures; Figure 12A is a stain analysis of the 293 cell lysate material that over-express insulin receptor, DDR1b or DDR2 in the absence or presence of 50 μg / ml soluble type I collagen, which binds to collagen covalently coupled to agarose; Figure 12B is a graph showing the amount of binding ligand of 293 cells transfected with DDR1a (frames), DDR1b (diamonds) or control plasmid (circles) after incubation with various concentrations of iodinated type I collagen; Figure 12C, is an analysis of antiphosphotyrosine antibody stains of cell lysates of 293 cells that over-express DDR2 stimulated with type I collagen deglycosylated with sodium m-periodate; Figure 13A is a protein stain analysis of lysates of 293 cells that overexpress DDR1a or DDR1b that bind to the GST fusion protein of a Shc PTB domain detected with an antibody against the C terminus of DDR1 . Figure 13B is a graphical representation of spot analysis of the binding of the GST-PTB domain of purified Shc to the medium T antigen phosphopeptide (LSLLSNPTpYSVMRSK) by resonance of surface plasmons in the presence of competing amounts of DDR1b phosphopeptide (ALLLSNPApYRLLLA, open circles) or NGF receptor phosphopeptide (Hl I IN PQpYFSD, closed circles) respectively; Figure 14 is a spot analysis with antibodies against MMP-1 of media conditioned from parental cells and HT 1080 that over-express DDR2 stimulated with type I collagen or TPA during the indicated time; Figure 15A is a spot immunoassay showing that DDR1a with the K618A mutation is no longer activated by collagen; Figure 15B is a spot immunoassay showing that DDR1 with the K618A mutation that is no longer activated by collagen; Figure 16 is a spot analysis showing that blocking antibodies to a1 or β1 integrins do not inhibit the activation of DDR1; Figure 17A is a spot analysis showing that DDR1 is activated by collagen in cells deficient in β1 integrins; Figure 17B is a spot analysis showing that DDR1 is activated by collagen in ßl integrin deficient cells; Figure 18 is an analysis showing that the activation of DDR1b in β1 integrin deficient cells is as slow as normal cells, indicating that the extensive activation of DDR1b is not due to the action of integrins; Figure 19A. it is a spot immunoassay that shows that the activation of the DDR1 and DDR2 receptor does not influence EGF-mediated activation of MAPK; Figure 19B is a spot immunoassay showing that DDR1 and DDR2 receptor activation does not influence EGF-mediated activation of MAPK; Figure 19C is a spot immunoassay showing that the activation of DDR1 and DDR2 does not influence the activation of MAPK mediated by EGF; Figure 20A shows the nucleotide and amino acid sequence of human DDR1 which is Figure 1 in Johnson et al., Proc. Nati Acad. Sci. USA 90: 5677, 1995; Figure 20B shows the amino acid sequence of DDR1 has GenBank Accession No. L20817; Figure 21 shows an alignment of the sequences of DDR1a, b and c where the NPXY motif in the DDR1 insertion region is underlined with a thin stripe and the putative SH3 site with solid bars, which is Figure 1 (c) in Alves et al. 1995, Oncogene 10: 609, 1995; Figure 22A shows the nucleotide and amino acid sequence of human DDR2 having Accession No. of GenBank X74764; and Figure 22B shows the amino acid sequence of human DDR2 having Accession No. of GenBank X74764. DETAILED DESCRIPTION OF THE INVENTION Discoidine Domain Receptor (DDR) Tyrosine Kinase and Collagens The term "discoidine domain receptor tyrosine receptor (DDR) -mediated tyrosine kinase signaling pathway", as used herein, refers to the interactions of a discoidine domain receptor tyrosine kinase protein with a collagen or a part thereof, to form a complex of collagen receptor tyrosine kinase proteins thus activating a series of regulatory pathways downstream of the cell that affect the cell, for example, controlling gene expression, cell division, cytoskeletal architecture, cell metabolism, migration cell-cell interactions, spatial placement, synthesis, degradation and remodeling of extracellular matrix, protein expression (e.g., upregulation of MMP-1) and / or cell adhesion. Examples of such downstream regulatory pathways are: the GAP / Ras pathway, the pathway that regulates the breakdown of polyphosphoinositides through phospholipase C (PLC) and the Src / tyrosine kinase and Ras pathways. The route includes the interactions of a DDR protein with intracellular signaling molecules that include Shc or proteins with PDZ domains. "Discoidine domain receptor tyrosine kinase (DDR)" proteins refers to the receptor tyrosine family that contains a discoidin I motif in its extracellular domains. The structure of the extracellular region determines the binding specificity of ligands. The intracellular regions contain the juxtamembrane and the catalytic kinase domain. The transduction of signals mediated by the receptor is initiated in the cell that expresses receptors by the binding of ligands to the extracellular domain, which facilitates dimerization of the receptor and autophosphorylation. The contrast marks of a discoidin domain receptor tyrosine kinase are exemplified by discoidin domain receptor I (DDR1) (Di Marco et al., 1993 J. Biol, Chem. 268: 24290; Johnson et al., Proc. Nati, Acad Sci USA, 90, 5677, 1993, Zerlin and others, 1993 Oncogene 8: 2731, Laval and others, 1994 Cell Growth Differ 5: 1173, Perez and others, 1994, Oncogene 9: 211; and others, 1994, Proc. Nati, Acad. Sci. USA 91: 1819, Alves, et al., Oncogene 10,609, 1995, Shelling et al., 1995, Genomics 25: 584, Valent et al., 1996, Humana Genet. 12) and the discoidin domain receptor 2 (DDR2) (Karn et al., 1993, Oncogene 8: 3433; Alves, et al., Oncogene 10, 609, 1995 Lai and Lemke et al., 1994). There are three different forms of DDR1, designated a, b and c, which represent alternative division variants of a common primary gene transcript. DDR1 and DDR2 have a high degree of similarity with an equalization of 78% within the amino-terminal discoidin I domain with approximately 150 amino acids long. DDR1 contains the consensual sequence RXRR at position 304-307 which represents a possible separation signal for the furine endopeptidase. The juxtamembrane domain of the DDR2 receptor comprises 148 amino acids. The DDR1a isoform comprises 139 amino acids in the juxtamembrane region, while isoform b differs from an isoform a by the incorporation of an additional extension of 37 amino acids in the juxtamembrane region encoded by an extra exon. The specific motif for isoform b contains the sequence LLSNPAY which serves as a coupling site for the phosphotyrosine binding domain (PTB) of the Shc adapter protein. Figures 20A and B show the nucleotide sequence and deduced amino acid sequence of human DDR1 cDNA (Figure 1 in Johnson et al., 1993, Supra). The sequence enclosed near the N terminus contains the domain similar to discoidin I and the box near the C terminus of the tyrosine kinase domain. The predicted signal peptide and the transmembrane domain are underlined and the glycine residues between the discoidin I-like domain and the tyrosine kinase domain are italic letters. The M symbols underline most of the connection region rich in proline and glycine. The juxtamembrane region is between amino acid 468 and amino acid 607. The DDR1 sequence can also be found in GenBank, with Accession Nos. L11315 or L20817. In Figure 21 an alignment of the sequences of the divided DDR1 isoforms is shown (Figure 1 of Alves et al., 1995). Figures 22A and 22B show the nucleotide sequence and chosen amino acid sequence of a human DDR2 (i.e., TKT). (GenBank Accession No.: X74764). The juxtamembrane region is between amino acid 422 and amino acid 570. It will be appreciated that the receptor tyrosine kinase protein for use in the present invention can be an isoform or a part of the protein. The isoforms contemplated for use in the methods of the invention are isoforms that have the same functional properties as the tyrosine kinase proteins of discoidin domain receptors. In a preferred embodiment, the part of the protein has at least 20 contiguous amino acids and preferably comprises an extracellular domain or the C-terminal region. The receptors can also be oligomerized, in particular the dimers and trimers are contemplated for use in the methods and compositions of the invention. A portion of a discoidine domain receptor tyrosine kinase protein includes a portion of the molecule that interacts directly or indirectly with a collagen or an intracellular molecule such as Shc, or a protein with a PDZ domain (i.e., a domain of Union). A binding domain can be a sequential portion of the molecule, i.e., a contiguous amino acid sequence, or it can be conformational, i.e., a combination of non-contiguous amino acid sequences that, when the molecule is in its native state, form a structure that interacts with another molecule in a complex of the invention. A part of a DDR protein contemplated herein includes a molecular entity that is identical or substantially equivalent to the native binding domain of a molecule in a complex of the invention (ie, DDR or part thereof and a collagen and a part of it). Peptides derived from the binding domains are discussed below. A DDR protein used in the invention can be a protein having a substantial sequence identity with the sequence of a discoidin domain receptor tyrosine kinase protein. The term "sequence having substantial identity" means the amino acid sequences that have slight sequence variations or that the sequence of the tyrosine kinase protein of discoidine domain receptor has no consequences. The variations can be attributed to local mutations or structural modifications. Suitable proteins may have more than 75%, preferably more than 85%, even more preferably more than 90% identity with a tyrosine kinase protein of discoidin domain receptor. A tyrosine kinase of discoidin domain receptor or part thereof, can be selected for use in the present invention based on the nature of the ligand to which it is directed or which is selected. The selection of a particular ligand and complementary discoidine domain receptor tyrosine kinase, provides specific complexes and in the methods of the invention allows identification of specific substances that affect a tyrosine kinase regulatory domain of discoidin domain. For example, a collagen type I, II can be interacted; III, IV or V with DDR1 in the complexes and methods of the invention.

Also, collagen type I or II and DDR2 can interact in the complexes and methods of the invention. A tyrosine kinase of discoidin domain receptor or part thereof can be isolated from cells that are known to express the proteins (e.g., DDR1 can be isolated from neuroepithelial cells during the embryonic development of mice or samples of ovarian cancers and human breast). Alternatively, the protein or a part of the protein can be prepared using recombinant DNA methods known in the art. By way of example, nucleic acid molecules having a sequence encoding a discoid domain domain tyrosine kinase protein, or a portion of the protein can be prepared and incorporated in a known manner into an appropriate expression vector that ensures a good expression of the protein or part of it. Possible expression vectors include, but are not limited to, cosmids, plasmids or modified viruses, while the vector is compatible with the host cell used. The tyrosine kinase protein of discoidin domain receptor or parts thereof can also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154) or synthesis in homogeneous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

A discoidine domain receptor tyrosine kinase protein or parts thereof, for use in the methods of the present invention, may be associated with a cell surface. Expression on cell surfaces of a tyrosine 5 kinase protein of discoidin domain receptor or parts thereof can be carried out using conventional methods. Conjugates of the protein, or parts thereof, with other molecules, such as proteins or polypeptides, can be prepared using the methods described herein. This can be achieved, for example, by the synthesis of N-terminal or C-terminal fusion proteins. Therefore, fusion proteins can be prepared by fusing, through recombinant techniques, the N-terminus or C-terminus of a discoidin domain receptor tyrosine kinase protein, or portions of the same, and the sequence of a protein selected from a protein T marker with a desired biological function. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins and parts thereof such as the constant region of an immunoglobulin and lymphokines such as interferon. range, tumor necrosis factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, GM-CSF, CSF-1 and G-CSF. The protein tyrosine kinase of discoidin domain receptor, isoforms or parts thereof, used in the invention, can be insolubilized. For example, the protein The receptor or part thereof, preferably the extracellular domain, can be attached to a suitable vehicle. Examples of suitable vehicles are agarose, cellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose polystyrene, paper filter, ion exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl acid copolymer. maleic, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The vehicle can be in the form of, for example, a tube, test plate, beads, discs, sphere, etc. The insolubilized receptor tyrosine kinase protein can be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, coupling of cyanogen bromide. Suitable collagens that can be used in the methods and compositions of the invention, include collagen types I, II, III, IV and V. The collagen can be obtained from a commercial source, or produced using conventional methods. A collagen is selected for the complexes and methods described herein that provide activation of a selected DDR. A part of the collagen can be used in the methods and compositions of the invention. In one embodiment of the invention, a carbohydrate portion of a collagen or a portion of this part, or a GXY repeat region having a triple helical conformation of a collagen, which is capable of binding to the extracellular domain, is used. of tyrosine kinase of discoidin domain receptor (preferably DDR2) and of activating the receptor. A collagen, or part thereof, used in the invention, can be insolubilized; for example, it can be attached to any suitable vehicle as described herein. Peptides The invention provides peptide molecules that bind to, and inhibit the interaction of, a DDR or part thereof and a collagen or part thereof, or a DDR and an intracellular molecule such as Shc, or a protein having a domain of PDZ. A peptide derived from a specific binding domain can encompass the amino acid sequence of a binding site that is present in nature, any portion of said binding site, or any different molecular entity that functions to bind to an associated molecule. A peptide derived from said binding domain will interact directly or indirectly with an associated molecule in such a manner that it mimics the native binding domain. Such peptides may include competitive inhibitors, enhancers, peptide mimics and the like. All of these peptides, as well as the molecules substantially homologous, complementary, or in some other way, functionally or structurally equivalent to these peptides, can be used for the purposes of the present invention. "Peptide mimics" are structures that serve as substituents for peptides in interactions between molecules (see Morgan et al. (1989), Ann. Reports Med. Chem. 24: 243-252 for a review) Imitations of peptides include synthetic structures which may or may not contain amino acids and / or peptide linkages but which retain the structural and functional aspects of a peptide, or enhancer or inhibitor of the invention. Imitations of peptides also include peptoids, oligopeptoids (Simón et al. (1972) Proc. Nati Acad. Sci. USA 89: 9367); and peptide libraries containing peptides of a designed length representing all possible amino acid sequences corresponding to a peptide of the invention. The peptides can be synthesized by conventional techniques. For example, peptides can be synthesized by chemical synthesis using solid phase peptide synthesis. These peptides employ solid phase or solution synthesis methods (see for example, JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford III. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol.2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques, and M. Bondansky, Principies of Peptide Synthesis , Springer-Verlag, Berlin 1984 and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for synthesis of classical solution). Peptide imitations can be designed based on the information obtained by the systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties and by systematic replacement by peptide bonds with amide junction replacements. . Local conformational constraints may also be introduced to determine the conformational requirements for the activity of a candidate peptide mimic. Imitations may include isomeric amide linkages, or D-amino acids to stabilize or promote reverse spin conformations and to help stabilize the molecule. Cyclic amino acid analogs can be used to restrict amino acid residues to particular conformational states. Imitations may also include imitations of secondary structures of inhibitory peptides. These structures can model the three-dimensional orientation of amino acid residues in the known secondary conformations of the proteins. Peptoids can also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules. The peptides of the invention can develop a biological expression system. The use of these systems allows the production of large banks of random peptide sequences and the screening of these libraries of peptide sequences to bind to particular proteins. Banks can be produced by the cloning of synthetic DNA encoding peptide sequences into appropriate expression vectors. (See Christian and others 1992, J. Mol. Biol. 227: 711; Devlin et al., 1990 Science 249: 404; Cwirla and others 1990. Proc. Nati Acad. Sci. USA 87: 6378). Banks can also be constructed by concurrent synthesis of overlapping peptides (see U.S. Patent No. 4,708,871). The peptides of the invention can be used to identify leading compounds to produce drugs. The structure of the peptides described herein can be easily determined by a number of methods such as NMR and X-ray crystallography. A comparison of peptide structures of similar sequence, but differing in biological activities, can produce white molecules that provide information about the activity relationship of the target structure. The information obtained from the examination of the activity relationships of the structure can be used to design modified peptides or other small molecules or leader compounds that can be tested for predicted properties related to the target molecule. The activity of the leader compounds is evaluated using similar analyzes those described herein. Information about the structure-activity relationships of the co-crystallization studies can also be obtained. In these studies, a peptide with a desired activity is crystallized in association with a target molecule and the structure of the complex is determined by X-rays. The structure can be compared to the structure of the target molecule in its native state and the comparison information it can be used to design compounds that are expected to have the desired activities.

Particular peptides that can be used in the invention, including peptides derived from the sites in a DDR (e.g., DDR1b) that binds to Shc, or that are derived from the PTB binding domain of Shc, derived peptides from sites in a DDR that bind to the insulin receptor substrate (IRS-1) or sites in IRS- that join a DDR, or sites in a DDR (eg, DDR1) that join proteins with a PDZ domain, or a PDZ domain. In one embodiment, the peptides comprising the amino acids XNPXpY are contemplated, where? is a hydrophobic amino acid including alanine, phenylalanine, isoleucine, leucine, methionine, proline and tryptophan, X is any amino acid, N is Asn, P is proline and pY is phosphotyrosine. Examples of specific peptides of the invention are LLSNPApY, ALLLSNPApYRLLA and AEDALNTV (amino acids 906 to 913 of DDR1). Complexes The complexes of the invention include the following: (a) an isolated complex comprising a DDR or an isoform or part thereof, and a collagen or part thereof; (b) an isolated complex comprising a DDR (e.g., DDR1b) and Shc or a PTB domain of Shc, and (c) an isolated complex comprising a DDR (e.g., DDR1) and a protein that contains a PDZ domain or a PDZ domain. The DDR in a complex can be oligomerized, can be conjugated to another protein and / or can be insolubilized. In addition, a collagen can be insolubilized in a complex of the invention. The complexes may comprise only the binding domains of the interacting molecules and other flanking sequences, as necessary, to maintain the activity of the complexes. Examples of complexes include DDR1 with collagen types I, II, III, IV and V, DDR2 with collagen types I and II, DDR1b and Shc, and DDR1 and a protein containing a PDZ domain. The invention also contemplates antibodies specific for complexes or peptides of the invention. The antibodies can be intact monoclonal or polyclonal antibodies and immunologically active fragments (e.g., a Fab or fragment of (Fab) 2), a heavy chain of antibodies and light chain of antibodies, a single molecule of chain Fv genetically treated (Ladner et al., U.S. Patent No. 4,946,778), or a chimeric antibody, for example, an antibody that contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies that include monoclonal and polyclonal antibodies, fragments and chimeras can be prepared using methods known to those skilled in the art. Antibodies specific for the complexes of the invention can be used to detect complexes in tissues and to determine their distribution in tissue. In vitro and in situ detection methods using the antibodies of the invention can be used to aid in the prognostic evaluation and / or diagnosis of conditions such as proliferative disorders. Antibodies specific for the complexes of the invention can also be used therapeutically as discussed in the present invention. Some genetic diseases may include mutations in the binding domain regions of the interaction molecules in the complexes of the invention. Therefore, if a complex of the invention is involved in a genetic disorder, it may be possible to use PCR to amplify DNA in the binding domains to quickly check whether a mutation is contained within one of the domains. The primers can be made corresponding to flanking regions of the domains and normal sequencing methods can be used to determine whether or not a mutation is present. This method does not require prior chromosome mapping of the affected gene and can save time by making obvious the sequencing of the entire gene encoding a defective protein. Substance Assessment and Identification The methods described herein can be used to identify substances that modulate a DDR tyrosine kinase-mediated signaling pathway and, in particular, that modulate the synthesis, degradation or remodeling of extracellular matrix. Novel substances are contemplated, which bind to molecules in the complexes of the invention, or bind to other molecules that interact with the molecules. Also contemplated are substances that interfere with, or increase, the interaction of the molecules in a complex of the invention or other proteins that interact with the molecules. Substances identified using the methods of the invention, include, but are not limited to, peptides such as peptides including fusion peptides formed with Ig, members of random peptide libraries and molecular banks derived from combination chemistry formed from amino acids of configuration D and / or L, phosphopeptides (including members of randomly or partially degenerated degenerate-directed phosphopeptide libraries), antibodies [eg, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single-chain antibodies, fragments, ( e.g., Fab, F (ab) 2 and fragments of Fab expression libraries and epitope binding fragments thereof) and small organic or inorganic molecules. The substance can be an endogenous physiological compound or it can be a natural or synthetic compound. The invention contemplates a method for evaluating a test substance for its ability to affect a DDR tyrosine kinase-mediated signaling pathway and in particular to modulate synthesis, degradation or remodeling of extracellular matrix by analyzing an agonist or antagonist (i.e., enhancer or inhibitor) of the binding of the molecules in a complex of the invention. The method generally involves preparing a reaction mixture containing the molecule in the complex and the test substance under conditions that allow the formation of complexes. The test substance can be added initially to the mixture, or it can be added subsequently to the addition of the molecules. The control reaction mixture is also prepared without the test substance or with a placebo. The formation of complexes is detected, and the formation of complexes in the control reaction, but not in the reaction mixture, indicates that the test substance interferes with the interaction of the molecules. The reactions can be carried out in the liquid phase, or the molecules or test substance can be immobilized as described herein. The substances identified using the methods of the invention can be isolated, cloned and sequenced, using conventional techniques. In one embodiment, a method is provided for identifying a substance that affects a signaling pathway mediated by the DDR tyrosine kinase-mediated signaling path, comprising the steps of: (a) reacting a collagen and at least one protein discoidin domain receptor tyrosine kinase, or an isoform or part of the protein, and a test substance, wherein the collagen and the discoid domain receptor tyrosine kinase protein are selected so that they bind to form a collagen-tyrosine kinase protein complex of discoidin domain receptor; and (b) compare it with a control in the absence of the substance to determine the effect of the substance.

In particular, a method is provided for identifying the substance that affects a DDR tyrosine kinase-mediated signaling pathway in a cell comprising: (a) reacting a collagen or part thereof, and at least one tyrosine protein kinase of the discoidin domain receptor, or an isoform or a part of the protein, and a test substance, wherein the collagen and the tyrosine kinase protein of the discoidin domain receptor are selected so that they bind to form a collagen-protein tyrosine kinase complex of discoidin domain receptor, under conditions that allow the formation of collagen-tyrosine kinase protein complexes of discoidin domain receptor, and (b) analyze the complexes, for the free substance, for collagens not complexed or for the activation of the protein. The conditions that allow the formation of the complexes can be selected considering the factors such as the nature and quantities of the substance and the ligand. The complexes, free substance or non-complexed ligand, can be isolated by conventional isolation techniques, for example, salt precipitation, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination or combinations thereof. . To facilitate the analysis of the components, an antibody against the substance, or a labeled collagen, or a labeled substance can be used. The antibodies, receptor protein or receptor substance, can be labeled with a detectable substance as described above. Activation of the protein can be analyzed by measuring tyrosine phosphorylation of the protein, oligomerization of the protein, binding of a PTB domain to the juxtamembrane domain of the tyrosine kinase protein of the discoidin domain receptor, or it can be analyzed by a biological effect on the cell, such as the inhibition or stimulation of proliferation, differentiation or migration. It will be understood that agonists and antagonists, i.e., inhibitors and enhancers that can be analyzed using the methods of the invention can act on one or more of the binding sites on the interaction molecules of the complexes including the agonist binding sites. , competitive antagonist binding sites, non-competitive antagonist binding sites or alloestheric sites. The invention also makes it possible to screen antagonists that inhibit the effects of an agonist from the interaction of molecules in a complex of the invention. Therefore, the invention can be used to analyze a compound that competes for the same binding site of a molecule in a complex of the invention. The invention also contemplates methods for identifying novel compounds that bind to proteins that interact with a molecule of a complex of the invention thus affecting a DDR signaling pathway. Protein-protein interactions can be identified using conventional methods such as co-immunoprecipitation, entanglement and co-purification through gradients or chromatographic columns. Methods that result in the simultaneous identification of genes encoding proteins by interacting with a molecule can also be employed. These methods include testing expression banks with labeled molecules. Additionally, crystallographic studies of X-rays can be used as means to evaluate interactions with substances and molecules. For example, recombinant molecules purified in a complex of the invention when crystallized in a suitable form accept the detection of intra-molecular interactions by X-ray crystallography. Spectroscopy can also be used to detect interactions and in particular, instrumentation can be used. of Q-TOF. Two hybrid systems can also be used to detect protein interactions in vivo. In general, plasmids encoding two hybrid proteins are constructed. A first hybrid protein consists of the DNA binding domain of a transcription activator protein fused to a molecule in a complex of the invention and the second hybrid protein consists of the activating domain of the transcription activating protein fused to an unknown protein encoded by a CDNA that has recombined in the plasmid as part of a cDNA library. The plasmids are transformed into a yeast strain (e.g., S. cerevisiae) which contains a reporter gene (e.g., lacZ, luciferase, alkaline phosphatase and horseradish peroxidase) whose regulatory region contains the binding site of the transcription activator. Hybrid proteins alone can not activate transcription of the reporter gene. However, the interaction of the two hybrid proteins reconstitutes the functional activator protein and results in the expression of the reporter gene, which is detected by an analysis for the reporter gene product. It will be appreciated that the fusion proteins and recombinant proteins can be used in the methods described above. It will also be appreciated that the complexes of the invention can be reconstituted in vitro using recombinant molecules and the effect of a test substance can be evaluated in the reconstituted system. Suitable reagents for applying the methods of the invention for the purpose of evaluating substances and compounds that affect or modulate a DDR receptor tyrosine kinase-mediated signaling path can be packaged in convenient equipment that provides the necessary materials packaged in suitable containers. The equipment may also include suitable supports useful for carrying out the methods of the invention. Compositions and Treatments The methods of the invention mentioned above, can be used to identify substances that affect a tyrosine kinase signaling pathway of discoidin domain receptors in a cell, particularly those involved in proliferation, metastasis or synthesis, degradation or remodeling of extracellular matrix. It will be appreciated that such substances will be useful as pharmaceuticals to modulate proliferation, metastasis, and / or synthesis, degradation or remodeling of extracellular matrix. The ability of the substances identified using the methods of the invention to affect proliferation and / or metastasis and other cellular processes can be confirmed in animal models. For example, the MDAY-D2 murine model can be used to confirm the utility of a substance as an anti-proliferative or anti-mestastatic agent. The invention provides a method for treating or avoiding a condition involving a discoidine domain receptor tyrosine kinase-mediated signaling pathway, which method comprises administering to a patient in need thereof an amount of a substance that is effective to interfere with (i.e., inhibiting or improving) the signaling pathway wherein the substance is (a) a discoidine domain receptor tyrosine kinase or part thereof; (b) a collagen or part thereof; (b) an isolated complex comprising a DDR or a part thereof and a collagen or part thereof; (c) peptides derived from the binding domain of a DDR that interacts with a collagen or part of a collagen, or with Shc or a protein having a PDZ domain; (d) a molecule derived from the collagen binding domain that interacts with a DDR or a part thereof, (e) antibodies specific for the peptide complexes: or (f) a substance first identified by (i) reacting a collagen, and at least one discoidine domain receptor tyrosine kinase protein, or an isoform or a part of the protein, and the test substance, wherein the collagen and the discoid domain receptor tyrosine kinase protein are selected so that they bind to form a collagen-protein tyrosine kinase complex of discoidin domain receptor; and (ii) compare it with a control in the absence of the substance to determine the effect of the substance. The invention also relates to a pharmaceutical composition comprising (a) a discoidine domain receptor tyrosine kinase or part thereof; (b) a collagen or part thereof, preferably a carbohydrate moiety; (b) an isolated complex comprising a DDR or a part thereof and a collagen or part thereof; (c) peptides derived from the binding domain of a DDR that interacts with a collagen or part of a collagen, or with Shc or a protein containing a PDZ domain; (d) a molecule derived from the collagen binding domain that interacts with a DDR or part thereof; (e) antibodies specific for the complexes, peptides and molecules of (b), (c) or (d); or, (f) a substance first identified by a method of the invention, in an amount effective to affect a tyrosine kinase-mediated signaling pathway of the discoidin domain receptor and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment of the invention, the composition may comprise an extracellular domain of a tyrosine kinase from the discoidin domain or the portion of the extracellular domain that binds to a repeat region of GXY or a carbohydrate portion of a collagen, or oligomers or imitations of them. The method and compositions of the invention can be used to alter proliferation or metastasis in a mammal, treat conditions involving structural or functional dysregulation of collagens such as Sickler's syndrome, treat conditions involving defects in collagens such as osteogenesis imperfecta, treat conditions that require modulation of extracellular matrix degradation or remodeling, improve wound healing and improve the formation of cartilage or bone. The compositions of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo. By "biologically compatible form suitable for administration in vivo" is meant a form of the protein that will be administered in which any toxic effects are exceeded by the therapeutic effects of the protein. The term subject is intended to include mammals. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. The administration of a therapeutically effective amount of the pharmaceutical compositions of the present invention is defined as an effective amount, at the doses and for periods of time necessary to achieve the desired result. For example, a therapeutically effective amount of an active substance may vary according to factors such as condition, age, sex and weight of the individual. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies in the therapeutic situation. The active compound (v.gr., protein), can be administered in a convenient form such as by injection (subcutaneous, intravenous, etc.), oral administration inhalation, transdermal application or rectal administration. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions that can inactivate the compound. The pharmaceutical compositions of the invention can be for local oral administration, inhalant or intracerebral administration. Preferably the pharmaceutical compositions of the invention are administered directly to the peripheral or central nervous system, for example by intracerebral administration. The pharmaceutical composition of the invention can be administered to a subject in an appropriate vehicle or diluent, co-administered with enzyme inhibitors or in an appropriate vehicle such as microporous beads or solid beads or liposomes. The term "pharmaceutically acceptable carrier", as used herein, is intended to include diluents such as saline solution and aqueous buffer solutions. Liposomes include emulsions of water in oil in water, as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27). The active compound can also be administered parenterally or intraperitoneally. Dispersions in glycerol, liquid polyethylene glycols and mixtures thereof and in oils can also be prepared. Under normal conditions of storage and use, these preparations may contain a preservative or prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where they are soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that they can be injected with a syringe. It can be stable under manufacturing and storage conditions and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be achieved by several antibacterial and antifungal agents, for example; parabens, chlorobutany, phenol, ascorbic acid, trimerosal and the like. In many cases, it will preferably be to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be carried out by including in the composition an agent retarding absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one, or a combination of, ingredients listed above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient (e.g., antibody) plus any additional desired ingredients of a previously sterile filtered solution thereof. When the active compound is adequately protected, as described above, the composition can be administered orally, for example, with an inert diluent or an edible assimilable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, sotonic and absorption retardation agents, and the like. The use of said agent means for pharmaceutically active substrates are well known in the art. Except that any conventional media or agent is compatible with the active compound, the use thereof in therapeutic compositions is contemplated. Also, supplementally active compounds can be incorporated into the compositions. It is also contemplated that the pharmaceutical compositions of the invention may comprise cells or viruses, preferably retroviral vectors, transformed with nucleic acid molecules encoding a purified or isolated discoidin domain receptor tyrosine kinase protein, or an isoform or part of the protein, a peptide of the invention, an antibody to a complex of the invention, or a substance identified using the methods of the invention, so as to express the protein, isoform or a part of the protein, preferably the extracellular domain, or substance in vivo. Suitable viral vectors for use in the present invention are well known in the art, including recombinant vaccine viral vectors (U.S. Patent Nos. 4,603,112 and 4,769,330), recombinant pox virus vectors (PCT Publication No. WO 89/01973 ) and preferably, retroviral vectors ("Recombinant Retrovirures with Arnhothotic and Ecotropic Host Ranges," PCT Publication No. WO 90/02806; "Retroviral Packaging Cell Lines and Processes of Using Same," PCT Publication No. WO 89/07150; and "Antisense RNA for Treatment of Retroviral Disease States," PCT Publication No. WO 87/03451). Compositions containing cells or viruses can be introduced directly into a subject. Nucleic acid molecules that encode a DDR or an isoform or part of the protein, a peptide of the invention, an antibody to a complex of the invention, or a substance identified using the methods of the invention, can also be introduced into a subject using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of nucleic acids in liposomes. They can also be supplied in the form of an aerosol or by washing. The following non-limiting examples are illustrative of the present invention. EXAMPLES Example 1 The inventors of the present have shown that the activated b-isoform of DDR1 (MCK 10), but not the isoform-a, is associated with Shc in vivo and binds to the PTB domain of Shc in vitro. This interaction is blocked by a phosphopeptide containing the LLSNPAY motif, which is found specifically in the juxtamembrane insert of the isoform-b. These results suggest that an alternative separation directly regulates the ability of DDR1 to interact with Shc by controlling the presence or absence of a PTB binding site. EXPERIMENTAL PROCEDURES Materials and cell lines - Cloning and expression of functional Shc domains have been described as glutathione S-transferase (GST) fusion proteins (13). In summary, PTB domain fusion comprises amino acids 1-225 for human p52 Shc and the construction of SH2 domains extends with amino acids 366-473. The expression vectors of DDR1 (also known as MCK10) have been previously described (1). The phosphopeptide ALLLSNPApYRLLA was synthesized using a Model 431A instrument from Applied Biosystems. Antibodies to Shc arose against a GST-SH2 fusion protein from Shc. Other antibodies were also purchased from Santa Cruz, Inc. (4G10 monoclonal antiphosphotyrosine antibody, and polyclonal rabbit serum against amino acid 894-913 of MCK10). Human embryonic kidney fibroblast 293 cells were obtained from American Tisusue Culture Collection (ATCC CRL 1573) and cultured under recommended conditions. Temporary Expression - The semi-effluent 293 cells were transfected by calcium-phosphate precipitation with a cytomegalovirus-based expression vector containing the a or b isoform of DDR1 (1). Sixteen hours later, the cells were transferred to a serum-free medium for an additional 24 hours, before lysis, the cells were stimulated with 1 mM orthovanadate (pH 10.0) for 90 minutes. For in vivo labeling of phosphorylated proteins, cells were developed with 0.5 mCi ml-1 inorganic phosphate [32 P] (NEN) for 4 hours. Immunoprecipitation, Western blot analysis and kinase analysis - Transfected 293 cells were used in NP40 buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% NP40, 10 mM of NaF, 1 mM of phenylmethylsulfonyl fluoride, 1 mM of orthovanadate and 10 μgml of aprotinin The cell lysates were centrifuged 10 minutes at 4 ° C and 13000 rpm and the aliquots of the supernatant were subjected to SDS-PAGE or were further analyzed by immunosuppression with specific antibodies for 3 hours at 4 ° C on a rotating wheel The immuno-complex was washed three times with NP40 buffer and analyzed by SDS-PAGE The proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) and immunoanalyzed with antibodies diluted 1: 500 in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.05% Triton X-100, 0.25% gelatin overnight. Western blots were developed using an antibody A secondary goat anti-protein coupled with horseradish peroxidase (Biorad) and increased chemiluminescence (Amersham). To retest, the membrane was separated in 70 mM Tris-HCl (pH 6.8), 2% SDS, 0.1% β-mechaptoethanol at 55 ° C for 30 minutes. For in vitro kinase assays, the immunoprecipitates were washed twice in 40 mM HEPES (pH 7.5), 20 mM MgCl 2, 2 mM MnCl 2, 10 μM orthovanadate, 5 μM d.ATP and incubated with μCu [ ? -32P] -ATP at 30 ° C for 20 minutes. The products of the kinase reaction were monitored by SDS-PAGE, transferred to an immobilon membrane (NEN) and autoradiography. Phosphopeptide Mapping - The proteins were cut from the membrane and labeled with Np-tosyl-L-lysine-chloromethyl ketone treated with trypsin (Sigma) for 16 hours, oxidized with performic acid for 1 hour, desalted in steps and concentrated by lyophilization. Equal cpm of radiolabeled peptides were separated in two dimensions, using electrophoresis in thin-film cellulose (CLF) plates (100 mm, Merck) with an HTLE 7000 apparatus (CBS Inc., Del Mar, CA, USA) in a buffer solution. of pH 1.9 (2.5% formic acid, 7.5% acidic acid) in the first dimension, and ascending chromatography (37.5% n-butanol, 25% pyridine, 7.5% acidic acid, 305 water) in the second dimension. The CLF plates were dried and exposed to X-ray film at -70 ° C using an intensification screen. Resonance of surface plasmons - experiments were performed with a BIAcore instrument (Pharmacia). A phosphopeptide corresponding to the sequence of NPXY in the polyomavirus intermediate T antigen (mT) (LSLLSNPTpYSVMRSK) was absorbed at the surface of a biosensor microcircuit. The binding of GST-Shc domain fusion protein of soluble PTB to the mT peptide was measured in the presence or absence of competing soluble phosphopeptides based on sequences around Tyr 513 of DDR1b or Tyr 490 of NGFR (HIIENPQpYFSD) (21) . RESULTS AND DISCUSSION To determine if DDR1 RTK interacts with Shc proteins, 293 cells of human embryonic kidney fibroblasts were transfected with expression vectors encoding the DDR1a and DDR1b isoforms. Activation of receptor kinase and autophosphorylation were achieved by treating the cells with 1 mM of orthovanadate 90 minutes before lysis. Orthovanadate treatment had previously been shown to stimulate DDR1 tyrosine phosphorylation in vivo (1). The Shc proteins were immunoprecipitated from lysates of these cells and the immunoprecipitates were immunographed with antiphosphotyrosine antibodies. A phosphorylated protein protein of approximately 125 kDa was co-precipitated with Shc from transfected DDR1b cells (Figure 1A). Retesting the analysis with the anti-DDR1 antibody showed that 125 kDa of Shc-associated protein is DDR1b (Figure 1B). An association between DDR1 and Shc has been observed in 293 cells, despite the fact that DDR1a itself was phosphorylated with tyrosine to an extent comparable to DDR1b (Figure 1D). To investigate in more detail the interaction of Shc with DDR1b. the PTB domains of Shc and SH "are expressed as GST fusion proteins These GST fusions were immobilized and incubated with lysates of 293 cells, which have been transfected with DDR1 or DDR1b soformas and incubated with orthovanadate to induce receptor tyrosine phosphorylation The DDR1 isoform b of the orthovanadate-treated cells specifically associated with the PTB domain of Shc in vitro, while no binding of the isoform b from unstimulated cells was observed (Figure 1E). In contrast, the PTB domain of Shc did not bind to DDR1a of the cells stimulated with orthovanadate, no binding of SH2 domain from Shc to DDR1a or DDR1b phosphorylated with tyrosine was detected.These results suggest that the PTB domain of Shc recognizes an autophosphorylation site that was specified for DDR1b and is absent from DDR1a, which agrees with the presence of the LLSNPAY motif in the juxtamembrane insert of DDR1b. The alternative apparently regulates the presence or absence of a PTB binding site of Shc in DDR1, and thus controls the ability of DDR1 to interact with this target downstream in vivo. The results presented above arise from the possibility that the PTB domain of Shc binds to phosphorylated Tyr 513, which lies within the LLSNPAY motif in the juxtamembrane insert of DDR1b. To test whether the phosphorylation of Tyr 513 can form a PTB binding site, a 14-mer phosphopeptide was synthesized with the sequence ALLLSNPApYRLLLA, corresponding to residues 505-518 of DDR1b. The ability of this phosphopeptide to bind to the PTB domain of Shc was analyzed using a proficiency analysis, in which the ability of the fusion protein to bind tyrosine-phosphorylated DDR1b to the lysates of transfected 293 cells was measured in the presence of increasing amounts of Tyr 513 phosphopeptide. 100 nM phosphopeptide inhibited the binding of the PTB domain from Shc to DDR1b and complete inhibition was achieved at a phosphopeptide concentration of 2 μM (Figure 2A). These results suggest that the phosphorylation of Tyr 513 creates a strong binding site for the PTB domain of Shc. To quantify this interaction more precisely, surface plasmon resonance technology was used in which soluble DDR1b phosphopeptides were used to inhibit the binding of the Shc PTB fusion protein to a polyomavirus medium T antigen phosphopeptide, immobilized in a biosensor microcircuit (21) As shown in Figure 2B, a concentration of approximately 800 nM phosphopeptide of DDR1b, which contains the sequence around Tyr 513, inhibits the binding of the PTB domain of Shc to the phospholipid of medium T antigen by 50% (ICso). These findings contrast with an IC5o value of approximately 100 nM found when a phosphopeptide around the NPXY motif of the NGF receptor was used in a comparable experiment (Figure 2B), but they are similar to the IC5o of the average T aptigene phosphopeptide (21). The experimental data analyze the interaction between the PTB domain of Shc and the phospho-peptide peptides, have emphasized the N-terminal importance of residues specific to the site of tyrosine phosphorylation. Phosphopeptides lacking Asn at position 3 are not effective for binding to the PTB domain of Shc, whereas Pro at position 2 seems significant, but not essential for binding (21-25). a substantial decrease was also observed in affinity to exchange the hydrophobic residue in position 5 to a polar amino acid, indicating that the hydrophobic residues bulky upstream sequence NPXY matrix contribute to recognition domain PTB Shc (13 , 18). The structural analysis of the PTB domain of Shc bound to the phosphopeptide of the NGF receptor, identified a hydrophobic bag that adapts residue 5, explaining the preference for a hydrophobic residue in this position (26). The sequence preceding Tyr 513 of DDR1b, exhibits a triplet of leucines at positions 5, 6 and 7, as well as an alanine at position 8, which agrees with the finding of this site binds to the PTB domain of Shc with high affinity This sequence is equated to the consensus not only for the PTB binding of Shc, but also to the interaction with the PTB domain of the insulin receptor substrate (IRS-1), which requires hydrophobic residues at positions 6 to 8 (27, 28 ), IRS-1 can be associated with DDR1b. Interestingly, three leucines have also been found in the +2 to +4 C-terminal positions for the NPXY motif in DDR1b. The importance of this highly symmetric sequence is not clear. Perhaps, the formation of an extended cycle, initiated by the turn b in the proline, is favored by these two blocks of triple leucines. The insert sequence of juxtamembrane is unique to isoform DDR1b contains two potential phosphorylation sites tyrosine, Tyr 513 and 520. The detailed experiments above show that phosphorylation of Tyr 513 can form a strong bond site for the PTB domain of Shc. To monitor the degree of phosphorylation of DDR1 isoforms, 293 cells were transfected with expression vectors encoding the a- or b isoforms of DDR1, and the transfected cells were metabolically labeled with [32P] -orfofosfato. After stimulation with orthovanadate for 90 minutes, the isoforms of DDR1a and DDR1b were immunoprecipitated from the cell lysates, purified by SDS-PAGE and subjected to tryptic digestion. The resulting tryptic phosphopeptides were separated into two dimensions. The two-dimensional phosphopeptide map of the receptor isoform showed complex patterns of spots, indicating phosphorylation at multiple sites. (Figures 3A and B). The composition of both maps indicated that all phosphopeptides derived DDR1a co-migrated with peptides DDR1b, while points c, e, i and I on the map tryptic phosphopeptides DDR1b were absent from the digestion DDR1a (Figures 3A , B and C). Among these specific phosphopeptides for DDR1b, point 1 was the most intense suggesting that it was derived from a main phosphorylation site in the cytoplasmic region of the receptor. This result is consistent with the possibility that the insert separated alternatively from DDR1b provides novel autophosphorylation sites, which are not found in DDR1a. The presence of multiple phosphopeptides specific for the isoform-b could be explained by the phosphorylation of multiple sites within the juxtamembrane insert, or by the production of different peptides containing a single phosphorylation site. To follow these results, anti-DDR1 immunoprecipitations were subjected to in vitro kinase reactions to induce autophosphorylation of the receptor. Tryptic phosphopeptide maps in DDR1a in vitro phosphorylated and DDR1b (Figures 3D and E) or a mixture of several isoforms (Figure 3F) showed that all phosphopeptides isoform-a were also present in the digestion of isoform-b, but also showed that the longer isoform had additional phosphopeptides. Two of the phosphopeptides specific for the isoform-b, the main point I and the minor point e, corresponded to single phosphopeptides for the isoform-b isolated from orthovanadate-treated cells labeled with 32P (Figures 3B and E). The kinase reaction in vitro also resulted in the phosphorylation of a DDR1b-specific peptide (p-point), which was not observed in labeled protein digestions in vivo, whereas the phosphorylation of peptide c and i only occurred under in vivo conditions. These data suggest that DDR1b contains at least one novel autophosphorylation site compared to DDR1a, which is phosphorylated in both an autokinase reaction in vitro and in cells expressing DDR1b following orthovanadate treatment. These results are compatible with the suggestion that the juxtamembrane insert of DDR1b modify the interaction of DDR1 with its targets, specifically by providing a coupling site for the PTB domain of Shc. Example 2 Up to now, a binding of DDR1 ligands to DDR2, or any peptide or protein, which could trigger the intrinsic tyrosine kinase activity of the DDRs has not been reported. Certain members of the collagen family have been identified as ligands for DDR1 and DDR2. It was found that collagen interacts directly with extracellular domains and collagen produced tyrosine phosphorylation of DDR in a manner that depends on time and concentration. Originally, it was found that a commercially available preparation of extracellular matrix proteins, called Matrigel, induces tyrosine phosphorylation of DDR1. When testing several components of Matrigel, it was identified that type IV collagen has ligand activity. Subsequently, almost all types of collagen commercially available from various organs and species (humans, rats, mice, bovines) were tested in the ligand analysis. Collagen types I, III, IV and V, in the same way was a good ligand for DDR1. Collagen types I and III are highly potent ligands for DDR2, collagen types II and V showed moderate and type IV showed no activity. When type i of collagen was added in the tissue culture medium of human embryonic kidney fibroblast 293 cells transfected with DDR1 or DDR2 cDNA, the activity of ligands was detected at a minimum concentration of ca. 250 ng of collagen per ml of the medium. Maximum tyrosine phosphorylation was observed 90 minutes after stimulation with 10 μg / ml type I collagen. It was previously found that T-47D, a line of human mammary carcinoma cells and A431, a human squamous cell carcinoma cell line , exhibit endogenous expression of DDR1. After stimulation with collagen type 1, the DDR1 protein was extracted by immunoprecipitation. In these immunoprecipitates, a significant increase in tyrosine phosphorylation of DDR1 was detected after the stimulation of collagen using Western analysis or in vitro kinase analysis techniques. The activity of the ligand was eliminated after the pre-treatment of type I collagen with collagenase, but not after the treatment with pepsin. After the removal of the portions of the type I carbohydrate portion of collagen using periodate as the oxidant, the activity of ligands for DDR2 was dramatically reduced. These data suggest that collagen glycosylation is essential for the activation of DDR. Figures 4, 5 and 6 and 12C illustrate the experimental results. The collagens are post-translationally bitten extensively, e.g., hydroxylated, glycosylated and disulfohydrilled. These modifications may be important or essential for the activity of the ligand. In the progress of carcinogenesis and metastasis, the initial steps in the growth of tumors is still a mystery.

Especially in metastasis, it is not clear what induces the separation of cells from a primary tumor, what factors are necessary for these cells to break the barrier of the basement membrane and connective tissues that surround the tumor and what factors are involved in the union at the site of metastasis. To initiate tumor invasion and metastatic growth, healthy membranes have to be separated from the base. The matrix metalloprotein family (MMP) is known to be involved in this process. The activation of DDR can be related to the MMP function. Because DDR1 is expressed on the surface of tumor cells, DDR2 is expressed in the surrounding stromal tissue and both are able to bind to the collagens, these receptors may be involved in tumor growth and metastasis. It is known that a pleiotropy of different human diseases is linked to the structural modification or functional dysregulation of collagens. The genetic basis of many hereditary connective tissue disorders is not resolved. Mutations in the ligand binding domain, in the kinase domain or in any other position of the DDR1 and DDR2 genes, can cause this class of disorders. DNA samples from patients with connective tissue disorders can be analyzed using Southern blot analysis and PCR technology to identify genetic mutations in the DDR1 and DDR2 genes. The human site for DDR1 is 6p21.3, for DDR2 it is 1q21-123. A database investigation indicated several diseases, which are mapped close to these sites and shown in bone, skin, or cartilage defects. A primary candidate close to the DDR1 site is Sickler's syndrome. Because the ubiquitous expression of collagens, other non-hereditary diseases could also be potentially linked to the poor function of DDR1 and DDR2. Example 3 The following are details for the experiments discussed in Example 2 which demonstrate that the collagen members are ligands for DDR. Reactive Methods, Cell Lines and Plasmids Matrigel was obtained from Collaborative Biomedical Products (Bedford, MA). All types of collagen and other reagents were purchased from Sigma (St. Louis, MO). Human embryonic kidney fibroblast 293 cells, human mammary carcinoma T-47D cells and human HT-fibrobrosarcoma HT 1080 cells were obtained from the American Tissue Cultive Collection and cultured under recommended conditions. The expression of the PTB domain of Shc as a fusion protein of bacterial glutathione S-transferase has been previously published (van der Geer et al., 1996). The DDR expression vectors were described before (1). The parts of the extracellular domains of DDR1 (amino acids 29-189) and DDR2 (amino acids 28-367) were cloned into the pET30a vector (Novagen, Madison, Wi) in frame with His-tag and expressed in low E. coli. the T7 promoter. The proteins were purified by NI affinity chromatography (Qiagen) and used to raise antibodies. The ALLLSNPAYRLLLATYARC peptide was used to raise antibodies against the b-isoform of DDR1 (amino acids 505-523). Antibodies to DDR1 (amino acids 894-913) and the insulin receptor (amino acids 1365-1382k) were purchased from Santa Cruz, Inc. (Santa Cruz, CA). The monoclonal antiphosphotyrosine antibody 4G10 was from Upstate Biotechnology, Inc. (Lake Placid, NY). Collagen Purification Adult mice were sacrificed and the collagen-containing tendon was excised from its tails using sterile forceps. The tendon was incubated in 500 mM acetic acid on a shaker at 4 ° C overnight. No non-soluble material was removed by centrifugation. The soluble material was dialyzed against 10 mM acetic acid. The purity and integrity of collagen was assessed by SDS-PAGE. Type IV collagen was extracted from Matrigel with a buffer solution containing 2 M guanidinium hydrochloride, 50 mM Tris (pH 7.5), 2 mM DTT (Kleinman et al., 1982). The soluble material was dialyzed against 500 mM acetic acid. Alternatively, matrigel was extracted with 500 mM acetic acid, 1% pepsin and soluble collagen dialyzed against 500 mM acetic acid (Timpl et al., 1979). Temporal Expression in 293 Cells and Western Blot Analysis Semi-confluent 293 cells were transfected by calcium phosphate precipitation with an expression vector based on cytomegalovirus. Sixteen hours later, the cells were transferred to serum-free medium for another 24 hours. Cells were stimulated with 10 μg / ml of collagen for 90 minutes and used with 1% Triton-X 100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl 2, 5 mM EGTA, 5 mM EDTA, 10% glycerol, 10 mM NaF, 1 mM phenylmethisulfonyl fluoride (PMSF), 1 mM Na-orthovanadate, 10 μg / ml aprotinin. The cell lysates were centrifuged 10 minutes at 4 ° C and 13,000 rpm and the aliquots of the supernatant were subjected to SDS-PAGE or further analyzed by immunoprecipitation with specific antibodies for 3 hours at 4 ° C on a rotating wheel. The immunocomplex was washed three times with 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton, 10% glycerol and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose membrane (Schleicher &; Schuell) and immunographed with antibodies diluted 1: 500 in 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.05% gelatin overnight. The Western plots were developed using secondary antibody coupled with horseradish peroxidase (Biorad) and enhanced chemiluminescence (Amersham). To retest, the membrane was separated in 70 mM Tris (pH 6.8), 2% SDS, 0.1% β-mercaptoethanol at 55 ° C for 30 minutes. For in vitro kinase assays, immunoprecipitates were washed twice with 40 mM HEPES (pH 7.5), 20 mM MgCl2, 2 mM MnCl2, 10 μM dATP and incubated with 5 μCi [? - 32 P] ATP at 30 ° C for 20 minutes. The products of the kinase reaction were monitored by SDS-PAGE and autoradiography. Binding assay 100 μg of mouse type I collagen was iodinated by the N-chloro-benzenesulfonamide method using 1 mCi of Na [125 l] (NEN) and an iodine bead (Pierce). The labeled collagen recovered by chromatography with Sephadex G50 (Pharmacia) was found to have a specific activity of 5 x 10s cpm / μg. Binding to the DDR receptors was measured by transfection of 293 cells in 24-well plates with expression plasmids for DDR1a, DDR1b or control plasmid. Cells were washed with ice-cold PBS supplemented with 1% BSA and 10 mM glucose and incubated on ice with increasing concentrations of radiolabelled collagen for 2 hours. The cells were washed three times with binding buffer and used with 100 μl of 200 mM NaOH, 1% SDS. The lysates were neutralized with 200 mM HCl and counted in a gamma scintillation counter (LKB 1282). Each value is the average of three measurements. Deglycosylation of collagen For deglycosylation, mouse type 1 collagen was incubated with 10 mM sodium m-periodate reaction prepared for 20 minutes at room temperature in the dark. The excess periodate was removed by adding 20 mM of sodium bisulfite. Collagen was dialyzed against 10 mM acetic acid overnight. Analysis for Expression of MMP-1 The full length DNA of DDR2 was stably expressed in 1080 HT cells using a retroviral expression (pLXSN). Neomycin resistant clones were tested for the expression of DDR2. Parenteral and DDR2 over-expression cells were cultured in serum-free medium and stimulated with 10 μg / ml of type I collagen for several periods. The conditioned medium was concentrated 20 times and analyzed for the presence of MMP-1 by Western analysis with the monoclonal antibody 41-IE5 (Oncogene Research Products, Cambridge, MA). Resonance of surface plasmons Experiments were performed with a BIAcore instrument (Pharmacia). A phosphopeptide corresponding to the sequence of NPXY in the polyomavirus medium T antigen (LSLLSNPTpYSVMRSK) was absorbed to the surface of the biosensor microcircuit. The binding of the GST-PTB domain fusion protein from Shc to the mean T peptide was measured in the presence or absence of competent soluble phosphopeptides based on the sequences around tyrosine 513 of DDR1b (ALLLSNPApYRLLLA) or tyrosine 490 of the NGF receptor ( HIIENPQpYFSD). CD spectroscopy The CD spectrum of mouse type I collagen was recorded with a AVIV 60DS spectropolarimeter. The melting curve was monitored at 221 nm. while the temperature was increased at a rate of 1 ° C / min. RESULTS: Ligand activity was found for DDR1 in matrigel To identify possible sources of the DDR1 ligand, an in vivo screening system was stabilized. 293 of human embryonic kidney fibroblast cells were temporarily transfected with expression plasmids encoding DDR1b. Cells with decreased growth were stimulated with several agents and monitored for receptor activation by anti-phosphotyrosine Western analysis of total cell lysates. An activity that strongly induced tyrosine phosphorylation of DDR1b was detected in matrigel, a commercially available preparation of mouse sarcoma-based membrane proteins from Engelbreth-Holm-Swarm (EHS). The increase in autophosphorylation of DDR1b depends on the concentration of matrigel added to the cells (Figure 7A). Maximal stimulation was observed with 250 μl of matrigel per ml of tissue culture medium and an approximately twice increase of receptor autophosphorylation was observed with 10 μl of matrigel per ml of the culture medium. Matrigel has been used repeatedly as a source of purification and characterization of extracellular matrix proteins. Therefore, the isolated matrix proteins fibronectin, laminin, SPARC, perlecan and collagen were tested for their ability to stimulate tyrosine phosphorylation of DDR1b. Of these, neither fibronectin nor laminin (Figure 7C, lines c and d), nor SPARC nor perlacan (data not shown) were able to induce autophosphorylation of DDR1b. To test type IV collagen this matrix protein was isolated from matrigel after the guanidinium hydrochloride extraction protocol of Kelinman et al. (1982) and used in the autophosphorylation analysis of DDR1b in vivo. This preparation of purified soluble collagen induced a greater increase in tyrosine phosphorylation of DDR1b than in Matrigel (Figure 7C, lines b and e). A similar result was obtained using collagen that was purified from matrigel using an alternative method based on extraction with acetic acid and digestion of pepsin (Timpl et al., 1979, Figure 7C, line f). In addition, type IV collagen commercially available from either EHS tumor or human placenta, induced tyrosine phosphorylation of DDR1b to a degree similar to matrigel purified collagen (Figure 7C, lines g and h). Collagen induces tyrosine phosphorylation of both DDR tyrosine kinase receptors To obtain collagen from a normal tissue, the collagen containing type I tendon was mechanically isolated from the tails of adult mice and the collagen was solubilized by extraction with 500 mM of acetic acid. This collagen preparation showed the same substantial activation of tyrosine phosphorylation of DDR1 as type IV collagen isolated from matrigel (Figure 8A). Using a concentration of 10 μg of collagen per ml of tissue culture medium in this analysis, the purified collagen induced similar levels of tyrosine phosphorylation on isoform a and b of DDR1 (Figure 8A). Soluble purified mouse tail collagen was also used to stimulate 293 cells transfected with cDNA for DDR2, the second member of the discoid domain subclass of RTK. Collagen induced an increase in tyrosine phosphorylation of DDR2 similar to that observed for DDR1 (Figure 8A). In contrast, 293 cells transfected with expression plasmids for the insulin receptor or the EGF receptor showed no increase in tyrosine phosphorylation of the receptor after collagen treatment (Figure 8 and data not shown). Kinetics of DDR1 and DDR2 activation by collagen Tyrosine receptor kinases are usually rapidly autophosphorylated by stimulation of an activating ligand. For example, an increase in autophosphorylation of receptors for EGF or normal insulin takes place in a matter of seconds. Maximal activation is usually achieved within a few minutes after stimulation and the receptor is then downregulated in a common manner through a variety of mechanisms, including internalization and receptor proteolysis. However, there are exceptions to this rule; for example, activation of the Eph family receptors by their cell surface ligands is a more recurrent aspect, requiring maximum receptor phosphorylation for at least one hour (Gale et al., 1996; Holland et al., 1997). . The kinetics of the activation of DDR1 and DDR2 by collagen was investigated using transfected 293 cells and no significant increase in autophosphorylation was found after 2 minutes of stimulation with 10 μg / ml of type 1 mouse collagen (approximately 30 nM) (Figure 9A and B). Activation of the receptor was delayed and arrived from 9 minutes to 2 hours after the stimulation. The tyrosine phosphorylation of both DDR1 and DDR2 was sustained for up to 18 hours after the addition of soluble collagen (Figure 9A and B). The isoforms of DDR1 and DDR2 showed identical activation kinetics. To investigate the effects of collagen on cells expressing endogenous DDR tyrosine kinases, the mammary carcinoma T-47D cell line was used. Previous results have shown that DDR1 is expressed lightly in human carcinoma cell lines, in particular, breast cancer cell lines BT-20, MDA-MB-175 and T-47D (1) (Pérez et al. nineteen ninety six). As shown in Figure 9C, DDR1b was inducibly tyrosine phosphorylated after incubation of 5-47D cells with collagen. The time course of tyrosine phosphorylation of endogenous DDR1b in T-47D cells was slower than in 293 cells, with maximal activation achieved only after stimulation for 18 hours. To analyze the effect of collagen on DDR1b autokinase activity, the receptor was immunoprecipitated from overexpression of 293 or T-47D cells after several periods of stimulation. The immunoprecipitates were subjected to in vitro kinase reactions and the incorporation of [32 P] -phosphate in the receptor was monitored by autoradiography. In each case, the degree of receptor kinase activity in vitro reflected the tyrosine phosphorylation status in vivo (Figure 9E and F). While DDR1b, which was over-expressed in 293 cells, reached maximal autokinase activity in vitro after stimulation for 2 hours, endogenous DDR1b of T-47D showed the highest activity after incubation overnight with collagen. Differential activation of DDR by various types of collagen To further investigate the role of different types of collagen in the activation of DDR1 and DDR2, collagen of types I, II, III and V purified from human placenta and collagen type were obtained. II of tracheal cartilage of bovines. To test its ability to induce receptor tyrosine phosphorylation, 293 cells were used that overexpress DDR1 or DDR2. After stimulation with 10 μg / ml of collagen for 90 minutes, DDR1 showed increased tyrosine phosphorylation with all types of collagen treated (Figure 10A). In contrast, DDR2 was highly activated only by two types of collagen types I and III, while collagen types II and V showed moderate activity (Figure 10C). Surprisingly, type IV collagen, which was originally identified as the ligand activity for DDR1 in matrigel, was not able to stimulate tyrosine phosphorylation of DDR2. The different types of collagen were also tested for their ability to stimulate tyrosine phosphorylation of endogenous DDR1b present in T-47D cells. To this end, DDR1b was immunoprecipitated from lysates of stimulated T-47D cells and analyzed by Western analysis with anti-phosphotyrosine antibodies. As shown in Figure 10E, collagen types I and IV caused a substantial increase in tyrosine phosphorylation of DDR1b. The level of activation induced by collagen types II, III and IV was lower, but can still be detected with unstimulated cells. As previously shown, tyrosine phosphorylation of DDR1 could also be activated by treating cells with 1 mM orthovanadate, a tyrosine phosphatase inhibitor. In addition, gelatin containing heat denatured collagen was tested for its ability to induce tyrosine phosphorylation of DDR1b. At a concentration of 10 μg / ml, gelatin does not induce an increase in tyrosine phosphorylation of DDR1b, indicating that the native collagen structure is essential for the activation of DDR1 receptor in the cell-based analysis (Figure 10E). Tyrosine phosphorylation of DDR1 and DDR2 can be activated by immobilized collagen. The experiments described above employed soluble collagen, whereas the collagen found in vivo was commonly immobilized as part of the extracellular matrix. The self-assembly of collagen solubilized in fribrilas is a spontaneous process that can be induced in vitro by the neutralization or evaporation of the solvent. To this end, the tissue culture plates were coated with collagen types I, III, IV and V. Transfected 293 cells that over-express DDR1b and DDR2 were re-suspended in PBS without trypsin / EDTA treatment and added to the plates coated with collagen for 90 minutes. The receptor phosphorylation analysis indicated that DDR1b was partially phosphorylated with tyrosine by removing the cells from the plate; however, the tyrosine phosphorylation of the receptor increased more after plating in collagen types I, II and V (Figure 10G). In contrast, no initial phosphorylation was observed after separation of the cells that over-express DDR2; Tyrosine phosphorylation of DDR2 was triggered by plating the cells in plaques on collagen types I and III and to a smaller extent in type V (Figure 10H). Type IV immobilized collagen was inactivated in this analysis, it was also observed for soluble collagen type IV (Figure 10C). The ligand activity for DDR activation is resistant to pepsin, but sensitive to collagenase. The predicted data suggested that collagen can stimulate tyrosine phosphorylation of DDR1 and DDR2. To investigate whether collagen is also the molecule responsible for activating these receptors, the unique structural properties of the members of the collagen family were exploited. The primary amino acid sequence of collagen contains the repeating extensions of Gly-X-Y that vary in length. By virtue of the glycine residue in each third position, three identical or highly similar chains of polypeptides are capable of being wound up with one another forming a left helix. The X or Y residues are frequently proline or 4-hydroxyproline, respectively, which allows additional stabilization of the triple helix due to the restrictions of chain flexibility and the formation of hydrogen bonds between the chains. This particular structure renders collagen resistant to protease separation, for example by pepsin, which is separated after large hydrophobic residues (phenylalanine, methionine, leucine, tryptophan) that are not found in the triple helix of collagen. In contrast, collagenase isolated from Clostridium histolyticum is specifically separated before each second or third glycine residue of the Gly-X-Y repeat. Type I collagen isolated from mouse tail or human placenta was preincubated with pepsin or collagenase for 30 minutes at 37 ° C. After protease treatment, an aliquot was analyzed by SDS-PAGE and tinsion with Coomassie Blue, while a second aliquot was used to stimulate DDR2 that over-expressed 293 cells, which were subsequently analyzed for DDR2 tyrosine phosphorylation. As shown in Figure 11A, mouse and human type I collagen was effectively digested by collagenase, but was resistant to the proteolytic activity of pepsin. In contrast, bovine serum albumin was degraded by collagenase, but was digested by treatment with pepsin. Analyzes of stimulated cells showed that collagenase treatment abrogated the ability of collagen to induce tyrosine phosphorylation of DDR2, while collagen treated with pepsin reduced its stimulatory activity (Figure 11B). This result indicates the ability of collagen preparation for DDR2 activity depends on the integrity of the collagen. The finding that the stimulatory activity is sensitive to the specific proteolytic enzyme collagenase, but resistant to the more general protease pepsin, supports the observation that the induction of DDR tyrosine phosphorylation is due to collagen rather than to a different protein associated non-covalently. Thermal denaturation of collagen that inhibits the stimulating activity for tyrosine phosphorylation of DDR As described above, the triple helical structure of collagen was stabilized by hydrogen bonds. At higher temperatures, therefore, the collagen polypeptide chains will melt and eventually denature in a random coil. To confirm the possibility that the ligand activity for DDR receptors is intrinsic to collagen, the mouse type I collagen was subjected to thermal denaturation and the circular dichroism spectrum was recorded at a wavelength of 221 nm. The absorption decreased quite a 40 ° C (Figure 11D, pictures), and the heat treatment resulted in the irreversible denaturation of collagen (Figure 11D, diamonds). It was calculated that the midpoint of thermal fusion transition is 41 ° C. To test the thermal denaturation effect on the ability of the collagen preparation to stimulate the tyrosine phosphorylation of DDR2, aliquots of type I collagen were incubated at various temperatures between 27 ° C and 45 ° C for 30 minutes and these samples then were incubated with DDR2 over-expressing 293 cells. As shown in Figure 11E, the ligand activity of the collagen preparation was markedly reduced after heat treatment at 39 ° C and almost completely eliminated at temperatures above 42 ° C. ° C. These data demonstrate that the activity of collagen preparation to stimulate DDR2 kinase activity decreases with the same temperature scale as the chimeric collagen structure unfolds due to coiled coil fusion. Therefore, the triple helical collagen configuration is essential for the complete activation of the DDR receptors of this analysis. The interaction between DDR and collagen is direct The ability of collagen to stimulate tyrosine phosphorylation of DDR, could be due to a direct association of collagen with the extracellular domain of the receptor, or could represent an indirect effect of collagen, for example, forming lumps of cell surface molecules. To explore whether collagen can be directly and specifically associated with DDR receptors, collagen linked covalently to agarose beads was used in an in vitro mixing experiment. Equal amounts of cell lysates of 293 cells that over-express DDR1, DDR2 or insulin receptors, were incubated with collagen-agarose in the absence or presence of type I soluble collagen. As shown in Figure 12A, the collagen-agarose was joined DDR1 and DDR2, but not the insulin receptor. This interaction of collagen immobilized with DDR1 and DDR2, competed with an excess of soluble collagen.

Consistently in that collagen interacts directly with DDR receptors, collagen capacity was shown to induce tyrosine phosphorylation of DDR that is eliminated by a lysine to alanine point mutation in a DDR receptor cDNA that destroys receptor kinase activity, but which was not inhibited with the treatment of cells with cycloheximide (data not shown). These results suggest that collagen directly induces DDR autophosphorylation and does not act by inducing a different DDR ligand. The expression of DDR1 and DDR2 increases cell surface binding sites for collagen. To investigate whether the expression of DDR tyrosine kinases increases the amount of cell surface receptors for collagen, mouse type I collagen was treated with iodine and incubated in varying concentrations with 293 cells that have been transfected with DDR1a, DDR1b or a control plasmid. As shown in Figure 12B, the binding of collagen to the cells transfected with DDR1 was approximately three times higher than that of the control cells. The binding of 125 I-collagen to DDR1 competed almost completely with a 100-fold excess of the cold ligand (data not shown). The interaction between DDR2 and collagen is sensitive to the carbohydrate portion of collagen. Multiple studies have shown that collagen is glycosylated extensively through carbohydrates linked with N and with O after its initial synthesis (for review see Kivirikko and Myllylá, 1982) . In particular, several Usinas were oxidized to hydroxylysine and then ligated to galactose and glucose. The monosaccharide composition of mouse tail collagen was analyzed using carbohydrate electrophoresis technology aided by fluorophores (Higgins &Friedman, 1995) and it was found that this collagen preparation contained high amounts of glucose and galactose, small amounts of fucose and mannose, but not sialic acid, N-acetyl-glucosamine or N-acetyl-galactosamine (data not shown). Therefore, the collagen was treated with m-periodate to partially remove the glyco-conjugate. The periodate treatment of type I collagen does not induce hydrolysis of the polypeptide base structure or denauraiizes the collagen triple helix (data not shown). In contrast, the ability of collagen to stimulate DDR2 in 293 over-expression cells was significantly reduced after deglycosylation (Figure 6C). Therefore, neither the N or O fraction linked to the collagen glycol fraction (or both), can be important for the activation of DDR2. Occasional commercial preparations of collagen have found that they do not give DDR receptor activation. This can be explained by a loss of native conformation or by the failure of a modification such as glycosylation. PTB domain of Shc binds to DDR1b after collagen stimulation Since collagen induces tyrosine phosphorylation of DDR1, it is possible that said autophosphorylation of the receptor may create phosphotyrosine recognition module binding sites, such as domains SH2 or PTB. In this regard, it is of interest that the DDR1b juxtamembrane insert contains the motif LSNPAY (including tyrosine 513), which corresponds to the consensual binding motif for the PTB domain of Shc. The PTB domain of Shc binds with high affinity to peptides containing phosphotyrosine with the sequence "XNPXpY (where? Is a hydrophobic residue) (23) (van der Geer et al., 1996). The possibility that Shc can interact with autophosphorylated DDR1b was tested. In addition, DDR1b bound to a GST fusion protein containing the PTB domain of Shc after collagen stimulation (Figure 13A). The PTB domain of Shc did not bind to the a-isoform of DDR1 and DDR2, which lacks the XNPXY motif found in DDR1b. This demonstrates that the stimulation of DDR1 collagen induces autophosphorylation of the receptor and the consequent formation of coupling sites for modular downstream signaling molecules. To quantify the interaction between DDR1b and Shc more preciselyResonance technology of surface plasmons was used in which a soluble phosphopeptide derived from the juxtamembrane region of DDR1b (residues 503-518) was used to inhibit the binding of the GST-PTB fusion protein of Shc to a phosphopeptide of polyomavirus medium T antigen, immobilized in a biosensor microcircuit. As shown in Figure 13B, a concentration of approximately 800 nM phosphopeptide of DDR1b, which contains the sequence around tyrosine 513, inhibited the binding of the PTB domain of Shc to the phosphopeptide of the mean T antigen by 50% (IC50 ). These findings contrast with a ICS0 value of 70 mM found when a phosphopeptide was used around the NPXY motif of the NGF receptor in an affordable experiment (Figure 13B), but are similar to the IC50 of the average T antigen phosphopeptide itself. Activation of DDR2 induces matrix metalloproteinase-1 expression Degradation and remodeling of extracellular matrix is controlled in large part by the activity of matrix metalloproteinases (MMPs). To test for any influences of DDR signaling on MMP expression, DDR2 was expressed stably in the HT 1080 human fibrosarcoma cell line, which does not show the detectable expression of DDR receptors. HT 1080 cells that over-express DDR2 and parenterals were stimulated for several periods with 10 μg / ml type I collagen. The amount of matrix metalloproteinase I (MMP-1) secreted by the cells was measured by Western blot analysis of conditioned medium. As shown in Figure 14, MMP-1 expression was up-regulated in HT 1080 cells that over-express DDR2 after collagen stimulation for 4 days. In contrast, MMP-1 was not induced in parental cells and HT 1080 in response to collagen. Expression of MMP-1 was induced both in parental cells and those that over-express DDR2 after treatment with phorbol myristate 13 (TPA) acetate, an MMP expression activator. These data suggest that the activation of the DDR receptor results in the secretion of collagenolytic activity, which could eventually lead to the decomposition of the extracellular matrix. Discussion Activation of collagen from discoidin domain receptors An investigation for ligands of the DDR sub-family of receptor tyrosine kinases unexpectedly revealed that collagen, one of the most abundant proteins in vertebrates, is capable of binding and activating both DDR receivers. The analysis showed that the activation of over-expressed DDR1 was triggered for the five tested collagens, while DDR2 was only activated by collagen types I and III and to a lesser extent by collagen types II and IV. In a human mammary carcinoma cell line of endogenous DDR1, it was strongly activated by type I and V collagen, and to a lesser degree by types II, III, IV. Therefore, there is some specificity in the interactions of DDR1 and DDR2 with the collagen molecules. Collagen types I, II, III, V, and XI have an uninterrupted Gly-X-Y repeat that expands over 1000 amino acids and forms a perfect triple helix structure. The individual helices are polymerized, thus generating fibers with high tensile strength. In contrast, type IV collagen is characterized by approximately 20 short interruptions of the triple helix, which provides more flexibility and allows the formation of network-like structures (Prockop & amp;; Kivirikko, 1995). Collagen type IV is the main component of the base membrane that surrounds various tissues and organs. Three lines of evidence support the conclusion that the binding epitopes for DDR1 and DDR2 are located in the repeat region of Gly-X-Y and that the triple helical conformation is essential for the activation and autophosphorylation of receptors. First, treatment with pepsin, a protease that separates collagen only in regions that are not helical at the N and C ends, does not affect this ligand activity. In contrast, bacterial collagenase, which specifically digests collagen in the Gly-X-Y repeat region, eliminated receptor activation. Second, the ability of collagen preparations to activate DDR receptors was sensitive to heat denaturation. The triple helix of collagen is mainly held together by non-covalent ligatures, making it sensitive to thermal denaturation. Several types of collagen were denatured between the scale at 37 ° C and 45 ° C, at which temperature twice as many triple helices were irreversibly destroyed (Niyibizi et al., 1984). The DDR stimulation activity of collagen preparations markedly decreased at the transitional point of thermal fusion of collagen, and that gelatin did not have the in vitro binding activity nor the ability to induce tyrosine phosphorylation in vivo. Third, although non-denatured collagen is not capable of activating DDR receptors, type I collagen that was denatured in 7 M urea and then doubled again by dialysis in a physiological solvent, regained its ability to induce phosphorylation of DDR1 and DDR2 tyrosine (data not shown). Several molecules associated with collagen, such as chondroitin sulfate A, B and C, decorin and heparin were tested and no effect on DDR activity was found. The results discussed above indicate that the collagen itself, or a closely linked molecule, acts as a stimulatory ligand for DDR receptors. The biological functions of proteins with discoidin motifs The discoidin domains of DDR1 and DDR2 have great homology (approximately 75%) to discoidin proteins of Dictyostelim discoideum. In mold, the discoidins are expressed and secreted during the formation of the drool and the body of the fruit and function as lecithins by binding to N-acetyl-galactosamine and galactose (Rosen et al., 1973). In Dictyostelim-deficient discoidin-1, the cells lose their ability to adhere and migrate onto the substrate, resulting in a defect in the aggregation of ordered cells (Springer et al., 1984). Activation of DDR receptors by collagen clearly requires the structure of collagen triple helix peptides, but also involves portions of N- or O-linked carbohydrates, as a collagen treatment with periodate that resulted from partial deglycosylation and marked reduction of activity of the ligands. If the ability to bind carbohydrates is conserved in the discoidin domains of mammalian DDR, it is possible that DDR1 and DDR2 recognize the binding of mono- or oligosaccharides to collagen. The additional specificity and high affinity could provide triple-helical conformation of the peptide base structure near the glycosylation site, which could also allow for the oligomerization and consequent transphosphorylation of bound DDR tyrosine kinases. In contrast to DDR2, however, treatment with periodate does not affect the ability of collagen to activate DDR1 (data not shown). Collagen as a signaling molecule The observation that DDR receptors bound to proteins are so abundant as collagen gives rise to an enigmatic aspect that is critical for surface signaling receptors for matrix components, namely, what way cytoplasmic signaling is regulated. The activation of DDR kinases by collagen follows a very delayed time course in relation to the conventional growth of factor receptors, which is consistent with the possibility that DDR receptors monitor the cell's relationship to the extracellular matrix instead of mediating an accurate signaling response. However, since the DDR1 receptor isolated from mouse embryos or adult brain contains little phosphotyrosine (Perez et al., 1996), there must be regular mechanisms that control the activation of DDR1. Although the expression of DDR1 RNA has been detected in the outer epithelial layer of the lung, kidney and colon in close proximity to the base membrane (1), it is possible that the location of the DDR receptors for sub-regions can be regulated. specific cell surface. In addition, the DDR1 signaling activity can potentially be controlled by the inclusion or exclusion of the juxtamembrane binding site for the PTB domain of Shc. In this respect it is interesting that DDR1 is the first example of an RTK whose coupling sites for the downstream targets are controlled directly by the alternative separation. Shc is apparently recruited to the α1ß1 integrin complex and may play a role in cell survival and proliferation by coupling this integrin (Wary et al., 1996). Since the PTB domain of Shc binds to the phosphorylated tyrosine 513 in the DDR1b isoform, it is possible that stimulated DDR1 and the activated integrin receptors converge on the same signaling pathways. However, the activation of the MAP kinase pathway has not been shown to suggest that Shc fulfills another function in DDR1 signaling. In this context, it is interesting that the Shc SH2 domain binds to the phosphorylated tail of cadherin, a transmembrane cell-cell receptor (Xu et al., 1997), with the possibility that Shc SH2 can bind different molecules of adhesion through its PTB and SH2 domains. Shc also couples to the cytoplasmic signaling pathways other than the MAP kinase pathway.

In embryogenesis, the appropriate expression of different types of collagen induces the correct formation of bone and cartilage (Mundlos &Olsen, 1997). A number of human genetic diseases caused by the aberrant expression of collagen or dot mutations in the primary collagen sequence results in skeletal malformations or hereditary osteoporosis (Prockop &Kivirikko, 1995). The early expression of DDR receptors in embryogenesis indicates that it may have a role to be a model of cartilage and bone formation. Interactions with collagen are also potentially important in controlling the shape and movement of cells, for example in the movement and joining of epithelial sheets during development. Recent studies have shown a high level of DDR1 and DDR2 in several primary human tumors (1). In particular, rapidly growing tumors, which originate from mammary, ovarian and lung epithelial cells, have high expression of DDR1. These tumors are characterized by their sive growth in nearby tissues and organs, leading to the metastasis of tumor cells. The initial stimulation necessary to induce the decomposition of the matrix barrier and migration of cells away from the tumor are largely unknown (Alves et al., 1995b). Elevated expression of matrix metalloproteinases, enzymes that specifically degrade collagens and elastin, has been found in several solid tumors, and therefore, binds this class of enzymes to the growth and metastasis of tumors (Stetler-Stevenson et al., 1996 ). For example, it was found that high levels of MMP-1 are associated with poor prognosis in cancer, colorectal (Murray et al., 1996). Because DDR1 and DDR2 are driven by collagen and because activated DDR2 promotes the expression of MMP-1, the two receptors may have a role in the activation of tumor cells and in the subsequent degradation of the matrix by metalloproteinases. One model places DDR receptors as sensors for collagen, as a major component for the extracellular matrix, on the surface of tumor cells. After ligand binding and receptor activity, the DDR signal induces the expression and secretion of MMP-1, which in turn degrades the collagen molecules surrounding the tumor allowing the tumor cells to migrate and metastasize. Normal cells, such as keratinocytes, MMP-1 expression is highly elevated after stimulation of type I collagen, raising the possibility that DDR receptor activity may be lved in healing wounds (Sudbeck et al., 1994, 1997). Mold is a simple model of metazoan development, since it exists in the form of unicellular amoebae that are inducibly added to multicellular structures that develop two different cell types: spores and petiole cells. Recent evidence has indicated that an SH2 domain signaling pathway, lving the tyrosine phosphorylation of a Dictyostelium Stat protein, plays a major role in mediating the differentiation of these two cell types in response to morphogenic DIF (Kawata and others, 1997). Here, the data establish that an extracellular domain originally identified in the lecithin discoidin of mold and known to be important for the aggregation of differentiation cells, is coupled in vertebrates for the signaling of collagen activated kinase kinase and is potentially important in the ordered movement in normal mammalian cells and the disordered migration of tumor cells. Example 4 Mutation of DDR1a with AK618A is no longer activated by collagen The experiment illustrated in Figures 15A and 15B shows that the tyrosine phosphorylation of DDR1a clearly depends on an intact catalytic domain. The activation of DDR1a by a collagen is eliminated by a dominant mutation of inactivation (negative domain) in the catalytic domain. It appears that other kinases are not lved in tyrosine phosphorylation in vivo. of DDR1a in response to collagen. Blocking antibodies for a1 or β1 integrinsdo not inhibit the activation of DDR1 The integrins of a1ß1 and a2ß1 have long been known to be receptors for collagen. Therefore, the tests have led to determine if these integrins are involved in some way in the activation of DDR1b. The mammary carcinoma cell line T-47D was used, which endogenously expresses the b-isoform of DDR1. Monoclonal antibodies directed against the extracellular domains of integrins can block collagen binding and thus signal integrins. T-47D cells were treated with antibody A2-IIE10 against a2-integrin and DE9 antibody against β1-integrin (both from Upstate Biotechnology) in the absence or presence of 10 μg / ml type I collagen overnight. DDR1b or Shc were immunoprecipitated from the cell lysates and analyzed for Western blot analysis with antiphosphotyrosine antibody. The experiment illustrated in Figure 16 shows that the activation of integrins is not necessary for the activation of DDR1b. The degree of tyrosine phosphorylation of DDR1b after stimulation with collagen in T-47D cells with blocked integrin receptors is identical to untreated cells. The binding of DDR1b to Shc is no longer altered after blocking the signaling of integrins DDR1 was activated by collagen in cells deficient in β1 integrin. The signaling of DDR1b in the cell line GD25 (Dr. R. Fasler, Martinsried, Germany), was proved to be derived from the elimination of β1 integrin in mice. In these cells, a functional integrin receptor for collagen is absent. The cDNA encoding DDR1b was transfected into GD25 cells using a retroviral transfer protocol. Overexpression of DDR1b and parenteral cells were stimulated with type 1 collagen overnight. DDR1b was immunoprecipitated from the cell lysates and analyzed by Western blot analysis with antiphosphotyrosine antibodies (Figure 17A). The analysis was retested with antibodies against DDR1 (Figure 17B). Using a genetically modified cell line, the experiment illustrated in Figures 17A and 17B shows that DDR1b can be reported in the absence of two integrin type collagen receptors. Slow activation of DDR1b in cells deficient in β1 integrin The generation of DDR1b which over-expresses GD25 cells is described in Figures 17A and 17B. These cells were stimulated with type I collagen for several periods of time. Immunoprecipitated DDR1b was analyzed in a Western blot analysis with antiphosphotyrosine antibody. The results shown in Figure 18 indicate that the activation of DDR1b in β1 integrin deficient cells is as slow as in normal cells, indicating that the extensive activation of DDR1b is not due to the action of integrins. Activation of the DDR1 and DDR2 receptor does not influence MAPK activation mediated by EGF T-47D or HT 1080 that over-expressed DDR2 cells were stimulated with PDGF or EGF for 5 minutes, with type I collagen during the night and with a combination of EGF / collagen or PDGF / collagen. Aliquots of cell lysates were separated by SDS-PAGE and tested with an antibody to MAPK (Figure 19A (T-47D) and Figure 19B (HT 1080-DDR2) .Activated MAPK show slower migration in SDS-PAGE than non-SDS-PAGE. activated MAPK is activated by treatment with EGF or EGF / collagen, but not by treatment with PDGF, collagen or PDGF / collagen The remaining lysates of T-47D cells were used to immunize DDR1b Western blot analysis with antiphosphotyrosine antibodies show that DDR1b was activated by collagen and not by EGF or PDGF (Figure 19C) .The combination of collagen with EGF does not decrease the degree of activation of MAPK or the tyrosine phosphorylation of DDR1b.The experiments illustrated in Figures 19A or 19C demonstrate that activation of DDR1 and DDR2 does not result in the activation of the MAPK pathway.Furthermore, the activation of MAPK by EGF was not influenced by simultaneous activation of DDR receptors. described with reference to what is currently considered to be the preferred examples, it should be understood that the invention is not limited to the described examples. On the contrary, it is intended that the invention cover several modifications and equivalent arrangements included between the spirit and scope of the appended claims.

All publications, patents and patent applications are hereby incorporated by reference in their entirety to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference in its entirety.

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DETAILED LEGENDS OF THE FIGURES Figures 1A to 1E. Co-immunoprecipitates of MCKIOb (DDR1b) with Shc and associated with the PTB domain of Shc. Figure 1A-1C, 293 human embryonic kidney fibroblast cells were transfected with expression plasmids encoding MCK10A or MCKIOb. After stimulation with 1 mM of orthovanadate for 90 minutes, Shc was immunoprecipitated from cell lysates. After the SDS-PAGE immunoassay was tested with antibodies to phosphotyrosine (Figure 1A). Subsequently, the blot was removed and tested again with antibodies against MCK10 (Figure 1B) and then against Shc (Figure 1C). The migration of MCKIOb and the Ubres templates of Shc, p66, p52 and p46 were indicated. Figure 1D. The aliquots of total cell lysates used in (Figure 1A) were analyzed by Western blot analysis with anti-phosphotyrosine antibodies. The migration of the precursors (prec.) And sub-units-b (sub-b) of MCKIOa and MCKIOb were indicated. Figure 1E, GST fusion proteins containing the Shc SH2 domain or the PTB domain of Shc were bound to glutathione agarose and incubated with lysates of 293 cells, which were transfected with MCKIOa or MCKIOb and stimulated with orthovanadate. The bound proteins were separated by SDS-PAGE, transferred to nitrocellulose with antibodies to MCK10. The molecular weight normals were indicated on the right. Figures 2A and 2B. Phosphorylation of Tyr 513 in the juxtamembrane insert of MCKIOb (DDR1b) forms a binding site for PTB of Shc. Figure 2A, GST-PTB domain fusion protein from Shc bound to glutathione beads was incubated with lysates of 293 cells that over-express MCKIOb in the absence or presence of increasing concentrations of a competing peptide, corresponding to the sequence ALLLSNPApYRLLLA around tyrosine 513 of MCKIOb. The binding protein was detected by immunoblots with antibodies to MCK10. Figure 2B, Analysis of the binding of GST-PTB domain of Shc purified in the phosphopeptide of average T antigen by the resonance of surface plasmons in the presence of increasing amounts of phosphopeptide of MCKIOb (ALLLSNPApYRLLLA, open circles) or phosphopeptide of receptor of NGF (HIIENPQpYFSD, closed circles), respectively. The percentage of the PTB domain of Shc bound to the surface of the microcircuit was plotted against the concentration of competing peptides. Figures 3A to 3F. MCKIOb (DDR1b) contains a primary autophosphorylation site that is absent from MCKIOa (DDR1a). Figures 3A and 3B, 293 cells were transfected with plasmids encoding a- or b-isoform of MCK10 and labeled in vivo with [32 P] -phosphate. After stimulation with 1 mM of orthovanadate for 90 minutes, MCK10 was immunoprecipitated and digested with trypsin. The two-dimensional tryptic phosphopeptide maps of the a-isoform (Figure 3A) and b-isoform (Figure 3B) were shown. Figures 3D-3F, MCKIOa or MCKIOb were immunoprecipitated from transfected cells and subjected to in vitro kinase analysis. The labeled protein of the a-isoform (Figure 3D) and the b-isoform (Figure 3E) were analyzed by tryptic mapping. Equal cpm of phosphopeptides from MCKIOa and MCKIOb were combined and analyzed in (Figure 3F). The origin was indicated by x. Figure 3C, schematic representation of the phosphopeptides in (Figure 3B) and (Figure 3F). Figures 4A, 4B and 4C - Temporary expression of DDR1a, DDR1b, DDR2 and trkA in 293 cells. Total cell lysates of spots: aPY. Use of collagens in 10 μg per ml of final concentration. Figures 5A, 5B and 5C - the stains of a-PY from total cell lysates: Stimulation that depends on the concentration with collagen i [C-7661 CBP: Collaborative Biomedical Research] and collagen IV [C-0543]. Figure 6 - Tyrosine phosphorylation of DDR1b in T47D, human mammary carcinoma cells. Immunoprecipitation of DDR1b with antibodies from alternative exons, analysis of PY. Stimulation of collagen I [C-766, Collagen IV [C-0543 for 30 minutes and 1 mM of orthovanadate for 90 minutes]. Figures 7A to D. Identification of type IV collagen as the ligand activity for DDR1 in matrigel. The human kidney fibroblast 293 cells were transfected with DDR1b by expression plasmid. Matrigel was added to the tissue culture medium at the indicated concentrations for 90 minutes. (Figure 7A) Cells were used and 10 μg of total cell protein was analyzed by SDS-PAGE and Western blot analysis with antiphosphotyrosine antibody. (Figure 7B) The spots were separated and retested with antibodies raised against DDR1. (Figure 7C) 293 cells that over-express DDR1b were treated with the following reagents: 400 M acetic acid (a), 50 μl / ml matrigel (b), 10 μg / m laminin type IV (c), 10 μg / ml of fibronectin (d), type IV collagen, partially purified from matrigel by extraction with guanidinium hydrochloride (e) or by extraction with acetic acid or pepsin (f), 10 μg / ml of type IV mouse collagen, sigma C-0543 (g), 10 μg / ml human collagen type IV, Sigma C-5533 (h). Equal amounts of total cell lysate were tested with antiphosphotyrosine antibody or (Figure 7D with anti-DDR1 antibody, Figures 8A and 8B.) DDR1 and DDR2 293 cells that specifically activate type I mouse collagen were transfected with plasmids encoding insulin receptor. Human (Ins-R), DDR1a, DDR1b or DDR2 Cells were stimulated with 10 μg / ml type I collagen, 100 nM insulin or left unstimulated (Figure 8A) Aliquots of cell lysates were analyzed by SDS- PAGE and Western analysis with antiphosphotyrosine antibody (Figure 8B) The blot was retested with a mixture of antibodies against DDR1, DDR2 and insulin receptor Figures 9A to 9F.DDR delayed activation in response to collagen DDR1b transfected and DDR2 in 293 cells and were stimulated with 10 g / ml of type I mouse collagen for different times (Figure 9A and Figure 9B). Total cell lysates were tested with antiphosphotyrosine antibody. Maximal tivation is observed 90 minutes after stimulation of DDR1b (Figure 9A) and DDR2 (Figure 9B). (Figure 9C) Human mammary carcinoma T-47D cells were grown in 10 cm plates for confluency and fasting in 0.5% serum overnight. The cells were stimulated with type I collagen for several periods and were used. DDR1b was immunoprecipitated from the lysates and analyzed by Western blots with antiphosphotyrosine antibody. The maximum phosphorylation of DDR1b is observed after stimulation for 18 hours. The tyrosine phosphorylated protein, unidentified with an apparent molecular weight of 115 kDa was co-purified in the immunoprecipitations. (Figure 9D). The stain was re-probed with an antibody specific for the C-terminus of DDR1 showed no reactivity with the 115 kDa protein. Prolonged stimulation with collagen increases the processing of DDR1b in T-47D cells, indicated by an additional band of 672 kDa. (Figures 9E and 9F) DDR1b was immunoprecipitated from over-expression of 293 cells (E) and T-47D cells (Figure 9F) and subjected to an in vitro kinase reaction. The incorporation of [32 P] -phosphate was monitored by SDS-PAGE followed by autoradiography. Figures 10A to 10H. The differential activation of DDR1 and DDR2 by various types of collagen. 293 cells were transfected with DDR1b or DDR2 and stimulated with 10 μg / ml human collagen types I, III, IV or V and bovine type II collagen. (Figures 10A and 10D). Total cell lysates were probed with antiphosphotyrosine antibodies (Figure 10A: DDR1b and Figure 10C: DDR2) and retested with antibodies specific for DDR1 (Figure 10B) or DDR2 (Figure 10D). DDR1b was immunoprecipitated from T-47D cells that were stimulated for 90 minutes with collagen types I, II, III, IV, V or gelatin or treated with 1 mM of orthovanadate. (Figure 10E) Immunoprecipitates were analyzed by Western blots with antiphosphotyrosine antibody and (Figure 10F) was retested with DDR1 with specific antibody. (Figure 10G and Figure 10H) 293 cells that had been transfected with DDR1b or DDR2 were washed from the plates with PBS by repeated pipetting and added to plates that had been coated with human collagen types I, III, IV and V. of the incubation at 37 ° C for 90 minutes, the cells were used. Total cell lysates were analyzed by Western blot analysis with antiphosphotyrosine antibody (Figure 10G: DDR1b and Figure 10H: DDR2). Figures 11A to 11E. The DDR ligand activity was destroyed by treatment with collagenase or thermal denaturation. Type I collagen isolated from mice or human tissue or BSA was treated with collagenase (from Clostridium histolyticum, 20 ng per μg of collagen) or pepsin (from pig mucosa, 2 ng per μg of collagen). (Figure 11A) Equal amounts were analyzed by SDS-PAGE and visualized by Coomassie tinsion (+ C: incubation with collagenase; + P: incubation with pepsin; a1 (l) and a2 (l): monomeric collagen chains; β and β: oligomeric collagen covalently crosslinked) or used to stimulate 293 cells that over-express DDR2. (Figure 11B) Aliquots of cell lysates were analyzed for spots with antiphosphotyrosine antibodies and (Figure 11C) was retested with DDR2-specific antibody. (Figure 11D) Mouse type I collagen (500 ng / ml in 10 mM acetic acid) was melted on a spectropolarimeter and the change in circular dichroism was recorded (frames). After denaturing with heat, the spectrum was measured again (diamonds). The average point of thermal transition (Tm) was indicated. (Figure 11E) Aliquots of type I collagen were incubated at various temperatures between 24 ° C and 45 ° C for 30 minutes and used to stimulate 293 cells that overexpress DDR2. Total cell lysates were analyzed by Western blot analysis with antiphosphotyrosine antibodies. Figure 12a to 12C. The binding of collagen to DDR1 and DDR2 was directed and activation of DDR2 was reduced after collagen deglycosylation. (Figure 12A) Collagen covalently coupled to agarose beads was incubated with lysates of 293 cells that over-express insulin-receptor, DDR1b or DDR2 in the absence or presence of 50 g / ml soluble collagen type I: the bound material was analyzed by SDS-PAGE and Western blots with a mixture of antibodies against insulin-receptor, DDR1 and DDR2 (Figure 12A). The lower glycosylated isoform of DDR2 showed stronger affinity of collagen beads. (Figure 12B) 293 cells transfected with DDR1a (squares), DDR1b (diamonds) or control plasmid (circles) were incubated with various concentrations of iodinated type I collagen and the amount of binding ligand determined by β-counting. (Figure 12C). The type collagen! it was de-glycolized with sodium m-periodate and used to stimulate 293 cells that over-express DDR2. The total cell lysates were analyzed with antiphosphotyrosine antibody stains. Figures 13A and 13B. Shc adapter protein binds to DDR1b. (Figure 13A) The PTB domain of Shc was expressed in E. coli as a GST fusion protein and incubated with lysates of 293 cells that overexpress DDR1a or DDR1b. The bound protein was detected with an antibody against the C-terminus of DDR1. (Figure 13B). Analysis of the binding of the GST-PTB domain of purified Shc to the phosphopeptide of medium T antigen (LSLLSNPTpYSVMRSK) by resonance of surface piasms in the presence of competing amounts of phosphopeptide DDR1b (ALLLSNPApYRLLLA, open circles) or phosphopeptide of NGF receptor ( HIIENPQpYFSD, closed circles), respectively. The percentage of PTB domain of Shc bound to the surface of the microcircuit was plotted against the concentration of competition peptide. Figure 14. Activation of DDR2 induces the expression of MMP-1. Parenteral and HT 1080 cells that over-express DDR2 were stimulated with type I collagen or TPA during the indicated periods. The conditioned media was concentrated and analyzed by Western blot analysis with antibodies against MMP-1. Figures 15A and 15B: DDR1a with K618A mutation no longer activated by collagen Sequence alignments showed that lysine 618 in the DDR1 protein is presumably essential for the catalytic function of the tyrosine kinase domain. Therefore, a point mutation was introduced into the cDNA encoding DDR1a, changing lysine 618 to alanine. The mutant cDNA was temporarily expressed together with the wild-type DDR1a cDNA in 293 embryonic kidney fibroblast cells. The cells were stimulated with 10 μg / ml type I collagen for 90 minutes or left untreated. Equal aliquots of total cell lysate were analyzed by SDS-PAGE and tested with antiphosphotyrosine antibody (Figure 15A). The spot analysis was retested with DDR1 antibodies (Figure 15B). This experiment shows that the tyrosine phosphorylation of DDR1a clearly depends on the intact catalytic domain. The activation of DDR1a by collagen was abolished by an inactivation mutation (dominant negative) in the catalytic domain. Other kinases do not appear to be involved in the in vivo tyrosine phosphorylation of DDR1a in response to collagen. Figure 16: Blocking antibodies for a1 or ß1 integrins do not inhibit the activation of DDR1. The integrins of the a1ß1 and a2ß1 type have been known to be receptors for collagen. Therefore, the involvement of integrins in the activation of DDR1b was investigated. The mammary carcinoma cell line of T-47D, which endogenously expresses the b-isoform of DDR1 was used. Monoclonal antibodies directed against the extracellular domains of integrins can block the binding to collagen and therefore the signaling of integrins. The T-47D cells were treated with the antibody A2-IIE10 against íhtegrin a2 and the antibody DE9 against the integrin ß1 (both of Upstate Biotechnology) in the absence or presence of 10 μg / ml of type collagen overnight. DDR1b or Shc were immunoprecipitated from cell lysates and analyzed by Western blot analysis with antiphosphotyrosine antibody. This experiment shows that the activation of integrins is not necessary for the activation of DDR1b. The degree of tyrosine phosphorylation of DDR1b after stimulation with collagen in T-47D cells with blocked integrin receptors is identical to untreated cells. The binding of DDR1b to Shc did not change after the integrin signaling block. Figure 17A and 17B: DDR1 was activated by collagen in cells deficient in β1 integrin. The signaling of DDR1b in the GD24 cell line, which was derived from mice deleted with β1 integrin, was tested. In these cells, a functional collagen integrin receptor is absent. The cDNA encoding DDR1b was transfected into GD25 cells using a retroviral transfer protocol. DDR1b and parenteral over-expression cells were stimulated with type I collagen overnight. DDR1b was immunoprecipitated from cell lysates and analyzed by Western blot analysis with antiphosphotyrosine antibodies (Figure 17A). The spot analysis was retested with antibodies against DDR1 (Figure 17B). Using a genetically modified cell line, this experiment shows that DDR1b can be signaled in the absence of two integrin type collagen receptors. Figure 18: Slow activation of DDR1b in cells deficient in β1 integrin. The generation of GD25 cells that over-express DDR1b was described in Figure 17. These cells were stimulated with type I collagen for several periods of time. Immunoprecipitated DDR1b was analyzed in a Western blot analysis with antiphosphotyrosine antibody. This result shows that the activation of DDR1b in cells deficient in β1 integrin is as slow as in normal cells, indicating that the activation of DDR1b is due to the action of integrins. Figures 19A to 19C: Activation of the DDR1 receptor and DDR2 did not influence the activation of MAPK mediated by EGF. The T-47D or HT 1080 cells that over-express DDR2 were stimulated with PDGF or EGF for 5 minutes, with type I collagen at night and with a combination of EGF / coagen or PDGF / collagen. The aliquots of cell lysates were separated by SDS-PAGE and tested with an antibody to MAPK (Figure 19a (T-47D) and Figure 19B (H 1080-DDR2).) Activated MAPK shows slower migration on SDS-PAGE that is not MAPK is activated by treatment with EGF or EGF / collagen, but not by treatment with PDGF, collagen or PDGF / collagen The remaining lysates of T-47D cells were used to immunoprecipitate DDR1b Western blot analysis with antiphosphotyrosine antibodies show , that DDR1b is activated by collagen and not by EGF or PDGF (Figure 19C) .The combination of collagen with EGF does not decrease the degree of activation of MAPK or the tyrosine phosphorylation of DDR1b This experiment shows that the activation of DDR1 and DDR2 it does not result in the activation of the MAPK route, and the activation of MAPK by EGF is not influenced by the simultaneous activation of DDR receivers.

Claims (21)

1. CLAIMS 1. An isolated complex comprising (a) a discoidine domain receptor tyrosine kinase or a part thereof and a collagen or part thereof; (b) a discoidine domain receptor tyrosine kinase or a part thereof and a Shc or PTB binding domain of Shc; or (c) a discoidine domain receptor tyrosine kinase or part thereof, and a PDZ domain-containing protein, or a PDZ domain.
2. 2. An isolated complex according to claim 1, comprising a discoidin domain receptor 1, or a part thereof, and a type I, II, III, IV or V collagen, or a part thereof.
3. 3. An isolated complex according to claim 1, comprising a discoidin domain receptor 2, or a part thereof, and a type I or III collagen or a part thereof.
4. 4. An isolated complex according to claim 1, wherein the tyrosine kinase of the discoidin domain receptor or a part thereof is an oligomer.
5. 5. An isolated complex according to claim 1, comprising a discoid domain domain 1b and Shc or PTB binding domain of Shc.
6. 6. A peptide derived from the tyrosine kinase binding domain of discoidin domain receptor that interacts with a collagen or interacts with Shc or a protein containing a PDZ domain
7. 7. A molecule derived from the collagen-binding domain that interacts with a tyrosine kinase of the discoidin domain receptor.
8. 8. An antibody specific for a complex according to claim 2.
9. 9. A method for modulating a discoidine domain receptor tyrosine kinase-mediated signaling pathway in a cell, comprising reacting a protein tyrosine kinase from discoidin domain receptor, or an isoform or part of the protein with a collagen or part of a collagen, or which reacts the cell with a complex according to claim 1, a peptide according to claim 6, a A molecule according to claim 7, or an antibody according to claim 8, thus modulating the signaling path in the cell.
10. A method according to claim 9, wherein the tyrosine kinase protein of the discoidin domain receptor is a discoidin domain receptor 1 or part thereof and the collagen is a type I, II, III, IV collagen or V, or part thereof.
11. 11. A method according to claim 9, wherein the díscoidin domain receptor tyrosine kinase protein is a discoidin domain receptor 2, or a part thereof, and the collagen is a type I or II collagen or part of it.
12. 12. A method for evaluating a compound for its ability to modulate a DDR-mediated signaling path, comprising the steps of (a) reacting a collagen and at least one discoidin domain receptor tyrosine kinase protein, or an isoform or part of the protein, and a test substance, wherein the collagen and the tyrosine kinase protein of discoidin domain receptor are selected so that they bind to form a collagen-tyrosine kinase receptor-domain protein complex of discoidin; and (b) compare it with a control in the absence of the substance to determine the effect of the substance.
13. 13. A method for identifying a substance that affects a DDR receptor tyrosine kinase-mediated signaling pathway in a cell, comprising (a) reacting a collagen or part thereof, and at least one protein tyrosine kinase from discoidin domain receptor, or an isoform or a part of the protein and a test substance, wherein the collagen and the discoid domain receptor tyrosine kinase protein are selected such that they bind to form a collagen complex Tyrosidine domain receptor tyrosine kinase protein, under conditions that allow the formation of collagen-protein complex tyrosine kinase receptor dystrophin complexes, and (b) analyze complexes, for free substances, for collagen not complexed, or for the activation of the protein.
14. 14. A method according to claim 13, wherein the activation of the protein is analyzed by measuring tyrosine phosphorylation of the protein, oligomerization of the protein, binding of a PTB domain to the juxtamembrane domain of the tyrosine kinase protein of the protein domain. juxtamembrane, or analyzing a biological effect on the cell.
15. A method according to claim 13, wherein the tyrosine kinase protein of the discoidin domain receptor is a discoidin domain receptor 1, or a part thereof and the collagen is a type I, II, III collagen , IV or IV or a part of it.
16. 16. A method according to claim 13, wherein the tyrosine kinase protein of the discoidin domain receptor is a discoidin domain receptor 2, or a part thereof and the collagen is a type I or III collagen or a part of it.
17. 17. A method according to claim 13, wherein the test substance is a carbohydrate portion of a collagen or an imitation thereof or a peptide derived from the domain of a DDR that binds to a collagen.
18. A method for treating or preventing a condition involving a tyrosidine receptor receptor tyrosine kinase signaling pathway, which method comprises administering to a patient in need thereof an amount of a substance that is effective to interfere with a pathway of where the substance is (a) a tyrosine kinase of discoidin domain receptor or part thereof; (b) a collagen or part thereof; (c) a substance identified with a method according to claim 13; (c) an isolated complex according to claim 1; (d) a peptide according to claim 6; (e) a molecule according to claim 7, or (f) an antibody according to claim 8.
19. 19. A composition comprising (a) an isolated and purified discoidin domain receptor tyrosine kinase or part thereof; (c) a substance identified first by a method according to claim 13, (c) an isolated complex according to claim 1 (d) a peptide according to claim 6; (e) a molecule according to claim 7, or (f) an antibody according to claim 8.
20. 20. A composition according to claim 19, comprising an extracellular domain of a tyrosine kinase of the domain receptor of discoidin, or the portion of the extracellular domain that binds to the carbohydrate portion of a collagen, or imitations thereof.
21. 21. A method for upregulating the expression of MMP-1 in a cell comprising administering a discoidin domain receptor 2 or an oligomer thereof.