Exploring the Collagen-binding Site of the DDR1 Tyrosine Kinase Receptor
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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 bindKeywords:
DDR1
Discoidin domain
Discoidin domain
DDR1
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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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Significance Statement The receptor discoidin domain receptor 1 (DDR1) is activated by collagen, upregulated in injured kidneys, and contributes to kidney fibrosis, but how DDR1 controls fibrosis is poorly understood. The authors show that upon collagen stimulation, DDR1 translocates to the nucleus. To do this, DDR1 must bind with SEC61B, a component of the Sec61 translocon, as well as with nonmuscle myosin IIA and β -actin. In the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. The study reveals a novel mechanism whereby collagen-activated DDR1 moves to the nucleus to increase the production of profibrotic molecules. Background The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. Methods To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. Results We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and β -actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. Conclusions These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.
DDR1
Discoidin domain
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DDR1
Discoidin domain
Receptor Protein-Tyrosine Kinases
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DDR1
Discoidin domain
Type I collagen
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The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx'GEx". The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.
Discoidin domain
DDR1
RGD motif
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Discoidin domain
DDR1
GPVI
Collagen fibril
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Citations (44)
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.
DDR1
Discoidin domain
Hydroxylysine
Structural motif
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Citations (175)
Interactions between the discoidin domain receptor 1 and β1 integrin regulate attachment to collagen
Summary Collagen degradation by phagocytosis is essential for physiological collagen turnover and connective tissue homeostasis. The rate limiting step of phagocytosis is the binding of specific adhesion receptors, which include the integrins and discoidin domain receptors (DDR), to fibrillar collagen. While previous data suggest that these two receptors interact, the functional nature of these interactions is not defined. In mouse and human fibroblasts we examined the effects of DDR1 knockdown and over-expression on β1 integrin subunit function. DDR1 expression levels were positively associated with enhanced contraction of floating and attached collagen gels, increased collagen binding and increased collagen remodeling. In DDR1 over-expressing cells compared with control cells, there were increased numbers, area and length of focal adhesions immunostained for talin, paxillin, vinculin and activated β1 integrin. After treatment with the integrin-cleaving protease jararhagin, in comparison to controls, DDR1 over-expressing cells exhibited increased β1 integrin cleavage at the cell membrane, indicating that DDR1 over-expression affected the access and susceptibility of cell-surface β1 integrin to the protease. DDR1 over-expression was associated with increased glycosylation of the β1 integrin subunit, which when blocked by deoxymannojirimycin, reduced collagen binding. Collectively these data indicate that DDR1 regulates β1 integrin interactions with fibrillar collagen, which positively impacts the binding step of collagen phagocytosis and collagen remodeling.
DDR1
Discoidin domain
CD49c
Vinculin
CD49b
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Citations (51)
Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). The DDR1 ligand is the extracellular matrix protein collagen. To identify receptor tyrosine kinases (RTKs) involved in control of growth and differentiation of human bronchial epithelial (HBE) cells, a polymerase chain reaction-based search for RTKs in HBE cells was performed. DDR1 was the most abundant clone identified. Northern analysis detected a 3.6 kb DDR1 messenger ribonucleic acid (mRNA) expressed in HBE cells and transformed HBE lines, BET-1A and BEAS-2B. In addition, fluorescence-activated cell sorter (FACS) analyses using an anti-DDR1 antibody showed that DDR1 was expressed on HBE cells and two HBE lines. Immunohistochemical staining using human bronchial tissue demonstrated that DDR1 was mainly expressed at the basolateral cell surface of the bronchial epithelium. Furthermore, immunostaining of type IV collagen, a major component of the basement membrane, clearly showed that the basement membrane was closely attached to the basal surface of the bronchial epithelium. Since collagen binds to and activates discoidin domain receptor 1 tyrosine kinase, colocalization of discoidin domain receptor 1 and its ligand type IV collagen demonstrates a potential interaction of discoidin domain receptor 1 on the bronchial epithelium with type IV collagen. Further study of this interaction may define the functional significance of the collagen-discoidin domain receptor 1 signalling pathway in health and in disease.
DDR1
Discoidin domain
Receptor Protein-Tyrosine Kinases
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