Identification of Disulfide-linked Dimers of the Receptor Tyrosine Kinase DDR1 (2007)

Discoidin domain receptor 1 (DDR1) is a transmembrane receptor tyrosine kinase activated by triple-helical collagen. So far six different isoforms of DDR1 have been described. Aberrant expression and signaling of DDR1 have been implicated in several human diseases linked to accelerated matrix degradation and remodeling, including tumor invasion, atherosclerosis, and lung fibrosis. Here we show that DDR1 exists as a disulfide-linked dimer in transfected as well as endogenously expressing cells. This dimer formation occurred irrespective of its kinase domain, as dimers were also found for the truncated DDR1d isoform. A deletion analysis of the extracellular domain showed that DDR1 mutants lacking the stalk region failed to form dimers, whereas deletion of the discoidin domain did not prevent dimerization. Point mutagenesis within the stalk region suggested that cysteines 303 and 348 are necessary for dimerization, collagen binding, and activation of kinase function. The identification of DDR1 dimers provides new insights into the molecular structure of receptor tyrosine kinases and suggests distinct signaling mechanisms of each receptor subfamily. Discoidin domain receptor 1 (DDR1) is a transmembrane receptor tyrosine kinase activated by triple-helical collagen. So far six different isoforms of DDR1 have been described. Aberrant expression and signaling of DDR1 have been implicated in several human diseases linked to accelerated matrix degradation and remodeling, including tumor invasion, atherosclerosis, and lung fibrosis. Here we show that DDR1 exists as a disulfide-linked dimer in transfected as well as endogenously expressing cells. This dimer formation occurred irrespective of its kinase domain, as dimers were also found for the truncated DDR1d isoform. A deletion analysis of the extracellular domain showed that DDR1 mutants lacking the stalk region failed to form dimers, whereas deletion of the discoidin domain did not prevent dimerization. Point mutagenesis within the stalk region suggested that cysteines 303 and 348 are necessary for dimerization, collagen binding, and activation of kinase function. The identification of DDR1 dimers provides new insights into the molecular structure of receptor tyrosine kinases and suggests distinct signaling mechanisms of each receptor subfamily. Discoidin domain receptors (DDRs) 3The abbreviations used are: DDR, discoidin domain receptor; ELISA, enzyme-linked immunosorbent assay; RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; HA, hemagglutinin. 3The abbreviations used are: DDR, discoidin domain receptor; ELISA, enzyme-linked immunosorbent assay; RTK, receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; HA, hemagglutinin. are a subfamily of transmembrane collagen-binding receptor tyrosine kinases (RTK). DDRs are distinguished from other RTKs by a discoidin domain in their extracellular region, which functions as a lectin in the slime mold Dictyostelium discoideum (1Johnson J.D. Edman J.C. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5677-5681Crossref PubMed Scopus (117) Google Scholar). There are two genes that code for DDRs in humans, DDR1 and DDR2 (2Vogel W.F. Abdulhussein R. Ford C.E. Cell. Signal. 2006; 18: 1108-1116Crossref PubMed Scopus (285) Google Scholar). Each DDR contains an extracellular region containing the ligand-binding discoidin domain, a stalk region, a single transmembrane region, as well as a cytoplasmic domain consisting of a juxtamembrane region and a tyrosine kinase domain. Through alternative splicing in the juxtamembrane or kinase domain of the human gene, at least five isoforms DDR1a– DDR1e are generated (3Alves F. Saupe S. Ledwon M. Schaub F. Hiddemann W. Vogel W.F. FASEB J. 2001; 15: 1321-1323Crossref PubMed Scopus (83) Google Scholar). The longest DDR1 transcript codes for the c-isoform with 919 amino acids. Compared with the c-isoform, the a-isoform lacks 37 amino acids in the juxtamembrane region, and the b-isoform lacks six amino acids in the kinase domain because of alternative splicing (4Playford M.P. Butler R.J. Wang X.C. Katso R.M. Cooke I.E. Ganesan T.S. Genome Res. 1996; 6: 620-627Crossref PubMed Scopus (24) Google Scholar, 5Alves F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618PubMed Google Scholar). The DDR1d and e-isoforms are truncated variants that either lack the entire kinase region (d-isoform) or parts of the juxtamembrane region and the ATP-binding site (e-isoform (3Alves F. Saupe S. Ledwon M. Schaub F. Hiddemann W. Vogel W.F. FASEB J. 2001; 15: 1321-1323Crossref PubMed Scopus (83) Google Scholar)). A sixth isoform lacking parts of the extracellular domain has been described from rat testis (6Mullenbach E. Walter L. Dressel R. Gene (Amst.). 2006; 372: 53-61Crossref PubMed Scopus (23) Google Scholar). DDR1 is expressed in a variety of cell types and tissues, specifically in the mammary gland, brain, kidney, lung, and colon mucosa and has been isolated from several carcinoma cell lines, including MCF7 mammary carcinoma cells, ovarian, lung, and esophageal cancer cells, and primary pediatric brain tumor samples (for review see Ref. 2Vogel W.F. Abdulhussein R. Ford C.E. Cell. Signal. 2006; 18: 1108-1116Crossref PubMed Scopus (285) Google Scholar). Immune cells, including macrophages, vascular smooth muscle cells, as well as oligodendrocytes also express DDR1 (7Kamohara H. Yamashiro S. Galligan C. Yoshimura T. FASEB J. 2001; 15: 2724-2726Crossref PubMed Google Scholar, 8Hou G. Vogel W. Bendeck M.P. J. Clin. Investig. 2001; 107: 727-735Crossref PubMed Scopus (185) Google Scholar, 9Avivi-Green C. Singal M. Vogel W.F. Am. J. Respir. Crit. Care Med. 2006; 174: 420-427Crossref PubMed Scopus (105) Google Scholar, 10Franco-Pons N. Virgos C. Vogel W.F. Urena J.M. Soriano E. del Rio J.A. Vilella E. Neuroscience. 2006; 140: 463-475Crossref PubMed Scopus (22) Google Scholar). From experiments with cultured cells, it was found that DDR1 regulates cell migration and branching morphogenesis within collagen-rich matrices (11Ferri N. Carragher N.O. Raines E.W. Am. J. Pathol. 2004; 164: 1575-1585Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 12Wang C.Z. Hsu Y.M. Tang M.J. J. Cell Physiol. 2005; 203: 295-304Crossref PubMed Scopus (49) Google Scholar). To address the role of DDR1 in an entire organism, gene targeting was used to generate DDR1-null mice. Homozygous mutant animals are smaller in size than control littermates and present with an altered renal basement membrane (13Vogel W.F. Aszodi A. Alves F. Pawson T. Mol. Cell. Biol. 2001; 21: 2906-2917Crossref PubMed Scopus (250) Google Scholar, 14Gross O. Beirowski B. Harvey S.J. McFadden C. Chen D. Tam S. Thorner P.S. Smyth N. Addicks K. Bloch W. Ninomiya Y. Sado Y. Weber M. Vogel W.F. Kidney Int. 2004; 66: 102-111Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The majority of DDR1-null females are unable to bear offspring because of defects in blastocyst implantation. Those null females that are able to give birth are unable to lactate, a defect caused by abnormal branching and differentiation of the mammary epithelium (13Vogel W.F. Aszodi A. Alves F. Pawson T. Mol. Cell. Biol. 2001; 21: 2906-2917Crossref PubMed Scopus (250) Google Scholar, 15Faraci-Orf E. McFadden C. Vogel W.F. J. Cell. Biochem. 2006; 97: 109-121Crossref PubMed Scopus (36) Google Scholar). The ligands for DDR1 include various types of native collagen (16Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar, 17Shrivastava 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 (442) Google Scholar). Maximal activation of DDR1 requires up to 18 h of collagen stimulation leading to phosphorylation of several cytoplasmic tyrosine residues (12Wang C.Z. Hsu Y.M. Tang M.J. J. Cell Physiol. 2005; 203: 295-304Crossref PubMed Scopus (49) Google Scholar, 18Wang C.Z. Su H.W. Hsu Y.C. Shen M.R. Tang M.J. Mol. Biol. Cell. 2006; 17: 2839-2852Crossref PubMed Scopus (88) Google Scholar, 19Vogel 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, 20Koo D.H. McFadden C. Huang Y. Abdulhussein R. Friese-Hamim M. Vogel W.F. FEBS Lett. 2006; 580: 15-22Crossref PubMed Scopus (41) Google Scholar). The presence of the discoidin domain in DDR1 has been shown to be the essential region for collagen binding, and specific residues within the discoidin domain that interact with collagen were identified (21Curat 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, 22Abdulhussein R. McFadden C. Fuentes-Prior P. Vogel W.F. J. Biol. Chem. 2004; 279: 31462-31470Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, the structure of the DDR2 discoidin domain has been solved by NMR studies, but such information is as yet not available for DDR1 (24Ichikawa O. Osawa M. Nishida N. Goshima N. Nomura N. Shimada I. EMBO J. 2007; 26: 4168-4176Crossref PubMed Scopus (88) Google Scholar). In extension of the current model of RTK signaling, it was hypothesized that activation of DDR1 also involves ligand-induced dimerization (25Schlessinger J. Cell. 1997; 91: 869-872Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). However, to date, the validity of this ligand-induced RTK dimerization model for DDR1 in particular has not been verified. In this study, we report the presence of disulfide-linked DDR1 dimers, which potentially contribute to receptor trafficking and ligand-induced receptor activation. We also assess the importance of the stalk region of DDR1, and we found that two cysteines in the stalk region are critical for dimerization. Cell Culture and Protein Expression—Human embryonic kidney 293 cells and human breast carcinoma MCF-7 and T-47D cells were purchased from the American Tissue Culture Collection and cultivated under the recommended conditions. MCF-7 cells were stably transfected with a mammalian expression vector pIRES-Neo containing the FLAG-tagged human cDNA of DDR1 isoforms a and d. Cells were transfected using Lipofectamine 2000 (Invitrogen) and stably expressing clones isolated. Semi-confluent 293 cells were transfected by calcium phosphate precipitation with 10 μg of plasmid DNA per 10-cm dish. The medium was replaced with serum-free Dulbecco's modified Eagle's medium 18 h after transfection. Twenty four hours later, cells were stimulated with 10 μg/ml rat tail type I collagen (BD Biosciences). Fifteen to 90 min after stimulation, cells were lysed using either Triton X-100 lysis buffer for Western blotting or Tween 20 lysis buffer for ELISA (each containing 1% detergent, 0.1% SDS, 150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl 10 mm 1 mm 1 mm 10 μg/ml 293 cells were transfected with for and stimulation, cells were and was at using an and Western to Western of cell was using the and of each were in either buffer mm Tris-HCl SDS, or were to were to membrane and using or or in mm Tris-HCl 150 mm NaCl, 5 mm EDTA, and Western were with and and Biosciences). the were in mm Tris-HCl SDS, 0.1% at 50 for min and for 1 h at were into the cDNA by using the mutagenesis Point were to the single amino and are available The to DDR1, including its were to the presence of the The deletion mutants lacking the discoidin domain or stalk region were described (21Curat 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). I collagen was in phosphate-buffered to a of 50 μg/ml and to were for 1 h at and with were with 150 of 1 in containing Tween 20 for 1 were with 150 of buffer in containing Tween 293 cells were lysed with Tween lysis and was to each well and at were for DDR1 expression by Western analysis to were six with of in were and for 90 were six with buffer and with for 90 was using of in 50 mm buffer mm and with min at the was using 50 of were in an ELISA at buffer were and were to analysis using as by are experiments were in cell of transfected 293 cells expressing type each mutant and cells were for DDR1 expression by Western blotting and at with in buffer mm 150 mm NaCl, 0.1% Triton were with in and to by Western was using and Cell cells were transfected with type or the mutants as Cells were with PBS, with in at for and with containing mm Cells were lysed in buffer containing and at at for μg of were with at were four with in and to by Western showed that the DDR1 isoforms a and are expressed as in a of different cell including breast cells F. Vogel W. Mossie K. Millauer B. Hofler H. Ullrich A. Oncogene. 1995; 10: 609-618PubMed Google Scholar, W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). To DDR1 disulfide-linked dimers, human embryonic kidney 293 cells were transfected with the b-isoform of DDR1 or a stimulated with type I collagen for 90 and by under or conditions. Western analysis was using an specific to the of DDR1 was expressed as single in the presence of an of was found in the of the of that of is found as with collagen did not the of To the dimer was present in cells expressing DDR1 from human breast carcinoma T-47D cells were under and conditions. In with the from 293 cells, DDR1 was as a under and as a and dimer under 293 cells were transfected with or an plasmid and stimulated with type I collagen for 90 an specific for the of DDR1, Western analysis of under as a whereas under was present as a and a dimer T-47D cells that endogenously express were stimulated with collagen for 18 h and by Western blotting under and The experiments were and are and were transfected in 293 cells or stably transfected into human breast carcinoma MCF-7 cells This was to DDR1 dimer formation is also found other than with the for dimers of and were by Western blotting of MCF-7 under the of to dimer for was to the of was of DDR1d found as dimer to or the of DDR1d to dimer was not altered collagen that the kinase domain, which is present in and but in is not necessary for and that DDR1 dimerization is by the extracellular To DDR1 form consisting of different and were using FLAG-tagged and We found that isoforms did not form linked did the to of each isoform not are in with that failed to show and truncated DDR1 isoforms (3Alves F. Saupe S. Ledwon M. Schaub F. Hiddemann W. Vogel W.F. FASEB J. 2001; 15: 1321-1323Crossref PubMed Scopus (83) Google Scholar). we the DDR1 is collagen To we transfected 293 cells with for and DDR1d and stimulated cells with collagen for 90 Western analysis of that the is stimulation To the of the we used from 293 cells transfected with and DDR1d for by Western blotting and in a with the with expression of or was by with an A and and by Western analysis of by To a particular region of DDR1 that be for dimer we deletion which either lack the discoidin domain or the stalk region (21Curat 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). receptor were at for at for the and at for deletion mutants have the to kinase activation (21Curat 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). under to dimers were for DDR1 and but not for the at mutant This suggests that a within the stalk region is necessary for the dimerization of To this we identified a of cysteines at and 348 in the stalk region are a of cysteines in the extracellular domain of cysteines are in the discoidin domain and are suggested to be disulfide-linked using molecular R. McFadden C. Fuentes-Prior P. Vogel W.F. J. Biol. Chem. 2004; 279: 31462-31470Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). mutagenesis was to single amino each in the stalk region to The mutant were expressed in 293 cells and to under or conditions. Western blotting of showed phosphorylation of type DDR1 and but was for the and mutants A and In the of DDR1 dimers were for type and for the mutant but not for and mutants that residues to the dimerization of the receptor we Western from that and were expressed than type DDR1 or that the cysteines at 303 and 348 are in dimerization ligand-induced receptor activation. To in the we the of the stalk region mutants to collagen in a collagen-binding was a collagen was with cell for of type DDR1, or We found that type and to collagen at whereas and mutants a to type DDR1 from transfected cells were with that are with type I collagen. and to whereas and mutants at or in the stalk region of DDR1 to the of the receptor it of to collagen. we that and mutants at a molecular than the type we to that mutants transfected cells with an of O. Biochem. Res. PubMed Scopus Google showed that mutants a with the type not this we that mutants be with type the of the and mutants to be activated by collagen be to we and found that cell of is with the type or mutant and In for the of a form of DDR1 in human cells, which is activated collagen We show that two cysteines in the stalk region of the receptor are necessary not for the formation of dimers but also for receptor are the to report that DDR1 exists as disulfide-linked dimers are found in cells transfected with DDR1 as well as in cells endogenously expressing The isoforms of DDR1, and dimerization. The of to dimers was and whereas DDR1d showed a of DDR1d not the tyrosine kinase domain, a role for this region in the of dimerization. In the DDR1 dimer formation in we found that dimers not form in the of the stalk analysis of the stalk region, of cysteines 303 or 348 to in a receptor that did not form dimers and was activated In and mutants were unable to collagen in either an ELISA or a the expression of two mutants was with or the type receptor altered the of DDR signaling a for this (21Curat 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 the role of the discoidin domain and the stalk region for DDR1 activation and collagen the discoidin domain was essential for the with of the stalk region in DDR1 able to to collagen. deletion of the discoidin domain in a receptor that to be activated by collagen. for DDR1 signaling was using an of the DDR1 B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). was shown that DDR1 collagen as a and not as a a for receptor dimerization in the expression of discoidin as well as in cells that the an to collagen than the discoidin domain R. McFadden C. Fuentes-Prior P. Vogel W.F. J. Biol. Chem. 2004; 279: 31462-31470Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). of this study, F. E. B. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google the formation of DDR1 dimers as and found DDR1 dimers and in a to this In to this study, failed to residues in the stalk region for dimer expression of the isolated DDR1 transmembrane region, F. E. B. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google that a within this region DDR1 dimer of the RTK are to to a activation the ligand-induced RTK dimerization and activation In this to the extracellular domain of the RTK to that and receptor dimerization, leading to kinase and of tyrosine residues in Ref. J. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). However, suggested that specific of RTKs of activation. the and receptor subfamily have been the of receptor to dimerization and activation is under E. K. 2006; PubMed Scopus Google Scholar, T.E. Biochem. Sci. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). is that and the other of the are in cells. of growth or leading to and activation of signaling J. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). is a and several mechanisms are in to or signaling in cells N. A. C. L. M. M.R. A. G. F. 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The are to different receptor of it is to that the receptors be to The presence of dimers be of particular importance in cells that express at the cell it is that the presence of DDR1 dimers signaling in of the necessary for receptors to the activation of DDR1 from the DDR1 phosphorylation and 18 h after collagen stimulation, whereas the phosphorylation than 1 min after the of DDR1 dimers from that of In to the EGFR, the receptor a linked receptor is into and of which the to the extracellular and whereas the contains a extracellular region, a single transmembrane domain, and a cytoplasmic region for M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A the two amino in the and in the formation two requires the presence of at least of the or J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. G. Mol. Biol. Cell. 1996; PubMed Scopus (23) Google Scholar). of the receptor in the as the has an and the and and are present G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, J. G. Y. J. Cell Biol. PubMed Scopus Google Scholar). The current model for receptor activation that a in the extracellular domain of the receptor that the two to into receptor kinase in the of DDR1 extracellular are critical for ligand-induced kinase activation. that of cysteines 303 or 348 the formation of dimers, we that of residues DDR1 dimer in a as in the However, because information the DDR1 stalk region is it is dimerization is by and or and the of the and with the mutant to to collagen in suggests that cysteines to be in an to domain trafficking to the the molecular of two we the that altered and contribute to the we In current model of DDR1 receptor suggests that and either to the and trafficking to the the in the stalk region of DDR1, as within the stalk domain. Compared with DDR1, of the receptor to the four cysteines for receptor dimerization and DDR1, the receptor was able to and in the presence of G. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). In with for DDR1, it is of that 10 expression has been for a mutant of the of the cysteines was not into a but a linked DDR1 dimers a that accelerated and formation of receptor which be necessary for receptor activation and phosphorylation of linked However, it also be that DDR1 a of activation. DDR1 has been shown to leading to cell migration G. Vogel W. Bendeck M.P. J. Clin. Investig. 2001; 107: 727-735Crossref PubMed Scopus (185) Google Scholar, 12Wang C.Z. Hsu Y.M. Tang M.J. J. Cell Physiol. 2005; 203: 295-304Crossref PubMed Scopus (49) Google Scholar). of the receptor by to linked dimers in of a with than of DDR1 dimer formation in cells to this as a role of A of and of the or the of receptor were for the of specific or which are in a of P. J. 1: PubMed Scopus Google Scholar, J. J. 2006; Google Scholar). a of the of dimerization, binding, and be for