Exploring the Collagen-binding Site of the DDR1 Tyrosine Kinase Receptor

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: Scholar). In this we to the collagen-binding in the discoidin domain of We two loops and two single amino acid residues within the loop that are essential Furthermore, we expressed DDR1 and DDR2 collagen-binding domains to the model of the human DDR1 discoidin domain was on the three-dimensional structures of factor V domain, 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 factor VIII domain, 18Pratt K.P. Shen B.W. Takeshima K. Davie E.W. Fujikawa K. Stoddard B.S. Nature. 1999; 402: 439-442Google and Npn-1 domain, C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure (Camb.). 2003; 11: 99-108Google at a and subjected to The model was the design of In we that of loop residues not formation of the central the loops the residues at the of the with all loop residues The of discoidin domains was and at a to the structural DDR1 the human DDR1 with the a site was into the the A the sequence was into the The was used to single point mutations and a with a single and The to DDR1 including its The and not to the presence of the used are upon cells with 20 of the (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar). The was with cells stimulated with type I collagen cells 1 1 and or 1 1 and Protein and DDR2 discoidin and extracellular domains from human into in an fusion with glutathione S-transferase The as the expression was with each into cells in with and at three in and the of used to cells in in the at 2 and 1 1 and with Cell to 2 at 4 and To 1 of glutathione 1 and was with the at 4 and from cells in and and with (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar) to protein 3 four with and in and on and subjected to a in and horseradish and and and with or and or from cells subjected to and by with the of DDR1 and respectively (11Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Google Scholar). The and with an membrane cells with 1 and and at The containing and transmembrane was isolated and at 1 The was in and with an three with and subjected to DDR1 was a I collagen was in to a of and to 1 at and with with of 1 in containing 20 1 with of in containing cells with and was to each well and at cell number by analysis with of C-terminal in and with and with protein was of in and with at the was of 3 in an at and subjected to analysis in the of binding to proteins to and at 3 with and of in 1 at protein was as loop ELISA, of from cells with DDR1 to each well with loop in to and at was C-terminal and DDR1 and DDR2 a of tyrosine kinase receptors because are activated not by growth factors by triple-helical Although work that the interaction is the molecular of this in the role of the discoidin domain, remains To study this we to the discoidin domain or the extracellular domain of DDR1 and DDR2 as proteins in insect The expressed as fusion proteins in insect cells (Fig. The of DDR1 and DDR2 to as proteins and the fusion proteins as proteins (Fig. and To test the of the fusion we of insect cell with type I collagen immobilized on We found specific binding of all four DDR fusion proteins to whereas showed no binding (Fig. and of the fusion proteins by affinity of all four of protein by glutathione and subjected to an binding assay. with type I collagen with of fusion and the of was by (Fig. a protein the to a affinity to collagen than We to that about a affinity to collagen than the in that binding a at a protein of compared with The showed a binding to that of with no in binding upon of the stalk In to the affinity type I and a in binding was all These DDR1 and DDR2 are by sequence within the stalk The the two is the stalk than the discoidin domains Furthermore, DDR1 a stretch of about amino the membrane that is not present in Whereas work indicated that the discoidin domain of DDR1 is essential binding to triple-helical the molecular of this binding remains (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar). To this we the homology the discoidin domains of human DDR and the three related human discoidin the C-terminal domain in coagulation factor the domain in factor VIII, and the discoidin domain in We these because their three-dimensional structures 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 than the strands the central β-barrel are well (Fig. on this and the structural we a three-dimensional model the (Fig. The N- and C-terminal residues and in a disulfide at the of the In model predicts that the of and are to a disulfide at the of the four loops protrude (Fig. loop consists of about amino acid and the of each loop within the sequence is DDR1 and discoidin domains in on their in the molecular model and the of the topologically equivalent of discoidin domains with P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Google we that these loops may involved in collagen-binding by To test this we and proteins with of the loop that not to the of the discoidin domain (Fig. In we mutated single amino acid residues in these loops to alanine (Fig. In we created and point within the of the DDR1 Although the of was created the DDR1 (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google we also an at the of the of expression We expressed individual in human cells, stimulated these cells with type I collagen and determined DDR1 tyrosine by followed by We found that the of loop DDR1 (Fig. whereas the Ser-175 to alanine in loop 4 its to collagen (Fig. single amino acid a on DDR1 Furthermore, of loop by the sequence stretch or by and residues in not of DDR1 activity (Fig. In of loop 2 not DDR1 function. In with the point a of loop 3 a on DDR1 To that all DDR1 proteins are within the we membrane of cells and showed of DDR1 wild-type and protein at the cell (Fig. of collagen binding by A and tyrosine of DDR1 point upon collagen expressed in cells, and DDR1 by DDR1 was as a protein. and with and to expression of membrane of several isolated and with an the to expression of To collagen-binding of DDR1 we used the with from DDR1 wild-type or protein was by a followed by a We found that the two point and which to activated by to to collagen as well (Fig. point as and binding in the activated by collagen by (Fig. of residues within of the four loops in of collagen affinity to was to that of loop 1 and or loop 3 or the binding of the DDR1 discoidin domain to whereas of loop 2 or loop 4 only to a To the of individual loop in binding we loops (Fig. We tested the of these to with the binding of DDR1 in to collagen in an a of we to of discoidin-collagen interaction a single or a of all three (Fig. Furthermore, of the was to interfere with DDR1 in cells with or DDR1 not In this by a of individual loop residues to we showed the that three neighboring, loops in the discoidin domain of DDR1 are critically involved in collagen binding. In we and tested with within these loops. We a at of the DDR1 discoidin domain as centrally involved in the This is by residues by loops 1 to and 3 to which protrude to each from the of the discoidin β-barrel (Fig. is that the the to a triple-helical collagen molecule with an of the of and to present we that collagen into this with its major molecular to the of the the the equivalent loop region of the factor V discoidin domain (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 is to that the loops of DDR1 DDR2) are in a to some structural upon collagen Although indicated that loops 1 and 3 are essential these to binding in an (Fig. A number of this The used or and loops the DDR1 this we the that are unable to the of the binding In of the of the of binding we a the the with proteins on the and DDR1 on the other, that the of the which by the is most collagen binding. In only with both of the (Fig. loops 1 and in collagen binding, and this the by the discoidin β-barrel. these a recent study to binding of from the factor to type I the site C. L. J. Biol. Chem. 2002; 277: Scholar). Our that the DDR1 residues and Ser-175 and Ser-175 at the of the are essential collagen binding. is that the of both residues in with specific collagen residues within the which in of the tyrosine kinase domain of 1 contains and four which bond formation to collagen (Fig. In loop 3 a by the residues and These three to residues within the collagen only point of not of or DDR1 in and binding and and the binding sequence within the collagen is DDR1 DDR2), that at collagen is involved in the binding is to that collagen binding by the integrin formation as In this the is not of the receptor is within the collagen sequence J. Cell. 2000; Scholar). The binding in DDR1 may have not only to the integrin also to the collagen-binding in the protein from M. A. D. M. J. Biol. Chem. 1999; Scholar, J. M. K. M. D. L. A. M. Biol. 1997; Scholar). In a of the DDR1 extracellular domain, we based on the sequence homology point in and the DDR1 discoidin sequence (13Curat C.A. Eck M. Dervillez X. Vogel W.F. J. Biol. Chem. 2001; 276: 45952-45958Google Scholar). These mutations within the strands of the β-barrel and the discoidin domain than the collagen-binding The molecular model to residues in which the are in the protein involved in with These residues mutated to alanine protein to the design of with of loop The of this is by the of of wild-type and proteins in membrane of cells (Fig. work by others the importance of the loop region in proteins with discoidin domains than In a recent of the to loops of DDR1 to loop which is both DDR, was not in this B. J. Biol. Chem. 2003; Scholar). is that residues in loop 4 are critically involved in the collagen-binding of DDR1 compared with have been in related domains. In both factor V and factor VIII, the C-terminal discoidin domain been found to within the membrane of in 19Fuentes-Prior P. Fujikawa K. Pratt K.P. Curr. Protein Pept. Sci. 2002; 3: 313-339Google Scholar). In recent indicated that the discoidin domain of factor V to the membrane as well S.W. J. Biol. Chem. 2002; 277: Scholar). The C-terminal discoidin domain of factor VIII also provides a binding site a protein. is that several hemophilia mutations are within the loop region of factor VIII, to its during and S.W. J. Biol. Chem. 2002; 277: Scholar). of the factor loop by showed that residues within loops 1 and 3 are critically as well as S.W. J. Biol. Chem. 2002; 277: Scholar, S.W. Quinn-Allen M.A. S. Fuentes-Prior P. Bode W. Kane W.H. 2000; Scholar, D.A. Ortel T.L. 2003; Scholar). The currently of a discoidin domain, the of Npn-1 a β-barrel with four major and two loops (16Lee C.C. Kreusch A. McMullan D. Ng K. Spraggon G. Structure (Camb.). 2003; 11: 99-108Google Scholar). of loops within Npn-1 an region that is to the binding site and the two of is that an cell in Npn-1 also to the loop within the discoidin domain M. F. H. J. Cell Biol. 2000; Scholar). A molecular model of the discoidin domain in been as well F. L. G. 2003; Scholar). Our that the extracellular domain of DDR1 a binding affinity to collagen than the discoidin domain Furthermore, we that a of is collagen-binding B. J. Biol. Chem. 2003; Scholar). the work by in that we to a of extracellular DDR proteins and we used a single to binding of proteins containing a affinity In the DDR2 discoidin domain from the stalk region and In discoidin domain which by the N- and C-terminal to the that residues of the discoidin domain to the collagen interaction we showed that the discoidin domain of DDR1 is collagen the extracellular stalk region of DDR1 an role in this We that the stalk region may as a the discoidin domain, to with its in a in and may to the the extracellular domain of DDR1 to collagen binding. the the stalk region as an binding The of in with the three-dimensional of the collagen-binding on DDR1 and DDR2 with the of developing that the and therefore have a in diseases as or

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