2D and 3D Matrices to Study Linear Invadosome Formation and Activity
Julie S. Di MartinoElodie HenrietZakaria EzzoukhryChandrani MondalJose Javier Bravo‐CorderoViolaine MoreauFrédéric Saltel
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Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.Keywords:
DDR1
Discoidin domain
Matrix (chemical analysis)
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Collagen is the most abundant component of tumor extracellular matrix (ECM). ECM collagens are known to directly interact with the tumor cells via cell surface receptor and play crucial role in tumor cell survival and promote tumor progression. Collagen receptor DDR1 is a member of receptor tyrosine kinase (RTK) family with a unique motif in the extracellular domain resembling Dictyostelium discoideum protein discoidin-I. DDR1 displays delayed and sustained activation upon interaction with collagen and recent findings have demonstrated that DDR1-collagen signaling play important role in cancer progression. In this review, we discuss the current knowledge on the role of DDR1 in cancer metastasis and possibility of a potential therapeutic approach of DDR1 targeted therapy in cancer.
DDR1
Discoidin domain
Receptor Protein-Tyrosine Kinases
Tumor progression
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C1q is known to perform several functions in addition to the role it plays in complement activation. C1q contains a collagen-like portion and DDR1 (discoidin domain receptor 1) is a well-known collagen receptor. Accordingly, we hypothesized C1q might be a novel ligand of DDR1. This study shows for the first time C1q directly induces the activation and upregulation of DDR1, and that this leads to enhanced migration and invasion of HepG2 cells. In addition, C1q was found to induce the activations of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/Akt signaling, and to increase the expressions of matrix metalloproteinases (MMP2 and 9). Our results reveal a relationship between C1q and DDR1 and suggest C1q-induced DDR1 activation signaling may be involved in the progression of hepatocellular carcinoma.
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DDR1
Complement
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The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site. The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site. receptor tyrosine kinase discoidin domain receptor discoidin homology transmembrane extracellular domain antibody monoclonal antibody bovine serum albumin phosphate-buffered saline bis(sulfosuccinimidyl)suberate Communication between cells and their environment is mediated by specific cell surface receptors that transduce signals from the outside of the cell to the inside. An important class of signaling receptors are receptor tyrosine kinases (RTKs),1 which play crucial roles in many fundamental cellular processes, including the cell cycle, differentiation, migration, and metabolism (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). RTKs are not only regulators of normal cellular processes but are also critically involved in the development and progression of human cancers, making them important targets for cancer intervention strategies (2Shawver L.K. Slamon D. Ullrich A. Cancer Cell. 2002; 1: 117-123Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). Most RTKs are activated by soluble proteins present in the blood or other body fluids. The two closely related receptors of the discoidin domain receptor (DDR) RTK subfamily, DDR1 and DDR2, are unusual in that they are activated by an extracellular matrix protein, triple-helical collagen (3Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 4Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). This activation is independent of the major cellular collagen receptors, औ1 integrins, as shown for DDR1 (5Vogel W. Brakebusch C. Fassler R. Alves F. Ruggiero F. Pawson T. J. Biol. Chem. 2000; 275: 5779-5784Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The two DDRs differ in their ligand specificities; whereas both are activated by fibrillar collagens (types I–III and V), only DDR1 can be activated by nonfibrillar collagens, such as type IV collagen. Another intriguing feature of DDRs is their unusually slow autophosphorylation upon stimulation by the ligand compared with typical RTKs (hours rather than seconds) (3Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 4Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Both DDRs are widely expressed in human and mouse tissues, with distinct distributions. DDR1 is mainly expressed in epithelial cells, whereas DDR2 is found in mesenchymal cells (6Alves F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618PubMed Google Scholar). The physiological functions of the DDRs have only begun to emerge, but it is clear that both receptors are involved in cell interactions with the extracellular matrix and control adhesion and cell motility. DDR1 signaling is essential for cerebellar granule differentiation (7Bhatt R.S. Tomoda T. Fang Y. Hatten M.E. Genes Dev. 2000; 14: 2216-2228Crossref PubMed Scopus (78) Google Scholar), arterial wound repair (8Hou G. Vogel W. Bendeck M.P. J. Clin. Invest. 2001; 107: 727-735Crossref PubMed Scopus (187) Google Scholar), and mammary gland development (9Vogel W.F. Aszodi A. Alves F. Pawson T. Mol. Cell. Biol. 2001; 21: 2906-2917Crossref PubMed Scopus (251) Google Scholar), whereas DDR2 regulates chondrocyte (10Labrador J.P. Azcoitia V. Tuckermann J. Lin C. Olaso E. Manes S. Bruckner K. Goergen J.L. Lemke G. Yancopoulos G. Angel P. Martinez A.C. Klein R. EMBO Rep. 2001; 2: 446-452Crossref PubMed Scopus (224) Google Scholar), hepatic stellar cell (11Olaso E. Ikeda K. Eng F.J. Xu L. Wang L.H. Lin H.C. Friedman S.L. J. Clin. Invest. 2001; 108: 1369-1378Crossref PubMed Scopus (251) Google Scholar), and fibroblast (12Olaso E. Labrador J.P. Wang L. Ikeda K. Eng F.J. Klein R. Lovett D.H. Lin H.C. Friedman S.L. J. Biol. Chem. 2002; 277: 3606-3613Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) proliferation. DDR1 mRNA is up-regulated in several malignant tumors (13Laval S. Butler R. Shelling A.N. Hanby A.M. Poulsom R. Ganesan T.S. Cell Growth Differ. 1994; 5: 1173-1183PubMed Google Scholar, 14Barker K.T. Martindale J.E. Mitchell P.J. Kamalati T. Page M.J. Phippard D.J. Dale T.C. Gusterson B.A. Crompton M.R. Oncogene. 1995; 10: 569-575PubMed Google Scholar, 15Perez J.L. Jing S.Q. Wong T.W. Oncogene. 1996; 12: 1469-1477PubMed Google Scholar, 16Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Crossref PubMed Scopus (116) Google Scholar, 17Weiner H.L. Huang H. Zagzag D. Boyce H. Lichtenbaum R. Ziff E.B. Neurosurgery. 2000; 47: 1400-1409Crossref PubMed Google Scholar), and DDR2 is present in stromal cells surrounding highly invasive DDR1-positive tumor cells (6Alves F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618PubMed Google Scholar). The elevated expression of DDRs in a number of fast growing invasive tumors suggests an important role of these matrix-activated RTKs in the proliferation and stromal invasion of tumors. DDR1 and DDR2 are composed of an N-terminal ∼150- amino acid discoidin homology (DS) domain (18Baumgartner S. Hofmann K. Chiquet-Ehrismann R. Bucher P. Protein Sci. 1998; 7: 1626-1631Crossref PubMed Scopus (169) Google Scholar), followed by a sequence of ∼220 amino acids unique to DDRs, a transmembrane (TM) domain, a large cytosolic juxtamembrane domain, and a C-terminal catalytic tyrosine kinase domain. The DDR DS domains are homologous to Dictyostelium discoideum discoidin I and to functionally important DS domains of known structure in a number of secreted (e.g. blood coagulation factors V and VIII) (19Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Crossref PubMed Scopus (223) Google Scholar, 20Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.L. Nature. 1999; 402: 439-442Crossref PubMed Scopus (285) Google Scholar) and membrane-bound mammalian proteins (e.g. neuropilins) (21Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure. 2003; 11: 99-108Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). No convincing data are available to define the location and nature of the collagen binding site(s) of DDRs. A recent study attempted to map DDR1 residues critical for collagen binding (22Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), but the results are inconclusive, since only highly conserved core residues in the DS domain were targeted by mutagenesis. To gain insight into the molecular basis of DDR-collagen signaling, I have studied an array of recombinant DDR proteins, obtained by eukaryotic expression, in collagen binding and cell-based receptor activation assays. I demonstrate for the first time that the isolated extracellular domains (ECDs) of DDR1 and DDR2 bind directly to collagen with high affinity and that binding requires these domains to be dimerized. Using deletion mutants, I show that the DS domain of DDR2 fully contains the collagen binding site. Finally, homology scanning of the DDR2 DS domain identified three spatially adjacent loop regions as essential for collagen binding and receptor activation. Human embryonic kidney 293 cells (ATCC, Manassas, VA), 293-EBNA cells (Invitrogen), and 293-T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium/F-12 nutrient mixture (Invitrogen) containing 107 fetal bovine serum. BSA, κ-casein, collagen I (acid soluble from rat tail; C-7661), collagen IV (acid-soluble from human placenta; C-5533), and fibronectin (0.17 solution from human plasma) were obtained from Sigma. EHS mouse tumor laminin was purchased from BD Biosciences (Oxford, UK). Bis(sulfosuccinimidyl)suberate (BS3) was from Pierce. Puromycin was obtained from Sigma, and zeocin was from Invitrogen. The antibodies (Abs) and their sources were as follows: anti-DDR1, rabbit anti-DDR1 Ig (sc-532 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-DDR2, goat anti-DDR2 Ig (sc-7554 from Santa Cruz Biotechnology); mouse anti-Myc tag, clone 9E10 from Upstate Biotechnology (Lake Placid, NY); peroxidase-conjugated goat anti-human Fc from Jackson ImmunoResearch Laboratories (West Grove, PA); anti-phosphotyrosine, clone 4G10, from Upstate Biotechnology. Secondary Abs were as follows: sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences); goat anti-rabbit Ig-horseradish peroxidase (Dako, Ely, UK); rabbit anti-goat IgG-horseradish peroxidase (Sigma). Restriction and modification enzymes were purchased from New England Biolabs (Hitchin, UK) or Promega (Southampton, UK). All PCR amplification reactions were performed with Pfu DNA polymerase according to the manufacturer's instructions (Stratagene, Amsterdam, The Netherlands). All PCR-derived sequences in the final constructs were verified by DNA sequencing. cDNA encoding full-length DDR1b (TrkE) in pBluescript vector was received from Dr. Michele de Luca (Istituto Dermopatico Dell'Immacolata, Rome, Italy). cDNA encoding full-length DDR2 in pBluescript vector was received from Dr. Michel Faure (SUGEN Inc., San Francisco, CA) as pBS-Tyro10. For expression in eukaryotic cells, these cDNAs were cloned into the mammalian expression vector pcDNA 3.1/Zeo (Invitrogen). His-tagged constructs were made by PCR amplification from cDNA clones using primers that introduced a novel EcoRI restriction site followed by a NheI restriction site on the 5′ end and a stop codon followed by aXhoI and a BamHI site on the 3′ end of the amplified cDNA fragment. The PCR products were cut withEcoRI and BamHI and subcloned into pSP72 vector (Promega). For episomal expression in eukaryotic cells, theNheI/XhoI fragment was cloned into a modified pCEP-Pu vector (23Kohfeldt E. Sasaki T. Gohring W. Timpl R. J. Mol. Biol. 1998; 282: 99-109Crossref PubMed Scopus (202) Google Scholar), which codes for a fusion protein containing at the N terminus the BM-40 signal sequence, a His6 tag, a Myc antigen, and an enterokinase cleavage site. In the DDR constructs, the enterokinase site is followed by the ECD fragments of DDR1, DDR2, or the various deletion constructs (Fig. 1A). For all DDR1 constructs, the first amino acid of the ECD fragments was Asp19, which is the first amino acid after the predicted signal peptide cleavage site. The C-terminal amino acid of the constructs was Thr416, which is the last residue before the predicted TM domain. Similarly, DDR2 constructs encompassed sequences between Lys22 and Thr398. Fc fusion proteins were constructed with the ECDs of DDR1, DDR2, and the various deletion constructs fused to the hinge, CH2, and CH3 domains of human IgG1 (24Fawcett J. Holness C.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar). The cDNAs encompassed coding sequences for the natural signal sequence up to Thr416 (DDR1) or up to Thr398 (DDR2) (Fig.1A). These cDNA sequences were isolated by PCR amplification from relevant cDNA constructs using primers that incorporated a 5′ EcoRI site and a 3′ BamHI site.EcoRI/BamHI-cut PCR products were cloned into the expression vector pcFc. pcFc was constructed by cloning theHindIII/NotI fragment encompassing the Fc sequences of the Fc expression vector pIG1 (24Fawcett J. Holness C.L. Needham L.A. Turley H. Gatter K.C. Mason D.Y. Simmons D.L. Nature. 1992; 360: 481-484Crossref PubMed Scopus (302) Google Scholar) into the expression vector pcDNA3.1/Zeo. All deletion mutants were constructed by overlap extension PCR (25Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (432) Google Scholar) from full-length cDNA clones. Two cDNA fragments (A and B) were amplified using specific primers such that the 3′ end of fragment A was complementary to the 5′ end of fragment B. Fragment A encompassed sequences 5′ of the deletion, including a natural unique restriction site, and fragment B encompassed sequences 3′ of the deletion, including another unique restriction site. The overlap was designed to result in the joining of the desired amino acid sequences, as detailed in Fig. 1B. Both fragments were purified by gel electrophoresis and fused via their overlapping sequences by a secondary PCR reaction. The amplified fused product was restriction digested and subcloned into vectors encoding the full-length DDRs after the corresponding wild-type fragment was removed. For expression in eukaryotic cells, deletion constructs encoding the TM receptors with intact cytoplasmic domains were cloned into pcDNA 3.1/Zeo, as above. DDR2 loop chimera cDNAs were constructed by overlap extension PCR, amplifying two cDNA fragments, A and B, in which the 3′ end of fragment A was complementary to the 5′ end of fragment B. For all fragments A the 5′ primer was 5′-CGGAATTCACAGAGAATGCTCTGCACCCGTT, which introduced a novel EcoRI restriction site 5′ relative to the start codon; for all fragments B, the 3′ primer was 5′-TGGTATTGACACTTGATGGCACTGG, which primes just 3′ of a uniqueAatII restriction site. The overlap was designed to result in the desired loop exchange. Table Idepicts the 3′ primers for fragments A and the 5′ primers for fragments B. Both fragments were purified by gel electrophoresis and fused via their overlapping sequences by a secondary PCR reaction. The amplified fused product was restriction-digested with EcoRI andAatII and subcloned into pBS-Tyro10 after the corresponding wild-type fragment was removed.Table IOligonucleotides used for the construction of DDR2 loop chimerasNameSequence of oligonucleotides 5′ to 3′L1AGGCGGACCAGTTCGTTGAGTACTGACTGGAAGCTGTGATGTCCL1BTACTCAACGAACTGGTCCGCCAAATATGGAAGGCTGGACTCL2AGCATTCACACGTCCCTGGGCGTCCAGCCTTCCATATTTGGCAGCL2BGCCCAGGGACGTGTGAATGCCTGGTGCCCTGAGATTCCL3ACTCCTTATTGTTGTTTGCCTCAGGGCACCAGGCTCCATCCL3BCCTGAGGCAAACAACAATAAGGAGTTTCTGCAGATTGACTTGCL4ACATTTCAGAGGACAGAGACTTGCACCCCTGGGTCCCCACCAGAGTL4BTGCAAGTCTCTGTCCTCTGAAATGTTTGCCCCCATGTACAAGATCAAT Open table in a new tab His-tagged proteins were produced from episomally transfected 293-EBNA cells; Fc-tagged proteins were produced from episomally transfected 293-T cells. Cells were transfected using Fugene reagent (Roche Applied Science). 24 h later, cells containing the episome were selected with either 1 ॖg/ml puromycin (293-EBNA cells) or 100 ॖg/ml zeocin (293-T cells). Resistant cells were allowed to grow to confluence and used for the collection of serum-free conditioned medium. Serum-free medium was collected from T150 tissue culture flasks after incubation for 2 days at 37 °C and again after another 2–3 days of culture at 37 °C. The harvested media (typically 0.5–1 liter) were pooled and cleared of detached cells by centrifugation, followed by filtration through a 5-ॖm pore filter. For the purification of His-tagged proteins, sodium phosphate buffer (500 mm, pH 7.4) was added to a final concentration of 50 mm. TALON metal affinity beads (Clontech), equilibrated in 50 mmsodium phosphate buffer, pH 7.4, 300 mm NaCl (binding buffer), were added to the medium. After incubation for 16 h at 4 °C on a magnetic stirrer, the beads were washed with binding buffer and transferred to a disposable column. After extensive washing, the His-tagged proteins were eluted with 150 mm imidazole, 300 mm NaCl, 50 mm sodium phosphate, pH 7.0. For the purification of Fc-tagged proteins, sodium phosphate buffer (500 mm, pH 7.0) was added to the clarified media to a final concentration of 20 mm. Protein A-Sepharose beads (Amersham Biosciences), equilibrated in PBS, were added and incubated for 16 h as above. The beads were washed with PBS and transferred to a disposable column. The Fc-tagged proteins were eluted with 100 mm citrate, pH 3.0, and immediately neutralized with 1m Tris, pH 9.0. All recombinant proteins were concentrated by ultrafiltration and dialyzed against PBS. Electrophoresis demonstrated a purity of >957. The yields were in the range of 3–7 mg/liter of conditioned medium. Collagen or other ligand proteins were diluted in PBS and coated in 50-ॖl aliquots onto 96-well microtiter plates (Maxisorp, Nalge NUNC International, Rochester, NY), overnight at room temperature. To denature collagen, samples were heated to 50 °C for 30 min prior to coating the wells. Wells were then blocked with 150 ॖl of 1 mg/ml BSA in PBS plus 0.057 Tween 20 (PBS-T) (DDR2 binding assays) or 0.04 mg/ml κ-casein in PBS-T (DDR1 binding assays) for 1 h at room temperature. After one wash with incubation buffer (0.5 mg/ml BSA in PBS-T for DDR2 binding assays; identical to blocking buffer for DDR1 binding assays), 50-ॖl aliquots of the recombinant DDR proteins diluted in incubation buffer were added for 3 h at room temperature. Wells were washed six times with incubation buffer, and 50-ॖl aliquots of mouse anti-Myc monoclonal Ab (mAb) (1:500 dilution; for His-tagged proteins) or goat anti-human Fc Ab coupled to horseradish peroxidase (1:5000 dilution; for Fc-tagged proteins) were added for 1 h at room temperature. After six washes as above, 50-ॖl aliquots of sheep anti-mouse horseradish peroxidase Ab (1:1000 dilution) were added for 1 h at room temperature (His-tagged proteins only), followed by six washes as above. Bound DDR proteins were detected with 75 ॖl/well of 0.5 mg/mlo-phenylenediamine dihydrochloride (Sigma) in 50 mm citrate-phosphate, pH 5.0, added for 3–5 min. The reaction was stopped with 50 ॖl/well of 3 mH2SO4. Plates were read in an enzyme-linked immunosorbent assay reader at a wavelength of 490 nm. All binding assays were carried out in duplicate and showed less than 157 difference on the same plate. The assays were highly reproducible with less than 157 variation between different experiments. 293 cells in 12-well tissue culture plates were transfected by calcium phosphate precipitation with the relevant DDR expression vectors. 24 h later, the cells were incubated in serum-free medium for 16 h. Cells were then stimulated with either 10 ॖg/ml collagen or 1 mm sodium orthovanadate for 90 min at 37 °C. After washing with PBS, cells were lysed in 17 Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 ॖg/ml aprotinin, 1 mm sodium orthovanadate, 5 mm NaF. The detergent-soluble fraction was recovered by centrifugation, and aliquots were analyzed by SDS-PAGE on 7.57 polyacrylamide gels, followed by blotting onto nitrocellulose membranes. The blots were probed with mouse anti-phosphotyrosine mAbs, followed by sheep anti-mouse horseradish peroxidase. Detection was by enhanced chemiluminescence (Amersham Biosciences). To reprobe the blots, the membranes were stripped in 62.5 mm Tris, pH 6.8, 27 SDS, 100 mm औ-mercaptoethanol at 55 °C for 30 min and probed with rabbit anti-DDR1 or goat anti-DDR2 Abs followed by goat anti-rabbit Ig-horseradish peroxidase or rabbit anti-goat IgG-horseradish peroxidase, respectively. Gel filtration chromatography was carried out at 4 °C using an Amersham Biosciences ÄKTA system and a Superdex S200 HR10/30 column. All experiments were done using PBS at a flow rate of 0.5 ml/min. Elution was monitored by UV absorbance at 280 nm. The S200 column was calibrated with the following molecular mass standards (Sigma): carbonic anhydrase (29 kDa), bovine albumin (66 kDa), and alcohol dehydrogenase (150 kDa). Up to 150 ॖl of DDR samples (1–2 mg/ml) were injected per run. Cross-linking was performed for 1 h at room temperature with the homobifunctional reagent BS3. 4 ॖg of DDR proteins were incubated in PBS with different concentrations of BS3 in a final volume of 15 ॖl. The reactions were stopped by the addition of 5× SDS sample buffer containing 107 औ-mercaptoethanol, followed by heating to 100 °C for 5 min and analysis by SDS-PAGE on 77 polyacrylamide gels. Human DDR1 and DDR2 are classified as collagen receptors (3Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 4Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), but a direct protein-protein binding assay that allows an estimation of binding strength has been lacking. I have developed a simple and robust enzyme-linked immunosorbent assay-type assay to measure the binding of soluble DDR fragments to immobilized collagens. Recombinant proteins corresponding to the ECDs of the DDRs, N-terminally tagged with a His tag and a Myc epitope (his-DDR1 and his-DDR2; Fig. 1A) were produced in stably transfected human 293-EBNA cells and purified from serum-free medium. his-DDR2 exhibited dose-dependent, saturable binding to rat tail collagen I, whereas his-DDR1 did not display any binding above background levels (Fig.2A). his-DDR2 binding to collagen I was of high affinity, with half-maximal binding at ∼10–20 nm his-DDR2. Collagen binding by his-DDR2 was specific, since only little binding occurred to denatured collagen I (Fig.2B). Furthermore, the ligand specificity of his-DDR2 mirrored that of the full-length receptor, as determined indirectly by autophosphorylation (3Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). Thus, no binding was detectable to fibronectin, collagen IV or laminin (Fig. 2B). To create recombinant DDR proteins with a different tag, the C termini of the ECDs were fused to a human IgG1 Fc sequence, which mediates covalent dimerization via disulfide bridges (DDR1-Fc and DDR2-Fc; Fig.1A). This approach was only successful for DDR1. Upon transient transfection of 293-T cells, only DDR1-Fc and no DDR2-Fc was secreted by the cells (data not shown). DDR1-Fc was produced and purified from the serum-free medium of stably transfected cells. In contrast to his-DDR1, which did not bind to collagen I, DDR1-Fc showed dose-dependent, saturable binding to collagen I, similar to his-DDR2 (Fig. 2C). In contrast to his-DDR2, DDR1-Fc showed binding to collagen IV, consistent with previous observations that full-length DDR1 can be activated by collagen IV (3Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 4Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar) (data not shown). Collagen binding by DDR1-Fc was also specific for triple-helical collagen as the binding to denatured collagens I or IV was greatly reduced. No binding was detected to laminin or fibronectin (data not shown). The ECD of DDR1 bound to collagen only when fused to a dimerizing Fc tag, suggesting that receptor dimerization is required for ligand binding. Since his-DDR2, whose oligomerization state is not determined by the tag, avidly bound to collagen, I suspected that his-DDR2 might already exist as a (noncovalent) dimer, whereas his-DDR1 might lack features responsible for dimerization. Indeed, his-DDR1 and his-DDR2 eluted at different positions from a gel filtration column (12.4- and 10.8-ml elution volume, respectively) (Fig.3A), suggesting that his-DDR2 forms higher oligomers in solution than his-DDR1. A small shoulder in the elution profile of his-DDR2 coincided with the elution volume of his-DDR1. Because of the presumed nonglobular shape of DDR ECDs, the elution volumes cannot be used to determine molecular masses. The most likely explanation of the gel filtration data, however, is that his-DDR1 is a monomer, whereas his-DDR2 is a noncovalent dimer, with a small monomer fraction. To further examine the oligomeric states of his-DDR1 and his-DDR2 I used chemical cross-linking (Fig. 3B). Dimers of his-DDR2 were readily detected at higher concentrations of cross-linker, whereas only the monomeric form was present for his-DDR1 across the entire range of cross-linker concentrations. No oligomers higher than dimers were seen for his-DDR2. Taken together, these data demonstrate that the ECD of DDR2, but not of DDR1, exists as a noncovalent dimer in solution, and that DDR dimerization is required for collagen binding. The ECDs of the DDRs are composed of an N-terminal DS domain followed by a ∼220-amino acid region of no homology to other proteins (Fig. 1). To establish which domains within the ECDs of the DDRs are involved in the binding to collagen, a set of deletion constructs were made (Fig.4A). For all constructs, cDNAs coding for His-tagged proteins and Fc fusion proteins were created. Unfortunately, not all constructs were secreted by the cells following transfection with the respective expression vectors (Fig.4A). Only full-length DDR1 and ΔDS1 Fc fusion proteins, but not DS1–1 and DS1–2, were obtained, whereas all His-tagged DDR1 constructs were produced, albeit with poor yields for his-DS1–1 and his-DS1–2 (data not shown). Conversely, only DS2 Fc fusion protein and not full-length DDR2 (see above) or ΔDS2 were secreted. All His-tagged DDR2 proteins except ΔDS2 were produced. The contribution of the DS domain to collagen binding by DDR1 could not be studied, because no DDR1 deletion construct containing the DS domain could be obtained as a Fc fusion protein. To study the contribution of the DS domain to collagen binding by DDR2, DS2-Fc and his-DS2 were purified from the serum-free medium of stably transfected cells. DS2-Fc showed strong and saturable binding to collagen I with the same specificity as full-length his-DDR2 (Fig. 4B; compare with Fig. 2), demonstrating that the DS domain of DDR2 is sufficient for high affinity binding to collagen I. His-DS2, in contrast, showed only limited binding to collagen I (data not shown). When analyzed by chemical cross-linking, his-DS2 was found to be mainly monomeric (data not shown). These results are in accord with the binding and oligomerization data described above and further emphasize that DDRs have to be dimerized in order to bind collagen. To be able to relate collagen binding to
Discoidin domain
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DDR1
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Role of microRNA-199a-5p and discoidin domain receptor 1 for human hepatocellular carcinoma invasion
Aims: Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be down-regulated in hepatocellular carcinoma (HCC). Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, is predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC. Material and Methods: Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and in five hepatoma cell lines by the use of TaqMan® real-time quantitative RT-PCR. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using the HepG2 cell line. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Results: A significant down-regulation of miR-199a-5p was observed in 65% of HCC tissues and in four of five cell lines as compared to adjacent non-tumor tissues and normal hepatocytes, respectively. In contrast, DDR1 was significantly overexpressed in 52% of HCC samples and in two of five cell lines. High DDR1 expression in HCC was associated with advanced tumor stage. Enhanced miR-199a-5p expression inhibited invasion of HepG2 cells in vitro by directly reducing DDR1 mRNA and protein levels. Conclusions: Altered expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.
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The Discoidin Domain Receptor 1 (DDR1) receptor tyrosine kinase performs pleiotropic functions in the control of cell adhesion, proliferation, survival, migration, and invasion. Aberrant DDR1 function as a consequence of either mutations or increased expression has been associated with various human diseases including cancer. Pharmacological inhibition of DDR1 results in significant therapeutic benefit in several pre-clinical cancer models. Here, we discuss the potential implication of DDR1-dependent pro-survival functions in the development of cancer resistance to chemotherapeutic regimens and speculate on the molecular mechanisms that might mediate such important feature.
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The discoidin domain receptors, (DDR)1 and DDR2, have been linked to numerous human cancers. We sought to determine expression levels of DDRs in human lung cancer, investigate prognostic determinates, and determine the prevalence of recently reported mutations in these receptor tyrosine kinases. Tumour samples from 146 non-small cell lung carcinoma (NSCLC) patients were analysed for relative expression of DDR1 and DDR2 using quantitative real-time PCR (qRT-PCR). An additional 23 matched tumour and normal tissues were tested for differential expression of DDR1 and DDR2, and previously reported somatic mutations. Discoidin domain receptor 1 was found to be significantly upregulated by 2.15-fold (P=0.0005) and DDR2 significantly downregulated to an equivalent extent (P=0.0001) in tumour vs normal lung tissue. Discoidin domain receptor 2 expression was not predictive for patient survival; however, DDR1 expression was significantly associated with overall (hazard ratio (HR) 0.43, 95% CI=0.22-0.83, P=0.014) and disease-free survival (HR=0.56, 95% CI=0.33-0.94, P=0.029). Multivariate analysis revealed DDR1 is an independent favourable predictor for prognosis independent of tumour differentiation, stage, histology, and patient age. However, contrary to previous work, we did not observe DDR mutations. We conclude that whereas altered expression of DDRs may contribute to malignant progression of NSCLC, it is unlikely that this results from mutations in the DDR1 and DDR2 genes that we investigated.
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The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.
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Hydroxylysine
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Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52–Thr-57) and loop 3 (Arg-105–Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function. Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52–Thr-57) and loop 3 (Arg-105–Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function. Collagens are the most abundant proteins found in the animal kingdom. Whereas some collagens are key structural components in load-bearing tissues, others are essential elements of basement membranes. Collagens have a pivotal role in regulating cellular differentiation and pattern formation during embryogenesis and postnatal development. Increased synthesis of fibrillar collagens or perturbed turnover correlates with a variety of human diseases, including liver fibrosis, glomerulonephritis, vascular diseases, or tumor angiogenesis (1Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Google Scholar). Three different types of collagen-receptors are currently known: the tyrosine kinases discoidin domain receptor 1 and 2 (DDR1 and DDR2), 1Abbreviations used are: DDR, discoidin domain receptors; DiscD, discoidin domain; Npn-1, neuropilin-1; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; HRP, horseradish peroxidase; ExD, extracellular domain. four integrin heterodimers containing the β1 subunit, and glycoprotein VI (2Vogel W.F. Eur. J. Dermatol. 2001; 11: 506-514Google Scholar). Although glycoprotein VI is only found on platelets, both integrins and DDR are widely expressed and trigger an array of signaling pathways upon collagen binding. DDR1 and DDR2 are characterized by a ∼155-amino acid discoidin homology domain (DiscD) in the extracellular region of the protein. The discoidin domain is followed by a 200-amino acid stretch termed the stalk region, a single transmembrane peptide, a juxtamembrane region, and the catalytic tyrosine kinase domain (Fig. 1A). DDR1 was isolated from a number of different human tissues and carcinoma cell lines (3Alves F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618Google Scholar, 4Nemoto T. Ohashi K. Akashi T. Johnson J.D. Hirokawa K. Pathobiology. 1997; 65: 195-203Google Scholar, 5Weiner H.L. Huang H. Zagzag D. Boyce H. Lichtenbaum R. Ziff E.B. Neurosurgery. 2000; 47: 1400-1409Google Scholar, 6Di Marco E. Cutuli N. Guerra L. Cancedda R. De Luca M. J. Biol. Chem. 1993; 268: 24290-24295Google Scholar, 7Laval S. Butler R. Shelling A.N. Hanby A.M. Poulsom R. Ganesan T.S. Cell Growth Differ. 1994; 5: 1173-1183Google Scholar, 8Sakamoto O. Suga M. Suda T. Ando M. Eur. Respir. J. 2001; 17: 969-974Google Scholar). RNA in situ hybridization analysis showed specific expression of human DDR1 in epithelial cells, particularly in the mammary gland, brain, kidney, lung and the mucosa of the colon (3Alves F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618Google Scholar). DDR2 is also widely expressed, particularly in skeletal and heart muscle, kidney, and skin. DDR1 knockout mice are consistently smaller than wild-type littermates, and most of the female knockout mice are unable to give birth because developing blastocysts do not implant (9Vogel W.F. Aszodi A. Alves F. Pawson T. Mol. Cell. Biol. 2001; 21: 2906-2917Google Scholar). Female mice that do successfully reproduce are unable to nourish their litters because the mammary gland epithelium fails to secret milk. DDR1 is therefore a key regulator of cell morphogenesis, differentiation, and collagen synthesis. DDR2-null mice are also smaller compared with their wild-type littermates, and healing of epidermal wounds (which is normal in the absence of DDR1) is significantly delayed in the absence of DDR2 (10Labrador J.P. Azcoitia V. Tuckermann J. Lin C. Olaso E. Manes S. Bruckner K. Goergen J.L. Lemke G. Yancopoulos G. Angel P. Martinez C. Klein R. EMBO Rep. 2001; 2: 446-452Google Scholar). Both activated DDR induce the expression of specific metalloproteases, including MMP1 and MMP2 (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar, 12Ikeda K. Wang L.H. Torres R. Zhao H. Olaso E. Eng F.J. Labrador P. Klein R. Lovett D. Yancopoulos G.D. Friedman S.L. Lin H.C. J. Biol. Chem. 2002; 277: 19206-19212Google Scholar). DDR1 autophosphorylation is stimulated by all collagens tested (types I–VI and VIII), whereas DDR2 is only activated by fibrillar collagens (types I–III and V). Heat-denatured collagen (gelatin), which is no longer triple-helical in structure, fails to induce DDR kinase activity (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar). The discoidin domain of DDR1 contains a functionally essential collagen binding site, yet the minimal binding sequence(s) on collagen remains elusive (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar). Discoidin domain repeats are also found in about 20 other, mostly secreted proteins (14Baumgartner S. Hofmann K. Chiquet-Ehrismann R. Bucher P. Protein Sci. 1998; 7: 1626-1631Google Scholar, 15Vogel W. FASEB J. 1999; 13: S77-S82Google Scholar). The structures of the C-terminal discoidin domain present in the two blood coagulation factors V and VIII and in the ectodomain of the neuronal receptor neuropilin-1 (Npn-1) have been determined by x-ray crystallography (16Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure (Camb.). 2003; 11: 99-108Google Scholar, 17Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Google Scholar, 18Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.S. Nature. 1999; 402: 439-442Google Scholar). The central feature of all three domains is an eight-stranded antiparallel β-barrel. Although most strands are connected by short turns, in each structure, several extended neighboring loops (or spikes) protrude from the "bottom" of the β-barrel (18Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.S. Nature. 1999; 402: 439-442Google Scholar, 19Fuentes-Prior P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Google Scholar). These loops are involved in the membrane binding of factors V and VIII (17Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Google Scholar, 19Fuentes-Prior P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Google Scholar) and most probably also in the binding of Npn-1 to its ligands, semaphorin 3A and vascular endothelial growth factor (20Mamluk R. Gechtman Z. Kutcher M.E. Gasiunas N. Gallagher J. Klagsbrun M. J. Biol. Chem. 2002; 277: 24818-24825Google Scholar). A disulfide bond links the N- and C-terminal ends in the three structures and is predicted in all mammalian proteins featuring discoidin domains. The importance of studying the structural aspects of discoidin domains is underscored by the fact that mutations within the discoidin domain have been linked to several human diseases: the two most prominent cases are hemophilia and retinoschisis. In human factor V and VIII, point mutations found in the discoidin domain cause parahemophilia and hemophilia A, respectively (21Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Google Scholar, 22Liu M.L. Shen B.W. Nakaya S. Pratt K.P. Fujikawa K. Davie E.W. Stoddard B.L. Thompson A.R. Blood. 2000; 96: 979-987Google Scholar). Point mutations in retinoschisin, a secreted protein that consists of a single discoidin domain, are the cause of retinoschisis, a genetic disorder characterized by macular deterioration and early blindness (23Grayson C. Reid S.N. Ellis J.A. Rutherford A. Sowden J.C. Yates J.R. Farber D.B. Trump D. Hum. Mol. Genet. 2000; 9: 1873-1879Google Scholar, 24Mooy C.M. Van Den Born L.I. Baarsma S. Paridaens D.A. Kraaijenbrink T. Bergen A. Weber B.H. Arch. Ophthalmol. 2002; 120: 979-984Google Scholar). Furthermore, recent work on the protein SED1 indicated that its discoidin domains are centrally involved in cell-matrix communication during sperm-egg binding (25Ensslin M.A. Shur B.D. Cell. 2003; 114: 405-417Google Scholar). In this study, we use site-directed mutagenesis to closely map the collagen-binding regions in the discoidin domain of DDR1. We identified two loops and two single amino acid residues within the loop regions that are essential for collagen-DDR1 recognition. Furthermore, we generated recombinantly expressed DDR1 and DDR2 collagen-binding domains to quantify the receptor-ligand affinity. Molecular Modeling—A preliminary model of the human DDR1 discoidin domain automatically generated using SWISS-MODEL software (www.expasy.org/swissmod/) was overlaid on the three-dimensional structures of factor V (C2 domain, PDB code 1CZT; Ref. 17Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Google Scholar), factor VIII (C2 domain, PDB code 1D7P; Ref. 18Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.S. Nature. 1999; 402: 439-442Google Scholar), and Npn-1 (b1 domain, PDB code 1KEX; Ref. 16Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure (Camb.). 2003; 11: 99-108Google Scholar), manually corrected at a few positions, and subjected to energy minimization using CNS software (cns.csb.yale.edu/v1.1/). The minimized model was employed for the rational design of mutations/deletions. In particular, we verified that limited deletion of loop residues would not affect formation of the central β-barrel; i.e. the shortened loops would still span the distance between conserved residues at the start/end of the barrel strands, with all loop residues adopting stereochemically favorable conformations. The alignment of discoidin domains was performed using ClustalW (www.ebi.ac.uk/clustalw/) and corrected at a few positions according to the structural information. DDR1 Mutagenesis—To tag the full-length human DDR1 cDNA with the HA sequence, a unique NheI site was introduced into the plasmid pRK5-DDR1b before the stop codon. A synthetic linker coding for the HA sequence was then inserted into the NheI site. The QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce single point mutations (W53A, D55A, S66A, D68A, R105A, H106A, L110A, K112A, V173A, M174A, and S175A), a multiple missense mutation with a single residue deletion (ΔW53-S54A-D55A-S56A), and multiple residue deletions (ΔS52-T57, ΔD68-D70, ΔG108-K112, ΔR105-K112, ΔD171-R172). The numbering refers to full-length DDR1 including its signal peptide. The mutants W53A, R105A, and L110A were not HA-tagged. All constructs were sequenced to verify the presence of the respective mutation. Primer sequences used for site-directed mutagenesis are available upon request. Transient Transfection Assay—Human embryonic kidney 293 cells were transfected with 20 μg of DNA per 10 cm dish using the calcium phosphate transfection procedure (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar). The medium was replaced with serum-free Dulbecco's modified Eagle's medium 18 h after transfection. Twenty-four hours after transfection, cells were stimulated with 10 μg/ml rat type I collagen (BD Biosciences). Ninety minutes after stimulation, cells were lysed using either Triton X-100-based lysis buffer (50 mm Tris, pH 7.5, 1% Triton X-100, 150 mm NaCl, 0.5 mm EDTA, 0.5 mm MgCl2, 10 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 10 mg/ml aprotinin) for immunoprecipitation or Tween 20-based lysis buffer (50 mm Tris, pH 7.5, 0.1% Tween 20, 150 mm NaCl, 0.5 mm EDTA, 0.5 mm MgCl2, 10mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 10 mg/ml aprotinin) for ELISA. GST Fusion Protein Purification—DDR1 and DDR2 discoidin and extracellular domains were amplified from human cDNAs. Inserts were subcloned in-frame into pAcG2T (BD Biosciences), resulting in an N-terminal fusion with glutathione S-transferase (GST). The pAcG2T constructs functioned as transfer vectors for the BaculoGold expression system (BD Biosciences). Attenuated BaculoGold DNA was co-transfected with each transfer vector into Sf9 (Spodoptera frugiperda) cells grown in TNM-FH media (Hyclone) with 10% heat-inactivated fetal calf serum, 2% glutamine, 50 μg/ml gentamicin, and 2.5 μg/ml Fungizone at 27 °C. Viruses were amplified for three rounds in sequentially larger monolayer culture dishes (25, 75, and 150 cm2). Five days after infection, viral supernatants were harvested for the next round of amplification. Recombinant viruses were used to infect HiV (Trichoplusia ni) cells grown in monolayer cultures in the above medium. Cells were incubated at 27 °C for 2 days and then lysed using SD buffer (50 mm Tris, pH 7.5, 500 mm NaCl, 0.5 mm EDTA, 0.5 mm MgCl2, 10 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, and 10 mg/ml aprotinin) with 2.5% Triton X-100. Cell lysates were added to glutathione-linked-agarose beads (BD Biosciences Clontech), incubated for 2 h at 4 °C and subsequently washed using SD buffer. To elute protein, 1 bead volume of glutathione elution buffer (100 mm glutathione, 500 mm NaCl, 1 mm EDTA, 10% glycerol, and 100 mm Tris, pH 8.5) was incubated with the beads at 4 °C for 15 min. Immunoprecipitation and Western Blotting—Lysates from 293 cells were diluted 1:1 (v/v) in HNTG buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% Triton X-100, and 10% glycerol) and incubated with anti-DDR1b antibody (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar) coupled to protein A-Sepharose beads (Amersham Biosciences) for 3 h. Beads were washed four times with HNTG buffer and denatured in Laemmli buffer (187.5 mm Tris, pH 6.8, 6% SDS, 30% glycerol, 0.01% bromphenol blue, and 15% 2-mercaptoethanol). Proteins were separated on 7.5% polyacrylamide gels and subjected to Western blotting. Samples were probed using a monoclonal anti-phosphotyrosine antibody (4G10; Upstate Biotechnology) diluted 1:500 in NET-Gel (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, and 0.05% gelatin). Western blots were developed using secondary horseradish peroxidase-linked anti-mouse IgG (Bio-Rad Laboratories) and enhanced chemiluminescence (Amersham Biosciences). Blots were stripped (65 mm Tris, pH 6.8, 2% SDS, and 50 μm 2-mercaptoethanol) and re-probed with anti-DDR1 polyclonal (Santa Cruz Biotechnology) or anti-HA monoclonal (Sigma) antibody and secondary HRP-linked anti-rabbit or -mouse IgG. Lysates from HiV cells were subjected to SDS-PAGE and analyzed by Western blotting with polyclonal antibodies raised against the N-terminal regions of DDR1 and DDR2, respectively (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar). The blots were stripped and re-probed with an anti-GST-linked-HRP monoclonal antibody (Santa Cruz). For membrane fractionation, cells were lysed with Tris-sucrose buffer (10 mm Tris, pH 7.4, 1 mm EDTA, and 0.1 mm sucrose), homogenized, and centrifuged at 3000 × g for 5 min. The supernatant containing cytoplasmic and transmembrane fractions was isolated and centrifuged at 100,000 × g for 1 h. The pellet was resuspended in Tris-sucrose buffer and immunoprecipitated overnight with an anti-DDR1b antibody. Beads were washed three times with Tris-sucrose buffer and subjected to SDS-PAGE. DDR1 was detected using a monoclonal anti-HA antibody (Sigma). Collagen-DDR1 ELISA—Type I collagen was diluted in phosphate-buffered saline to a concentration of 50 μg/ml and added to 96-well microtiter plates (50 μl/well). Plates were incubated for 1 h at room temperature and washed twice with phosphate-buffered saline. Wells were blocked with 150 μl of 1 mg/ml BSA in phosphate-buffered saline containing 0.05% Tween 20 for 1 h. Wells were washed once with 150 μl of wash buffer (0.5 mg/ml BSA in phosphate-buffered saline containing 0.05% Tween 20). DDR1-transfected 293 cells were lysed with Tween 20-based lysis buffer, and lysate was added to each well and incubated at room temperature overnight (lysates were normalized for cell number by Western blot analysis before ELISA). Wells were washed six times with wash buffer. 50-μl aliquots of diluted anti-DDR1 C-terminal antibody (1:500 dilution in wash buffer) were added and incubated for 90 min. Wells were washed six times with wash buffer and incubated with HRP-linked anti-rabbit secondary antibody for 90 min. Bound protein was detected using 75 μl/well of 0.5 mg/ml o-phenylenediamine dihydrochloride (Sigma-Aldrich) in 50 mm citrate-phosphate buffer (50 mm sodium citrate and 0.1 m sodium phosphate, pH 5.0) with 0.012% hydrogen peroxide. After 30 min at room temperature, the reaction was stopped using 50 μl/well of 3 m sulfuric acid. Plates were analyzed in an ELISA reader at 490 nm, buffer only-values were subtracted, and normalized values were subjected to statistical analysis using Microsoft Excel. All experiments were performed in triplicate. For the ELISA of GST-DDR binding to collagen, purified recombinant proteins were added to collagen-coated wells and incubated at room temperature for 3 h. Wells were washed six times with wash buffer, and 50-μl aliquots of anti-GST monoclonal antibody (diluted 1:1000 in wash buffer) were added for 1 h at room temperature. After six washes, bound protein was detected as above. For collagen-DDR1 loop peptide ELISA, 100 μl of lysate from 293 cells transfected with DDR1 were added to each collagen-coated well along with loop peptides in 1250-fold molar excess (relative to collagen) and incubated at room temperature overnight. Binding was detected using polyclonal rabbit anti-DDR1 C-terminal antibody and secondary HRP-linked anti-rabbit IgG antibody. DDR1 and DDR2 constitute a unique pair of tyrosine kinase receptors because they are activated not by soluble growth factors but by native, triple-helical collagen. Although previous work has shown that the DDR-collagen interaction is direct, the molecular basis of this interaction, in particular the precise role of the discoidin domain, remains unclear. To study this interaction, we decided to produce the discoidin domain or the entire extracellular domain (ExD) of DDR1 and DDR2 as recombinant proteins in insect cells. The constructs were expressed as GST fusion proteins in HiV insect cells (Fig. 1B). The ExD of DDR1 and DDR2 fused to GST were detected as 73-kDa proteins and the DiscD fusion proteins as 47-kDa proteins (Fig. 1, C and D). To test the functionality of the fusion proteins, we mixed equal amounts of insect cell lysates with native type I collagen immobilized on agarose beads. We found specific binding of all four DDR fusion proteins to collagen, whereas GST alone showed no binding (Fig. 1, E and F). Purification of the GST fusion proteins by glutathione-agarose affinity chromatography yielded significant amounts of all four constructs (0.1–1.0 mg of protein per 108 cells). Proteins were eluted by glutathione displacement and were subjected to an ELISA-based binding assay. Plates coated with 50 μg/ml type I collagen were incubated with various concentrations of purified GST fusion proteins, and the amount of bound material was quantified by anti-GST ELISA (Fig. 2). At a 100 nm protein concentration, the DDR1-DiscD fused to GST had a 14-fold higher affinity to collagen than GST alone. We were surprised to find that DDR1-ExD had about a 10-fold higher affinity to collagen than the DiscD alone, in that DDR1-ExD binding reached a plateau at a protein concentration of ∼10 nm compared with 100 nm for DDR1-DiscD. The DDR2-DiscD showed a binding profile similar to that of DDR2-ExD, with no significant increase in binding capacity upon addition of the stalk region. In contrast to DDR1-ExD, the DDR2-ExD had much lower affinity for type I collagen, and a linear increase in binding was observed through all concentrations measured (60–180 nm). These differences between DDR1 and DDR2 are possibly caused by diverse sequence motifs within the stalk region. The overall identity between the two sequences is much lower for the stalk regions than for the discoidin domains (44% versus 58%). Furthermore, DDR1 shows a unique stretch of about 15 amino acids near the plasma membrane (amino acids 385–399) that is not present in DDR2. Whereas previous work indicated that the discoidin domain of DDR1 is essential for binding to triple-helical collagen, the molecular composition of this binding epitope remains unknown (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar). To address this question, we exploited the homology between the discoidin domains of human DDR and the following three related human discoidin domains: the C-terminal domain in coagulation factor V, the homologous domain in factor VIII, and the first discoidin domain in Npn-1. We selected these domains, because their three-dimensional structures were recently resolved by x-ray crystallography (16Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure (Camb.). 2003; 11: 99-108Google Scholar, 17Macedo-Ribeiro S. Bode W. Huber R. Quinn-Allen M.A. Kim S.W. Ortel T.L. Bourenkov G.P. Bartunik H.D. Stubbs M.T. Kane W.H. Fuentes-Prior P. Nature. 1999; 402: 434-439Google Scholar, 18Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.S. Nature. 1999; 402: 439-442Google Scholar). Whereas all five sequences share less than 20% identity, the eight strands forming the central β-barrel are well conserved (Fig. 3A). Based on this alignment and the structural information, we generated a three-dimensional model for the DDR1-DiscD (Fig. 3B). The conserved N- and C-terminal cysteine residues (Cys-31 and Cys-185 in DDR1, respectively) form a disulfide bridge located at the top of the barrel. In addition, our model predicts that the Sγ atoms of Cys-74 and Cys-177 are optimally positioned to form a second disulfide bridge. More importantly, at the bottom of the barrel, four prominent, finger-like loops protrude (Fig. 3B). Each loop consists of about 7–11 amino acid residues, and the position of each loop within the primary sequence is conserved between DDR1 and other discoidin domains (as highlighted in Fig. 3A). Based on their exposed character in the molecular model and the relevance of the topologically equivalent regions of other discoidin domains for interactions with diverse ligands (19Fuentes-Prior P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Google Scholar), we hypothesized that these loops may be involved in collagen-binding by DDR1. To test this hypothesis, we designed and produced mutant proteins with partial deletions of the loop sequences that were expected not to compromise the folding of the discoidin domain (Fig. 4A). In addition, we systematically mutated single amino acid residues located in these loops to alanine (Fig. 4B). In total, we created six deletion mutants and 11 point mutants within the context of the full-length DDR1 sequence. Although the initial set of mutants was created using the unmodified DDR1 cDNA (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar), we also introduced an HA tag at the C terminus of the sequence, thereby allowing more accurate normalization of expression levels. We expressed individual mutants in human embryonic kidney 293 cells, stimulated these cells with type I collagen for 90 min, and determined DDR1 tyrosine phosphorylation by immunoprecipitation followed by Western blotting. We found that the mutation of Arg-105 (in loop 3) almost completely abolished DDR1 activation (Fig. 5A), whereas the Ser-175 to alanine mutation in loop 4 entirely lost its responsiveness to collagen (Fig. 5C). No other single amino acid exchange had a measurable effect on DDR1 phosphorylation. Furthermore, modification of loop 1, by either deleting the sequence stretch Ser-52–Thr-57 or by deleting Trp-53 and alanine-mutating residues 54–56 resulted in significant but not complete ablation of DDR1 activity (Fig. 5C). In contrast, deletion of loop 2 (Δ68–70) did not perturb DDR1 function. In agreement with the R105A point mutant, a deletion of loop 3 (Δ105–112) had a detrimental effect on DDR1 phosphorylation. To show that all DDR1 mutant proteins are properly processed within the cell, we prepared membrane fractions of transfected 293 cells and showed equal amounts of DDR1 wild-type and mutant protein localized at the cell surface (Fig. 5E).Fig. 5Identification of collagen binding epitope by site-directed mutagenesis. A and C, tyrosine phosphorylation of DDR1 point mutants upon collagen stimulation. Individual mutants were expressed in 293 cells, and DDR1 immunoprecipitates were analyzed by antiphosphotyrosine (αPY) Western blotting. Phosphorylated DDR1 was detected as a 125-kDa protein. Blots were stripped and reprobed with anti-DDR1 antibodies (B and D) to verify equal expression of mutant proteins. E, membrane fractions of several mutants were isolated and probed with an antibody against the HA-tag to verify surface expression of DDR1.View Large Image Figure ViewerDownload (PPT) To directly quantify collagen-binding of various DDR1 mutants, we used the ELISA-based readout described above. Collagen-coated plates were incubated with lysates from DDR1 wild-type or mutant overexpressing cells. Bound protein was detected by a primary DDR1-specific antibody followed by a secondary horseradish-conjugated antibody. We found that the two point mutants R105A and S175A, which failed to be activated by collagen, largely failed to bind to collagen as well (Fig. 6). Several other point mutants, such as H106A, V173A, and M174A, had markedly reduced binding in the ELISA although they were still activated by collagen when assessed by antiphosphotyrosine Western blotting (Fig. 5). Deletion of residues within any of the four loops resulted in loss of collagen affinity to varying degrees. It was interesting to note that deletion of loop 1 (Δ52–57 and Δ53/54–56A) or loop 3 (Δ105–112 or Δ108–112) largely abolished the binding of the DDR1 discoidin domain to collagen, whereas deletion of loop 2 (Δ68–70) or loop 4 (Δ171–172) led only to a partial reduction. To further define the contribution of individual loop regions in binding collagen, we generated synthetic cyclic peptides mimicking loops 1–3 (Fig. 7A). We tested the ability of these peptides to compete with the bind
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