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• W2023458566 abstract "Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper. Receptor tyrosine kinases of the discoidin domain family, DDR1 and DDR2, are activated by different types of collagen and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. In a previous study, we found that collagen binding by the discoidin domain receptors (DDRs) requires dimerization of their extracellular domains (Leitinger, B. (2003) J. Biol. Chem. 278, 16761-16769), indicating that the paradigm of ligand-induced receptor dimerization may not apply to the DDRs. Using chemical cross-linking and co-immunoprecipitation of differently tagged DDRs, we now show that the DDRs form ligand-independent dimers in the biosynthetic pathway and on the cell surface. We further show that both the extracellular and the cytoplasmic domains are individually dispensable for DDR1 dimerization. The DDR1 transmembrane domain contains two putative dimerization motifs, a leucine zipper and a GXXXG motif. Mutations disrupting the leucine zipper strongly impaired collagen-induced transmembrane signaling, although the mutant DDR1 proteins were still able to dimerize, whereas mutation of the GXXXG motif had no effect. A bacterial reporter assay (named TOXCAT) showed that the DDR1 transmembrane domain has a strong potential for self-association in a biological membrane and that this interaction occurs via the leucine zipper and not the GXXXG motif. Our results demonstrate that the DDRs exist as stable dimers in the absence of ligand and that receptor activation requires specific interactions made by the transmembrane leucine zipper. Receptor tyrosine kinases (RTKs) 5The abbreviations used are: RTK, receptor tyrosine kinase; ECD, extracellular domain; DDR, discoidin domain receptor; DS, discoidin homology; TM, transmembrane; HEK, human embryonic kidney; BS3, bis(sulfonsuccinimidyl) suberate; GpA, glycophorin A; MBP, maltose-binding protein; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; mAb, monoclonal antibody.5The abbreviations used are: RTK, receptor tyrosine kinase; ECD, extracellular domain; DDR, discoidin domain receptor; DS, discoidin homology; TM, transmembrane; HEK, human embryonic kidney; BS3, bis(sulfonsuccinimidyl) suberate; GpA, glycophorin A; MBP, maltose-binding protein; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; mAb, monoclonal antibody. control many fundamental cellular processes, such as cell proliferation, differentiation, migration, and metabolism. RTK activity normally is under tight control, and dysregulated RTK activation is associated with most cancers, making RTKs important targets for cancer therapy (1Krause D.S. Van Etten R.A. N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1158) Google Scholar). RTKs allow the cell to respond to external cues; ligand binding to the extracellular domain (ECD) of RTKs results in transphosphorylation of their cytoplasmic domains, which in turn leads to downstream signaling. The prevailing model of RTK activation states that receptors are monomeric in the absence of ligand but become dimerized upon ligand binding; dimerization brings the cytoplasmic domains in close proximity, favoring transphosphorylation (2Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3502) Google Scholar). However, some studies have found dimerized RTKs in the absence of ligand, suggesting that activation may involve ligand-induced conformational changes within a dimeric receptor (e.g. Refs. 3Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (276) Google Scholar, 4Yu X. Sharma K.D. Takahashi T. Iwamoto R. Mekada E. Mol. Biol. Cell. 2002; 13: 2547-2557Crossref PubMed Scopus (177) Google Scholar, 5Martin-Fernandez M. Clarke D.T. Tobin M.J. Jones S.V. Jones G.R. Biophys. J. 2002; 82: 2415-2427Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The discoidin domain receptor (DDR) family of RTKs consists of two members, DDR1 and DDR2, that are characterized by the presence of an extracellular discoidin homology (DS) domain. Uniquely among RTKs, the DDRs are activated by a major extracellular matrix component, triple-helical collagen (6Shrivastava 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 (438) Google Scholar, 7Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar). Several collagen types bind to and activate the DDRs, with the two receptors displaying different specificities toward certain collagen types (6Shrivastava 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 (438) Google Scholar, 7Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar, 8Leitinger B. Steplewski A. Fertala A. J. Mol. Biol. 2004; 344: 993-1003Crossref PubMed Scopus (78) Google Scholar). The DDRs are widely expressed in normal and malignant tissues and control developmental processes; DDR1 is essential for mammary gland development in the mouse (9Vogel W.F. Aszodi A. Alves F. Pawson T. Mol. Cell. Biol. 2001; 21: 2906-2917Crossref PubMed Scopus (250) Google Scholar), and DDR2 controls bone growth through chondrocyte proliferation (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 (217) Google Scholar). Both receptors regulate cell proliferation, adhesion, and motility and control remodeling of the extracellular matrix by regulating the expression and activity of matrix metalloproteinases (11Hou G. Vogel W. Bendeck M.P. J. Clin. Investig. 2001; 107: 727-735Crossref PubMed Scopus (185) Google Scholar, 12Olaso E. Ikeda K. Eng F.J. Xu L. Wang L.H. Lin H.C. Friedman S.L. J. Clin. Investig. 2001; 108: 1369-1378Crossref PubMed Scopus (245) Google Scholar, 13Olaso 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 (194) Google Scholar, 14Ferri N. Carragher N.O. Raines E.W. Am. J. Pathol. 2004; 164: 1575-1585Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). With respect to human disease, the DDRs are associated with cancer (15Dejmek J. Leandersson K. Manjer J. Bjartell A. Emdin S.O. Vogel W.F. Landberg G. Andersson T. Clin. Cancer Res. 2005; 11: 520-528PubMed Google Scholar, 16Heinzelmann-Schwarz V.A. Gardiner-Garden M. Henshall S.M. Scurry J. Scolyer R.A. Davies M.J. Heinzelmann M. Kalish L.H. Bali A. Kench J.G. Edwards L.S. Vanden Bergh P.M. Hacker N.F. Sutherland R.L. O'Brien P.M. Clin. Cancer Res. 2004; 10: 4427-4436Crossref PubMed Scopus (177) Google Scholar, 17Ongusaha P.P. Kim J.I. Fang L. Wong T.W. Yancopoulos G.D. Aaronson S.A. Lee S.W. EMBO J. 2003; 22: 1289-1301Crossref PubMed Scopus (146) Google Scholar, 18Evtimova V. Zeillinger R. Weidle U.H. Tumour Biol. 2003; 24: 189-198Crossref PubMed Scopus (34) Google Scholar, 19Wall S.J. Werner E. Werb Z. DeClerck Y.A. J. Biol. Chem. 2005; 280: 40187-40194Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Ram R. Lorente G. Nikolich K. Urfer R. Foehr E. Nagavarapu U. J. Neurooncol. 2006; 76: 239-248Crossref PubMed Scopus (93) Google Scholar), fibrotic diseases of the lung and liver (12Olaso E. Ikeda K. Eng F.J. Xu L. Wang L.H. Lin H.C. Friedman S.L. J. Clin. Investig. 2001; 108: 1369-1378Crossref PubMed Scopus (245) Google Scholar, 21Matsuyama W. Watanabe M. Shirahama Y. Oonakahara K. Higashimoto I. Yoshimura T. Osame M. Arimura K. J. Immunol. 2005; 174: 6490-6498Crossref PubMed Scopus (34) Google Scholar), atherosclerosis (14Ferri N. Carragher N.O. Raines E.W. Am. J. Pathol. 2004; 164: 1575-1585Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and osteoarthritis (22Xu L. Peng H. Wu D. Hu K. Goldring M.B. Olsen B.R. Li Y. J. Biol. Chem. 2005; 280: 548-555Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). DDR1 and DDR2 share the same domain architecture: an ECD consisting of an N-terminal DS domain followed by a unique sequence of ∼200 amino acids; a single-span transmembrane (TM) domain; an unusually large cytosolic juxtamembrane domain; and a C-terminal tyrosine kinase domain. In a previous study, we found that DDR activation, manifested by receptor autophosphorylation, is a consequence of collagen binding to a specific site within the DDR DS domain (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We also observed that collagen binding by the DDRs requires dimerization of their ECDs (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), but how this observation relates to the oligomeric state of full-length DDRs within the cell membrane and the activation mechanism remained unclear. In the present study, we have analyzed the oligomerization state of full-length DDRs in their natural environment, the mammalian cell membrane. We show that the DDRs form ligand-independent dimers, both in the endoplasmic reticulum and at the cell surface. Neither the ECD nor the cytoplasmic domain of DDR1 is required for this interaction. In contrast, a leucine zipper motif in the DDR1 TM domain mediates strong self-association in a bacterial cell membrane and is essential for DDR1 activation in mammalian cells. Our findings demonstrate that the activation mechanism of DDRs is unlikely to involve ligand-induced receptor dimerization. Cell Culture—Human embryonic kidney (HEK) 293 cells were cultured as described (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Chemicals and Reagents—Rat tail collagen I (C-7661) was from Sigma (Poole, UK). Bis(sulfonsuccinimidyl) suberate (BS3) and sulfo-NHS-LC-biotin were from Pierce. Protein A and protein G-Sepharose beads were from Amersham Biosciences UK (Chalfont St. Giles, UK). The antibodies and their sources were as follows: rabbit anti-DDR1 (sc-532), goat anti-DDR2 (sc-7554), and rabbit anti-Myc from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); mouse anti-FLAG, M2, from Sigma; rabbit anti-maltose-binding protein (MBP) from New England Biolabs (Hitchin UK); anti-phosphotyrosine, clone 4G10, from Upstate Biotechnology (Lake Placid, NY). The anti-DDR1 Ab 74A, against the ECD, was a kind gift from Dr Michel Faure, SUGEN Inc, San Francisco, CA. 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 (Zymed Laboratories Inc., San Francisco, CA). DNA Constructs—Restriction and modification enzymes were purchased from New England Biolabs or Promega (Southampton, UK). All PCR amplification reactions were performed with Pfu DNA polymerase (Stratagene, Amsterdam, The Netherlands). All PCR-derived sequences were verified by DNA sequencing. All constructs for expression in HEK293 cells were subcloned into the mammalian expression vector pcDNA3.1/zeo (Invitrogen). PCR primers used for generating the mutant constructs can be obtained on request. The generation of the DDR1 deletion constructs DS1-1, DS1-2, and ΔDS1 was described previously (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The same method was used for the generation of the ΔPG construct, eliminating residues Asn-370-Ala-411. The cytoplasmic DDR1 deletion construct MDN (DDR1b truncated after Arg-525) was a gift from Dr Michel Faure, SUGEN Inc. The cytoplasmic deletion construct ECTM was made by PCR amplification from full-length DDR1b, introducing a STOP codon after Arg-445. The same PCR primers were used to generate the two constructs DS1-2ΔCYT and ΔDS1ΔCYT, but amplification was from the DS1-2 and ΔDS1 constructs, respectively. The TM mutations were constructed by overlap extension PCR using mutagenic primers that introduced the desired point mutations. The TOXCAT constructs were made as follows. The expression vectors pccKAN, pccGpA-wt, and pccGpA-83I and the Escherichia coli malE− strain MM39 (24Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (323) Google Scholar) were a kind gift of Dr. Donald Engelman (Yale University, New Haven, CT). The genes coding for the chimeric proteins with DDR1 TM domains were generated by annealing of custom-designed, complementary oligonucleotides, which were subsequently cloned inframe into a NheI-BamHI digested pccKAN vector. The resulting ToxR′(DDR1-TM)MBP constructs were verified by DNA sequencing and transformed into MM39 cells. Chemical Cross-linking of Surface-expressed Receptors— Cross-linking was carried out for 30 min at room temperature using the homobifunctional reagent BS3. Different concentrations of BS3 in a final volume of 500 μl of PBS were added to confluent cells in 12-well tissue culture plates. The reaction was quenched by the addition of 20 mm Tris, pH 7.4, and incubating at room temperature for 15 min followed by cell lysis. Co-immunoprecipitation—HEK293 cells at 50% confluency were transfected by calcium phosphate precipitation, as described (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Cells were co-transfected with plasmid vectors containing DDR-Myc and DDR-FLAG. Single transfections, with either one of the plasmids alone, were also carried out as controls. 48 h later, cells were lysed on ice in 1% Nonidet P40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin. The detergent soluble fraction was recovered by centrifugation at 15,000 × g, and supernatants were subjected to immunoprecipitation with either rabbit anti-Myc or mouse anti-FLAG Abs. Immune complexes were isolated with protein A or protein G conjugated to Sepharose beads, respectively. The immunoprecipitates were washed four times with lysis buffer. The proteins were analyzed by SDS-PAGE on 7.5% polyacrylamide gels followed by blotting onto nitrocellulose membranes. The blots were probed with either rabbit anti-Myc or mouse anti-FLAG followed by relevant secondary Abs conjugated to horseradish peroxidase. Detection was by enhanced chemiluminescence (Amersham Biosciences). Cell Surface Biotinylation—Cell surface biotinylation of HEK293 cells expressing DDR1 constructs was carried out as follows. The cells were incubated with sulfo-NHS-LC-biotin in PBS for 30 min on ice. The reaction was quenched by washing twice with 100 mm glycine in cold PBS. The cells were lysed in Nonidet P-40 lysis buffer (as above), and one aliquot of cell lysate was incubated for 2 h with streptavidin-Sepharose beads to bind biotinylated cell surface receptors. The beads were pelleted by centrifugation, and the recovered supernatant was incubated for another 2 h with fresh streptavidin-Sepharose beads to remove any remaining biotinylated cell surface receptors. The beads from the first and second incubation were pooled and washed four times in Nonidet P-40 lysis buffer and then boiled in reducing SDS sample buffer to release bound protein. The supernatant from the second incubation was recovered and incubated with anti-DDR1 Ab to immunoprecipitate the intracellular DDR receptor pool. To analyze the total DDR protein in the sample, another aliquot of starting lysate of equal volume was incubated with anti-DDR1 Ab for DDR immunoprecipitation. As a control for an intracellular protein, ERK2 was chosen. Samples were separated by 7.5% SDS-PAGE and analyzed by Western blotting with anti-DDR1 or anti-ERK2 Ab. Collagen-dependent DDR Autophosphorylation—This assay was performed as described (23Leitinger B. J. Biol. Chem. 2003; 278: 16761-16769Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Briefly, HEK293 cells in 12-well plates were transfected by calcium phosphate precipitation with the relevant DDR expression vectors. 24 h later, the cells were incubated with serum-free medium for 16 h. Cells were then stimulated with 10 μg/ml collagen for 90 min at 37 °C. Cells were lysed in 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 1 mm sodium orthovanadate, 5 mm NaF. Aliquots of the lysates were analyzed by SDS-PAGE followed by blotting onto nitrocellulose membranes. The blots were probed with mouse anti-phosphotyrosine mAbs followed by sheep anti-mouse Ig horseradish peroxidase. Detection was by enhanced chemiluminescence (Amersham Biosciences). To strip the blots, membranes were incubated in either 62.5 mm Tris, pH 6.8, 2% SDS, 100 mm β-mercaptoethanol at 55 °C for 30 min or antibody stripping solution (Alpha Diagnostic International, San Antonio, TX) for 10 min at room temperature. The blots were reprobed with rabbit anti-DDR1 or goat anti-DDR2 Abs followed by goat anti-rabbit Ig-horseradish peroxidase or rabbit anti-goat Ig-horseradish peroxidase. Chloramphenicol Acetyltransferase (CAT) ELISA—To generate cell-free extracts for the CAT ELISA, single colonies of transformed MM39 cells were inoculated into 5 ml of M9 minimal medium containing 0.4% maltose as a unique source of carbon and 100 μg/ml ampicillin. The bacterial cultures were grown at 37 °C with vigorous shaking to A600 of 0.6-0.7 and then harvested by pelleting 1 ml of culture at 250 × g for 10 min at 4 °C. The cell pellets were washed twice with PBS at 4 °C and finally resuspended in 1 ml of lysis buffer (Roche Applied Science). The cells were lysed for 30 min at room temperature, and 75 μl of the resulting cell-free extracts were assayed for CAT concentration using a CAT ELISA kit (Roche Applied Science) according to the manufacturer's instructions. Expression of TOXCAT Proteins—Single colonies of transformed MM39 cells were grown in LB broth with 100 μg/ml ampicillin to A600 of ∼0.6. Cells were pelleted from 0.5 ml of bacterial culture and resuspended in PBS. Cells were lysed by the addition of SDS sample buffer and boiling for 5 min. The samples were separated by 10% SDS-PAGE, blotted, and probed with anti-MBP as primary Ab followed by anti-rabbit Abs conjugated to horseradish peroxidase. Detection was by enhanced chemiluminescence (Amersham Biosciences). Ligand-independent DDR Dimerization—To investigate whether the DDRs exist as preformed oligomers in the absence of collagen, we performed chemical cross-linking experiments. HEK293 cells were transiently transfected with human DDR1 (DDR1b isoform) or DDR2 and incubated in the presence or absence of the membrane-impermeable, homobifunctional cross-linker BS3. The DDRs were detected by Western blotting of cell lysates. In the absence of cross-linker, DDR1 is observed to migrate as two species of ∼120 and 125 kDa (Fig. 1A). The lower molecular weight species represents the high mannose, biosynthetic precursor of the receptor, whereas the upper band represents complex glycosylated DDR1 (data not shown and Ref. 25Curat 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). In the presence of BS3, an ∼240-kDa band, corresponding to the size of a DDR1 dimer, was detected by the anti-DDR1 Ab. Likewise, for DDR2, which migrates as a mixture of three species, with the upper two forms representing complex glycosylated mature forms (data not shown), a band appeared in the presence of BS3, which corresponds to the size of dimeric DDR2. These results indicate that the DDRs, or at least a sizeable fraction of molecules, exist as dimers on the cell surface in the absence of ligand. To confirm the formation of DDR homodimers, we performed co-immunoprecipitation studies, using two differently epitope-tagged DDR constructs. Both DDR1 and DDR2 were tagged at the C terminus with either a Myc tag or a FLAG tag. Upon transient transfection into HEK293 cells, these receptor constructs were expressed with the same efficiency as their wild-type counterparts, exhibited the same pattern of protein bands, and showed collagen-dependent autophosphorylation indistinguishable from the wild-type receptors (data not shown). Following transient expression in HEK293 cells, Myc- and FLAG-tagged DDR1 could be immunoprecipitated, respectively, with anti-Myc (Fig. 1B, lane 4) and anti-FLAG Ab (data not shown). The immunoprecipitation was specific since anti-Myc and anti-FLAG Ab did not precipitate, respectively, DDR1-FLAG (Fig. 1B, lane 1) and DDR1-Myc (data not shown). For co-immunoprecipitation assays, DDR1-Myc and DDR1-FLAG were co-transfected into HEK293 cells. Cell lysates were immunoprecipitated with anti-Myc Ab, and the precipitated material was analyzed by Western blotting with anti-Myc or anti-FLAG Ab. Fig. 1B, lane 2, shows that DDR1-FLAG co-precipitated with the anti-Myc Ab, indicating that DDR1-Myc and DDR1-FLAG associate with one another. Importantly, co-precipitation only occurred in lysates from cells co-transfected with both types of tagged DDR1 but not when lysates of singly transfected cells were mixed before immunoprecipitation (Fig. 1B, lane 3, asterisk). This indicates that co-precipitation requires the tagged receptors to be expressed on the same cell membrane and rules out nonspecific receptor association after cell lysis. It is notable that both the precursor (high mannose) and the mature forms of DDR1-FLAG co-precipitate with DDR1-Myc expressed in the same cell (Fig. 1B, lane 2). Similar results were obtained with DDR2 (Fig. 1B). We obtained equivalent results when the protocol was reversed, and immunoprecipitation of cells co-expressing the two epitope-tagged forms of the DDRs was performed with the anti-FLAG Ab followed by Western blotting with the anti-Myc Ab (data not shown). From these experiments, we conclude that DDR1 and DDR2 associate to form homodimers and that dimerization already takes place during biosynthesis, preceding the appearance of DDRs on the cell surface. We also investigated whether collagen binding increases the amount of DDR dimers on the cell surface. Cells co-expressing both types of tagged DDRs were subjected to co-immunoprecipitation analysis in the presence or absence of collagen stimulation. For both DDR1 and DDR2, there was no increase in the amount of DDR co-immunoprecipitation (Fig. 2 and data not shown), indicating that collagen binding does not increase DDR dimerization on the cell surface. Neither the Extracellular nor the Cytoplasmic Domain of DDR1 Is Essential for DDR1 Dimerization—We next wished to determine which structural domains in DDR1 are responsible for homodimerization. We made a set of deletion constructs and subjected them to dimerization analysis by both cross-linking and co-immunoprecipitation. The extracellular domain of DDR1 is composed of an N-terminal DS domain followed by a domain unique to DDRs. The last ∼40 residues of the DDR1 ECD are rich in Pro and Gly. The cytoplasmic domain is composed of an unusually long juxtamembrane domain (176 amino acids in DDR1b) followed by the tyrosine kinase domain. Notably, neither the cytoplasmic domain nor individual subdomains of the DDR1 ECD were found to be essential for dimerization as none of the deletion constructs was defective in the co-immunoprecipitation assay (Fig. 3A and data not shown). The cytoplasmic deletion constructs were readily cross-linked by BS3, with prominent bands corresponding to the expected molecular weights of the shortened proteins (Fig. 3B and Supplemental Fig. 1; see the legend for Supplemental Fig. 1 for molecular size determination). The appearance of cross-linked bands corresponding to the expected dimer sizes of these truncated proteins provides strong evidence that the cross-linking assay detects DDR homodimers rather than hetero-oligomers with another unknown protein. Not all of the extracellular deletion mutants could be cross-linked (Fig. 3A and data not shown). We therefore used cell surface biotinylation to establish which of the deletion mutants were localized to the cell surface. There was a strict correlation between the ability of the truncated DDR1 proteins to be cross-linked by BS3 and their surface localization (Fig. 3, A and E), showing that the cross-linking assay indeed detects only surface-localized DDR1. To rule out that DDR1 dimerization is due to multiple interactions along the entire ECD (in which case individual domain deletions within a homodimer might be tolerated), we co-expressed two different ECD deletion mutants that are not expected to be able to interact via their ECD. The DS1-2 construct retains the DDR1 DS domain but places it in an unnatural position close to the TM domain. The co-expressed ΔDS1 construct, on the other hand, is missing the DS domain and retains the rest of the DDR1 ECD. Co-immunoprecipitation studies (Fig. 3C) indicated no impairment in the ability of these proteins to associate, showing that the entire DDR1 ECD is dispensable for DDR1 dimerization. A construct bearing only the DS domain in the ECD with the entire cytoplasmic domain deleted also showed homodimeric interaction in the co-immunoprecipitation assay (Fig. 3D), indicating that, in the absence of the cytoplasmic domain, the DS domain and the TM domain are sufficient for DDR1 dimerization. The Role of the DDR1 TM Domain in Dimerization and Signaling—As the DDR1 TM domain is common to all of the deletion constructs tested in our dimerization assays, it seemed likely that the TM domain contributes to interactions between the DDR1 monomers. The DDR1 TM domain contains two putative dimerization motifs (Fig. 4A). One is a GXXXG motif (GXXXA in the DDRs) (26Sternberg M.J. Gullick W.J. Protein Eng. 1990; 3: 245-248Crossref PubMed Scopus (129) Google Scholar, 27Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (782) Google Scholar), which is the principal component of the dimerization interface of the strongly dimerizing TM domain of glycophorin A (GpA) (28MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (869) Google Scholar). GXXXG motifs are also important for TM interactions of ErbB receptors (29Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In addition to the GXXXG motif, the DDR1 TM sequence also contains heptad motifs of (iso)leucine residues, which are thought to mediate TM domain dimerization in a similar manner as in leucine zippers (30Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31Gurezka R. Langosch D. J. Biol. Chem. 2001; 276: 45580-45587Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A dimeric leucine zipper is a parallel coiled-coil composed of two α-helices interacting through apolar residues in positions a and d of a 7-residue helical repeat (32Mason J.M. Arndt K.M. Chembiochem. 2004; 5: 170-176Crossref PubMed Scopus (538) Google Scholar). To test which (if any) of the potential dimerization motifs are important for DDR1 dimerization or TM signaling, we created three DDR1 TM mutants (Fig. 4A). To disrupt association via the leucine zipper, we introduced Gly-Pro residues (mutants TM1 and TM2), similarly to previous work on E-cadherin (33Huber O. Kemler R. Langosch D. J. Cell Sci. 1999; 112: 4415-4423Crossref PubMed Google Scholar) and the erythropoietin receptor (34Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The 5-residue GXXXG motif tolerates only the smallest ami" @default.
• W2023458566 created "2016-06-24" @default.
• W2023458566 creator A5009745073 @default.
• W2023458566 creator A5012151196 @default.
• W2023458566 creator A5018180229 @default.
• W2023458566 creator A5022146196 @default.
• W2023458566 creator A5034134005 @default.
• W2023458566 date "2006-08-01" @default.
• W2023458566 modified "2023-10-16" @default.
• W2023458566 title "A Transmembrane Leucine Zipper Is Required for Activation of the Dimeric Receptor Tyrosine Kinase DDR1" @default.
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