Molecular Analysis of Collagen Binding by the Human Discoidin Domain Receptors, DDR1 and DDR2

IDENTIFICATION OF COLLAGEN BINDING SITES IN DDR2*

Birgit LeitingerDagger

From the Sackler Institute for Muscular Skeletal Research, Department of Medicine, University College London, 5 University St., London WC1E 6JJ, United Kingdom

Received for publication, February 7, 2003, and in revised form, February 27, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Communication between cells and their environment is mediated by specific cell surface receptors that transduce signals from the outside of the cell to the inside. An important class of signaling receptors are receptor tyrosine kinases (RTKs),1 which play crucial roles in many fundamental cellular processes, including the cell cycle, differentiation, migration, and metabolism (1). RTKs are not only regulators of normal cellular processes but are also critically involved in the development and progression of human cancers, making them important targets for cancer intervention strategies (2). Most RTKs are activated by soluble proteins present in the blood or other body fluids. The two closely related receptors of the discoidin domain receptor (DDR) RTK subfamily, DDR1 and DDR2, are unusual in that they are activated by an extracellular matrix protein, triple-helical collagen (3, 4). This activation is independent of the major cellular collagen receptors, beta 1 integrins, as shown for DDR1 (5). The two DDRs differ in their ligand specificities; whereas both are activated by fibrillar collagens (types I-III and V), only DDR1 can be activated by nonfibrillar collagens, such as type IV collagen. Another intriguing feature of DDRs is their unusually slow autophosphorylation upon stimulation by the ligand compared with typical RTKs (hours rather than seconds) (3, 4).

Both DDRs are widely expressed in human and mouse tissues, with distinct distributions. DDR1 is mainly expressed in epithelial cells, whereas DDR2 is found in mesenchymal cells (6). The physiological functions of the DDRs have only begun to emerge, but it is clear that both receptors are involved in cell interactions with the extracellular matrix and control adhesion and cell motility. DDR1 signaling is essential for cerebellar granule differentiation (7), arterial wound repair (8), and mammary gland development (9), whereas DDR2 regulates chondrocyte (10), hepatic stellar cell (11), and fibroblast (12) proliferation. DDR1 mRNA is up-regulated in several malignant tumors (13-17), and DDR2 is present in stromal cells surrounding highly invasive DDR1-positive tumor cells (6). The elevated expression of DDRs in a number of fast growing invasive tumors suggests an important role of these matrix-activated RTKs in the proliferation and stromal invasion of tumors.

DDR1 and DDR2 are composed of an N-terminal ~150- amino acid discoidin homology (DS) domain (18), followed by a sequence of ~220 amino acids unique to DDRs, a transmembrane (TM) domain, a large cytosolic juxtamembrane domain, and a C-terminal catalytic tyrosine kinase domain. The DDR DS domains are homologous to Dictyostelium discoideum discoidin I and to functionally important DS domains of known structure in a number of secreted (e.g. blood coagulation factors V and VIII) (19, 20) and membrane-bound mammalian proteins (e.g. neuropilins) (21). No convincing data are available to define the location and nature of the collagen binding site(s) of DDRs. A recent study attempted to map DDR1 residues critical for collagen binding (22), but the results are inconclusive, since only highly conserved core residues in the DS domain were targeted by mutagenesis.

To gain insight into the molecular basis of DDR-collagen signaling, I have studied an array of recombinant DDR proteins, obtained by eukaryotic expression, in collagen binding and cell-based receptor activation assays. I demonstrate for the first time that the isolated extracellular domains (ECDs) of DDR1 and DDR2 bind directly to collagen with high affinity and that binding requires these domains to be dimerized. Using deletion mutants, I show that the DS domain of DDR2 fully contains the collagen binding site. Finally, homology scanning of the DDR2 DS domain identified three spatially adjacent loop regions as essential for collagen binding and receptor activation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Cell Lines

Human embryonic kidney 293 cells (ATCC, Manassas, VA), 293-EBNA cells (Invitrogen), and 293-T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium/F-12 nutrient mixture (Invitrogen) containing 10% fetal bovine serum.

Chemicals and Reagents

BSA, kappa -casein, collagen I (acid soluble from rat tail; C-7661), collagen IV (acid-soluble from human placenta; C-5533), and fibronectin (0.1% solution from human plasma) were obtained from Sigma. EHS mouse tumor laminin was purchased from BD Biosciences (Oxford, UK). Bis(sulfosuccinimidyl)suberate (BS3) was from Pierce. Puromycin was obtained from Sigma, and zeocin was from Invitrogen. The antibodies (Abs) and their sources were as follows: anti-DDR1, rabbit anti-DDR1 Ig (sc-532 from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-DDR2, goat anti-DDR2 Ig (sc-7554 from Santa Cruz Biotechnology); mouse anti-Myc tag, clone 9E10 from Upstate Biotechnology (Lake Placid, NY); peroxidase-conjugated goat anti-human Fc from Jackson ImmunoResearch Laboratories (West Grove, PA); anti-phosphotyrosine, clone 4G10, from Upstate Biotechnology. Secondary Abs were as follows: sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences); goat anti-rabbit Ig-horseradish peroxidase (Dako, Ely, UK); rabbit anti-goat IgG-horseradish peroxidase (Sigma).

DNA Constructs

Restriction and modification enzymes were purchased from New England Biolabs (Hitchin, UK) or Promega (Southampton, UK). All PCR amplification reactions were performed with Pfu DNA polymerase according to the manufacturer's instructions (Stratagene, Amsterdam, The Netherlands). All PCR-derived sequences in the final constructs were verified by DNA sequencing.

Transmembrane Receptor Constructs-- cDNA encoding full-length DDR1b (TrkE) in pBluescript vector was received from Dr. Michele de Luca (Istituto Dermopatico Dell'Immacolata, Rome, Italy). cDNA encoding full-length DDR2 in pBluescript vector was received from Dr. Michel Faure (SUGEN Inc., San Francisco, CA) as pBS-Tyro10. For expression in eukaryotic cells, these cDNAs were cloned into the mammalian expression vector pcDNA 3.1/Zeo (Invitrogen).

His-tagged Constructs-- His-tagged constructs were made by PCR amplification from cDNA clones using primers that introduced a novel EcoRI restriction site followed by a NheI restriction site on the 5' end and a stop codon followed by a XhoI and a BamHI site on the 3' end of the amplified cDNA fragment. The PCR products were cut with EcoRI and BamHI and subcloned into pSP72 vector (Promega). For episomal expression in eukaryotic cells, the NheI/XhoI fragment was cloned into a modified pCEP-Pu vector (23), which codes for a fusion protein containing at the N terminus the BM-40 signal sequence, a His6 tag, a Myc antigen, and an enterokinase cleavage site. In the DDR constructs, the enterokinase site is followed by the ECD fragments of DDR1, DDR2, or the various deletion constructs (Fig. 1A). For all DDR1 constructs, the first amino acid of the ECD fragments was Asp19, which is the first amino acid after the predicted signal peptide cleavage site. The C-terminal amino acid of the constructs was Thr416, which is the last residue before the predicted TM domain. Similarly, DDR2 constructs encompassed sequences between Lys22 and Thr398.

Fc-tagged Constructs-- Fc fusion proteins were constructed with the ECDs of DDR1, DDR2, and the various deletion constructs fused to the hinge, CH2, and CH3 domains of human IgG1 (24). The cDNAs encompassed coding sequences for the natural signal sequence up to Thr416 (DDR1) or up to Thr398 (DDR2) (Fig. 1A). These cDNA sequences were isolated by PCR amplification from relevant cDNA constructs using primers that incorporated a 5' EcoRI site and a 3' BamHI site. EcoRI/BamHI-cut PCR products were cloned into the expression vector pcFc. pcFc was constructed by cloning the HindIII/NotI fragment encompassing the Fc sequences of the Fc expression vector pIG1 (24) into the expression vector pcDNA3.1/Zeo.

Deletion Constructs-- All deletion mutants were constructed by overlap extension PCR (25) from full-length cDNA clones. Two cDNA fragments (A and B) were amplified using specific primers such that the 3' end of fragment A was complementary to the 5' end of fragment B. Fragment A encompassed sequences 5' of the deletion, including a natural unique restriction site, and fragment B encompassed sequences 3' of the deletion, including another unique restriction site. The overlap was designed to result in the joining of the desired amino acid sequences, as detailed in Fig. 1B. Both fragments were purified by gel electrophoresis and fused via their overlapping sequences by a secondary PCR reaction. The amplified fused product was restriction digested and subcloned into vectors encoding the full-length DDRs after the corresponding wild-type fragment was removed. For expression in eukaryotic cells, deletion constructs encoding the TM receptors with intact cytoplasmic domains were cloned into pcDNA 3.1/Zeo, as above.

DDR2 Loop Chimeras-- DDR2 loop chimera cDNAs were constructed by overlap extension PCR, amplifying two cDNA fragments, A and B, in which the 3' end of fragment A was complementary to the 5' end of fragment B. For all fragments A the 5' primer was 5'-CGGAATTCACAGAGAATGCTCTGCACCCGTT, which introduced a novel EcoRI restriction site 5' relative to the start codon; for all fragments B, the 3' primer was 5'-TGGTATTGACACTTGATGGCACTGG, which primes just 3' of a unique AatII restriction site. The overlap was designed to result in the desired loop exchange. Table I depicts the 3' primers for fragments A and the 5' primers for fragments B. Both fragments were purified by gel electrophoresis and fused via their overlapping sequences by a secondary PCR reaction. The amplified fused product was restriction-digested with EcoRI and AatII and subcloned into pBS-Tyro10 after the corresponding wild-type fragment was removed.


                              
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Table I
Oligonucleotides used for the construction of DDR2 loop chimeras

Production and Purification of Recombinant Proteins

His-tagged proteins were produced from episomally transfected 293-EBNA cells; Fc-tagged proteins were produced from episomally transfected 293-T cells. Cells were transfected using Fugene reagent (Roche Applied Science). 24 h later, cells containing the episome were selected with either 1 µg/ml puromycin (293-EBNA cells) or 100 µg/ml zeocin (293-T cells). Resistant cells were allowed to grow to confluence and used for the collection of serum-free conditioned medium. Serum-free medium was collected from T150 tissue culture flasks after incubation for 2 days at 37 °C and again after another 2-3 days of culture at 37 °C. The harvested media (typically 0.5-1 liter) were pooled and cleared of detached cells by centrifugation, followed by filtration through a 5-µm pore filter. For the purification of His-tagged proteins, sodium phosphate buffer (500 mM, pH 7.4) was added to a final concentration of 50 mM. TALON metal affinity beads (Clontech), equilibrated in 50 mM sodium phosphate buffer, pH 7.4, 300 mM NaCl (binding buffer), were added to the medium. After incubation for 16 h at 4 °C on a magnetic stirrer, the beads were washed with binding buffer and transferred to a disposable column. After extensive washing, the His-tagged proteins were eluted with 150 mM imidazole, 300 mM NaCl, 50 mM sodium phosphate, pH 7.0. For the purification of Fc-tagged proteins, sodium phosphate buffer (500 mM, pH 7.0) was added to the clarified media to a final concentration of 20 mM. Protein A-Sepharose beads (Amersham Biosciences), equilibrated in PBS, were added and incubated for 16 h as above. The beads were washed with PBS and transferred to a disposable column. The Fc-tagged proteins were eluted with 100 mM citrate, pH 3.0, and immediately neutralized with 1 M Tris, pH 9.0. All recombinant proteins were concentrated by ultrafiltration and dialyzed against PBS. Electrophoresis demonstrated a purity of >95%. The yields were in the range of 3-7 mg/liter of conditioned medium.

Collagen Binding Enzyme-linked Immunosorbent Assays

Collagen or other ligand proteins were diluted in PBS and coated in 50-µl aliquots onto 96-well microtiter plates (Maxisorp, Nalge NUNC International, Rochester, NY), overnight at room temperature. To denature collagen, samples were heated to 50 °C for 30 min prior to coating the wells. Wells were then blocked with 150 µl of 1 mg/ml BSA in PBS plus 0.05% Tween 20 (PBS-T) (DDR2 binding assays) or 0.04 mg/ml kappa -casein in PBS-T (DDR1 binding assays) for 1 h at room temperature. After one wash with incubation buffer (0.5 mg/ml BSA in PBS-T for DDR2 binding assays; identical to blocking buffer for DDR1 binding assays), 50-µl aliquots of the recombinant DDR proteins diluted in incubation buffer were added for 3 h at room temperature. Wells were washed six times with incubation buffer, and 50-µl aliquots of mouse anti-Myc monoclonal Ab (mAb) (1:500 dilution; for His-tagged proteins) or goat anti-human Fc Ab coupled to horseradish peroxidase (1:5000 dilution; for Fc-tagged proteins) were added for 1 h at room temperature. After six washes as above, 50-µl aliquots of sheep anti-mouse horseradish peroxidase Ab (1:1000 dilution) were added for 1 h at room temperature (His-tagged proteins only), followed by six washes as above. Bound DDR proteins were detected with 75 µl/well of 0.5 mg/ml o-phenylenediamine dihydrochloride (Sigma) in 50 mM citrate-phosphate, pH 5.0, added for 3-5 min. The reaction was stopped with 50 µl/well of 3 M H2SO4. Plates were read in an enzyme-linked immunosorbent assay reader at a wavelength of 490 nm. All binding assays were carried out in duplicate and showed less than 15% difference on the same plate. The assays were highly reproducible with less than 15% variation between different experiments.

Collagen-dependent Phosphorylation

293 cells in 12-well tissue culture plates were transfected by calcium phosphate precipitation with the relevant DDR expression vectors. 24 h later, the cells were incubated in serum-free medium for 16 h. Cells were then stimulated with either 10 µg/ml collagen or 1 mM sodium orthovanadate for 90 min at 37 °C. After washing with PBS, 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. The detergent-soluble fraction was recovered by centrifugation, and aliquots were analyzed by SDS-PAGE on 7.5% polyacrylamide gels, followed by blotting onto nitrocellulose membranes. The blots were probed with mouse anti-phosphotyrosine mAbs, followed by sheep anti-mouse horseradish peroxidase. Detection was by enhanced chemiluminescence (Amersham Biosciences). To reprobe the blots, the membranes were stripped in 62.5 mM Tris, pH 6.8, 2% SDS, 100 mM beta -mercaptoethanol at 55 °C for 30 min and probed with rabbit anti-DDR1 or goat anti-DDR2 Abs followed by goat anti-rabbit Ig-horseradish peroxidase or rabbit anti-goat IgG-horseradish peroxidase, respectively.

Gel Filtration

Gel filtration chromatography was carried out at 4 °C using an Amersham Biosciences ÄKTA system and a Superdex S200 HR10/30 column. All experiments were done using PBS at a flow rate of 0.5 ml/min. Elution was monitored by UV absorbance at 280 nm. The S200 column was calibrated with the following molecular mass standards (Sigma): carbonic anhydrase (29 kDa), bovine albumin (66 kDa), and alcohol dehydrogenase (150 kDa). Up to 150 µl of DDR samples (1-2 mg/ml) were injected per run.

Chemical Cross-linking Analysis

Cross-linking was performed for 1 h at room temperature with the homobifunctional reagent BS3. 4 µg of DDR proteins were incubated in PBS with different concentrations of BS3 in a final volume of 15 µl. The reactions were stopped by the addition of 5× SDS sample buffer containing 10% beta -mercaptoethanol, followed by heating to 100 °C for 5 min and analysis by SDS-PAGE on 7% polyacrylamide gels.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Soluble DDR ECDs Bind to Immobilized Collagen with High Affinity-- Human DDR1 and DDR2 are classified as collagen receptors (3, 4), but a direct protein-protein binding assay that allows an estimation of binding strength has been lacking. I have developed a simple and robust enzyme-linked immunosorbent assay-type assay to measure the binding of soluble DDR fragments to immobilized collagens. Recombinant proteins corresponding to the ECDs of the DDRs, N-terminally tagged with a His tag and a Myc epitope (his-DDR1 and his-DDR2; Fig. 1A) were produced in stably transfected human 293-EBNA cells and purified from serum-free medium. his-DDR2 exhibited dose-dependent, saturable binding to rat tail collagen I, whereas his-DDR1 did not display any binding above background levels (Fig. 2A). his-DDR2 binding to collagen I was of high affinity, with half-maximal binding at ~10-20 nM his-DDR2. Collagen binding by his-DDR2 was specific, since only little binding occurred to denatured collagen I (Fig. 2B). Furthermore, the ligand specificity of his-DDR2 mirrored that of the full-length receptor, as determined indirectly by autophosphorylation (3). Thus, no binding was detectable to fibronectin, collagen IV or laminin (Fig. 2B).


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Fig. 1.   Schematic representation of recombinant protein constructs. A, ECD constructs. The ECDs of DDR1 and DDR2 both contain an N-terminal DS domain followed by ~200 amino acids of unique sequence. Sequence numbers refer to the amino acid positions within the full-length DDRs with respect to the start Met (M1). The predicted signal sequences for DDR1 and DDR2 end at Gly18 and Ala21, respectively. The last amino acid before the TM domain is Thr416 for DDR1 and Thr398 for DDR2. In the His-tagged proteins, the last three vector-derived amino acids ( ... KLA) are followed by the DDR-specific protein sequences encompassing Asp19-Thr416 (DDR1) or Lys22-Thr398 (DDR2). The Fc fusion proteins contain Met1-Thr416 (DDR1) or Met1-Thr398 (DDR2) followed by vector-derived sequences (TGK ... ). B, TM receptor constructs. All TM receptor constructs start at Met1 and contain the natural TM and cytoplasmic sequences of DDR1b or DDR2. DDR1 contains a Pro/Gly-rich sequence close to its TM domain. Deletions were made as indicated. For example, in DS1-1, the deletion encompasses residues Thr201-Val369, thereby joining residues Gln200-Asn370.


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Fig. 2.   Collagen binding by the ECDs of DDR1 and DDR2. Solid phase binding assays were carried out with recombinant DDR ECDs. A, binding of his-DDR1 (triangle ) and his-DDR2 (black-triangle) to immobilized rat tail collagen I and binding of his-DDR1 to BSA (black-square). B, binding of his-DDR2 to different immobilized proteins: rat tail collagen I (black-triangle), heat-denatured collagen I (triangle ), human fibronectin (), human placental collagen IV (black-square), mouse EHS laminin (open circle ), and BSA (). C, binding of DDR1-Fc to different immobilized substrates: rat tail collagen I (black-triangle), human placental collagen IV (), heat-denatured collagen I (triangle ), heat-denatured collagen IV (black-square), and kappa -casein (). The experiments were carried out at least three times with very similar results.

To create recombinant DDR proteins with a different tag, the C termini of the ECDs were fused to a human IgG1 Fc sequence, which mediates covalent dimerization via disulfide bridges (DDR1-Fc and DDR2-Fc; Fig. 1A). This approach was only successful for DDR1. Upon transient transfection of 293-T cells, only DDR1-Fc and no DDR2-Fc was secreted by the cells (data not shown). DDR1-Fc was produced and purified from the serum-free medium of stably transfected cells. In contrast to his-DDR1, which did not bind to collagen I, DDR1-Fc showed dose-dependent, saturable binding to collagen I, similar to his-DDR2 (Fig. 2C). In contrast to his-DDR2, DDR1-Fc showed binding to collagen IV, consistent with previous observations that full-length DDR1 can be activated by collagen IV (3, 4) (data not shown). Collagen binding by DDR1-Fc was also specific for triple-helical collagen as the binding to denatured collagens I or IV was greatly reduced. No binding was detected to laminin or fibronectin (data not shown).

Dimerization of DDR ECDs Is Required for Collagen Binding-- The ECD of DDR1 bound to collagen only when fused to a dimerizing Fc tag, suggesting that receptor dimerization is required for ligand binding. Since his-DDR2, whose oligomerization state is not determined by the tag, avidly bound to collagen, I suspected that his-DDR2 might already exist as a (noncovalent) dimer, whereas his-DDR1 might lack features responsible for dimerization. Indeed, his-DDR1 and his-DDR2 eluted at different positions from a gel filtration column (12.4- and 10.8-ml elution volume, respectively) (Fig. 3A), suggesting that his-DDR2 forms higher oligomers in solution than his-DDR1. A small shoulder in the elution profile of his-DDR2 coincided with the elution volume of his-DDR1. Because of the presumed nonglobular shape of DDR ECDs, the elution volumes cannot be used to determine molecular masses. The most likely explanation of the gel filtration data, however, is that his-DDR1 is a monomer, whereas his-DDR2 is a noncovalent dimer, with a small monomer fraction.


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Fig. 3.   The ECDs of His-tagged DDR1 and DDR2 exist in different oligomeric states. A, gel filtration chromatography. The elution profiles of purified his-DDR1 and his-DDR2 on a Superdex S200 HR10/30 column are shown. The arrows indicate the peak elution volumes of molecular mass standards in kDa. B, chemical cross-linking analysis. His-DDR1 and his-DDR2 were incubated with the indicated concentrations (µM) of BS3. The samples were resolved on a 7.5% polyacrylamide gel under reducing conditions followed by staining with Coomassie Blue. The positions of molecular mass markers (in kDa) are indicated. The experiment was carried out twice with very similar results.

To further examine the oligomeric states of his-DDR1 and his-DDR2 I used chemical cross-linking (Fig. 3B). Dimers of his-DDR2 were readily detected at higher concentrations of cross-linker, whereas only the monomeric form was present for his-DDR1 across the entire range of cross-linker concentrations. No oligomers higher than dimers were seen for his-DDR2. Taken together, these data demonstrate that the ECD of DDR2, but not of DDR1, exists as a noncovalent dimer in solution, and that DDR dimerization is required for collagen binding.

The Collagen Binding Site of DDR2 Is Contained within Its DS Domain-- The ECDs of the DDRs are composed of an N-terminal DS domain followed by a ~220-amino acid region of no homology to other proteins (Fig. 1). To establish which domains within the ECDs of the DDRs are involved in the binding to collagen, a set of deletion constructs were made (Fig. 4A). For all constructs, cDNAs coding for His-tagged proteins and Fc fusion proteins were created. Unfortunately, not all constructs were secreted by the cells following transfection with the respective expression vectors (Fig. 4A). Only full-length DDR1 and Delta DS1 Fc fusion proteins, but not DS1-1 and DS1-2, were obtained, whereas all His-tagged DDR1 constructs were produced, albeit with poor yields for his-DS1-1 and his-DS1-2 (data not shown). Conversely, only DS2 Fc fusion protein and not full-length DDR2 (see above) or Delta DS2 were secreted. All His-tagged DDR2 proteins except Delta DS2 were produced.


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Fig. 4.   The DS domain of DDR2 binds collagen I with high affinity. A, schematic drawing summarizing the recombinant production of DDR deletion mutants as soluble ECD fragments. cDNAs encoding the deletion mutants as Fc fusion or His-tagged proteins were used to transiently transfect 293-T or 293-EBNA cells, respectively. After 2 days in culture, serum-free medium was collected and subjected to protein A (Fc fusion proteins) or TALON (His-tagged proteins) affinity purification. +, detection of recombinant protein production; -, no observation of protein production. For DS1-1-Fc, DS1-2-Fc, DDR2-Fc, Delta DS2-Fc, and his-Delta DS2, no protein products were detectable by SDS-PAGE and staining with Coomassie Blue. B, binding of DS2-Fc to different immobilized substrates in a solid phase binding assay: rat tail collagen I (black-triangle), heat-denatured collagen I (triangle ), human fibronectin (black-square), human placental collagen IV (black-diamond ), mouse EHS laminin (open circle ), and BSA (). The experiment was carried out three times with very similar results.

The contribution of the DS domain to collagen binding by DDR1 could not be studied, because no DDR1 deletion construct containing the DS domain could be obtained as a Fc fusion protein. To study the contribution of the DS domain to collagen binding by DDR2, DS2-Fc and his-DS2 were purified from the serum-free medium of stably transfected cells. DS2-Fc showed strong and saturable binding to collagen I with the same specificity as full-length his-DDR2 (Fig. 4B; compare with Fig. 2), demonstrating that the DS domain of DDR2 is sufficient for high affinity binding to collagen I. His-DS2, in contrast, showed only limited binding to collagen I (data not shown). When analyzed by chemical cross-linking, his-DS2 was found to be mainly monomeric (data not shown). These results are in accord with the binding and oligomerization data described above and further emphasize that DDRs have to be dimerized in order to bind collagen.

The DS Domain of DDR2, but Not of DDR1, Is Sufficient for Collagen-induced Receptor Autophosphorylation-- To be able to relate collagen binding to DDR activation, the deletions described above were also introduced in the context of TM receptor constructs with intact cytoplasmic domains (for details, see Fig. 1B). Following transient expression in 293 cells, I examined the collagen-induced autophosphorylation of wild-type and mutant DDR1 and DDR2.

For DDR1, the full-length receptor protein was phosphorylated in response to collagen I, as expected (Fig. 5A). This protein also showed enhanced autophosphorylation with the addition of 1 mM orthovanadate. Although, compared with mock-transfected cells, additional phosphorylated bands were detected for the DDR1 deletion mutants upon orthovanadate addition, none of these corresponded in size to the deletion mutants. Similarly, no phosphorylation in response to collagen I could be detected in any of the DDR1 deletion mutants, indicating that in DDR1 both the DS domain and the region following it are required for receptor activation. These results are in agreement with data by Curat et al. (22), who could not detect collagen I-induced autophosphorylation of a construct similar to DS1-2.


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Fig. 5.   DDR1 and DDR2 have different sequence requirements for collagen-induced autophosphorylation. Full-length DDRs and their deletion mutants were transiently expressed in 293 cells. After stimulation with 10 µg/ml collagen or 1 mM orthovanadate, cell lysates were analyzed by SDS-PAGE and Western blotting. A, DDR1 constructs. V, orthovanadate; C, collagen I. The blots were probed with anti-phosphotyrosine mAb 4G10 (upper panel) and reprobed with an Ab against the C terminus of DDR1 (lower panel). B, DDR2 constructs. V, orthovanadate; CI, collagen I; CIV, collagen IV. The blots were probed with anti-phosphotyrosine mAb 4G10 (upper panel) and reprobed with an Ab against the C terminus of DDR2 (lower panel). The positions of molecular markers (in kDa) are indicated. The experiments were carried out three times with very similar results.

For DDR2, the full-length receptor showed collagen I and orthovanadate-dependent autophosphorylation but no phosphorylation in response to collagen IV, as expected (Fig. 5B). In contrast to the situation with DDR1, the DS2 construct showed robust collagen I and orthovanadate-induced autophosphorylation, whereas Delta DS2 was not phosphorylated. These results indicate that in DDR2, the DS domain (potentially in combination with a sequence close to the TM domain) is sufficient for collagen I-induced autophosphorylation. Furthermore, the data imply that DDR1 and DDR2 have different requirements for receptor activation.

Design of DDR2 Loop Chimeras-- Because DS2-Fc, which contains little more than the DS domain of DDR2, binds collagen with high affinity and because collagen can induce autophosphorylation in a DDR2 mutant lacking the extracellular region following the DS domain, it is safe to assume that the functionally important collagen binding site is contained within the DS domain. To identify these sites in more detail, mutagenesis of the DDR2 DS domain was performed by homology scanning, whereby putative surface-exposed loops were exchanged for equivalent loops of another, functionally unrelated DS domain.

My approach was based on the crystal structures of the C2 domains of the blood coagulation factors V and VIII (19, 20), which were the only three-dimensional structures of the DS domain fold available at the time of planning the experiments. The C2 domain structures consist of a beta -sandwich core with three prominent loops (L1, L2, and L4 in Fig. 6A). These loops protrude like spikes from the top of the beta -barrel and are opposite the polypeptide chain termini, which are connected by a disulfide bridge. The C2 domains bind to phospholipids, with the region delimited by the spikes constituting the primary membrane binding site (19, 20, 26).


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Fig. 6.   Design of DDR2 DS domain loop chimeras. A, crystal structure of human blood coagulation factor V C2 domain (19). Three prominent loops project from the top of the beta -sandwich and are labeled L1, L2, and L4. Another, less prominent but spatially adjacent loop is labeled L3. The individual beta -strands are numbered, and the N and C termini are indicated. B, sequence alignment of the DS domains from human factors V and VIII (FA5 and FA8; C2 domains), human neuropilin-1 (NRP1; b1 domain), and human DDR1 and DDR2. Strictly conserved residues are shown in boldface type. beta -Strands (S1-S8) are defined as in the factor V structure. Residues corresponding to loops L1-L4 of the factor V structure are boxed and labeled. In each box, the residues that substitute for the respective DDR2 sequences in the loop chimeras are underlined. DDR1 residues mutated by Curat et al. (22) are marked by an asterisk.

Sequence alignment of several representative DS domains indicates a high degree of conservation of amino acids forming the beta -sandwich core, as expected, and identifies several variable regions, three of which (loops 1, 2, and 4) correspond to the prominent spikes seen in the factor V crystal structures. A fourth loop, L3, is a short turn in the factor V structure, but is predicted to be more prominent in the DDR DS domains. The strict conservation among DS family members of amino acids that are part of the beta -sandwich and the greater variability of the protruding loops may indicate a common binding role for the spike region.

To test whether the spike region of the DDR2 DS domain is involved in collagen binding, I exchanged the predicted loops L1-L4 with amino acid residues of the corresponding loop regions from two other DS domains (for details, see Fig. 6B). L1 was exchanged with the equivalent region from the first DS domain (b1) of neuropilin 1, since this preserved the length of the loop and avoided the introduction of exposed hydrophobic residues present in the corresponding loops of the factor V and VIII C2 domains. L2-L4 were replaced with corresponding sequences of the C2 domain of factor V. The loop replacements were introduced in the context of the full-length DDR2 receptors as well as in the soluble ECD constructs.

Loop Replacements in the DDR2 DS Domain Abolish Collagen Binding-- When made as His-tagged, full-length ECD constructs, all four DDR2 loop chimeras were secreted by the 293-EBNA cells in similar amounts as wild-type protein that was expressed in parallel, strongly suggesting that the mutations did not impair protein folding. Wild-type and mutant DDR2 proteins were purified from the serum-free medium of stably transfected cells and examined for their ability to bind collagen I (Fig. 7A). Only one loop chimera, L3, bound collagen, albeit with somewhat reduced affinity compared with wild-type protein. In contrast, the three remaining chimeras had lost the ability to recognize collagen I. To exclude the possibility that the inactive loop chimeras did not bind to collagen I because they were defective in dimerization, I analyzed their oligomeric state by chemical cross-linking (Fig. 7B). For all four DDR2 loop chimeras, dimers were detected in the presence of cross-linker in equivalent amounts to the wild-type DDR2 protein. Taken together, the data implicate three spatially adjacent loop regions in the DDR2 DS domain (L1, L2, and L4) in collagen binding.


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Fig. 7.   Three loops within the DS domain of DDR2 are essential for collagen binding. Wild-type DDR2 (DDR2-wt) and DDR2 loop chimeras (DDR2-L1 to -L4) were produced as His-tagged ECD fragments. A, binding of His-tagged DDR2 proteins to immobilized collagen I in a solid phase binding assay: DDR2-wt (black-diamond ), DDR2-L3 (triangle ), DDR2-L4 (), DDR2-L2 (), and DDR2-L1 (black-triangle). No significant background binding to BSA was detected for any DDR2 proteins. B, chemical cross-linking analysis. His-tagged DDR2 loop chimeras were incubated with or without 0.5 mM BS3. The samples were resolved on a 7.5% polyacrylamide gel under reducing conditions followed by staining with Coomassie Blue. The positions of molecular mass markers (in kDa) are indicated. The experiments were carried out twice (B) or three times (A) with very similar results.

The Same Loop Replacements Also Abolish Collagen-induced DDR2 Activation-- The DDR2 loop replacements were also introduced in the context of full-length TM receptor constructs. Upon transfection of 293 cells, all four mutant receptor proteins were detectable by Western blotting (Fig. 8A) and showed the characteristic higher molecular weight forms of DDR2, which most likely represent the glycosylated mature protein. This observation again argues for the correct folding of the mutant DDR2 proteins. Unfortunately, due to the lack of available Abs against the DDR ECDs that recognize native DDR receptors on the cell surface, the correct location at the plasma membrane of the mutant receptors could not be tested, raising the formal possibility that some of the mutant receptors may not be in the proper location or orientation at the plasma membrane.


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Fig. 8.   Loop chimeras defective in collagen binding are also defective in receptor activation. Full-length wild-type DDR2 (DDR2-wt) and loop chimera receptor constructs (DDR2-L1 to -L4) were transiently expressed in 293 cells. A, Western blotting analysis of protein expression using an Ab against the C terminus of DDR2. B, cells were incubated with or without 10 µg/ml collagen I (Coll). Aliquots of cell lysates were analyzed by SDS-PAGE and Western blotting with anti-phosphotyrosine mAb 4G10. The positions of molecular markers (in kDa) are indicated. The experiment was carried out three times with very similar results.

When collagen I-induced autophosphorylation of the loop chimera receptors was analyzed, only one mutant, L3, was still capable of responding to collagen I with increased phosphorylation (Fig. 8B). The other mutants showed only base-line phosphorylation of a band that corresponded to mature DDR2. Thus, the lack of collagen-induced autophosphorylation in mutants L1, L2, and L4 exactly mirrored the results of the collagen binding assay. In response to 1 mM orthovanadate, all mutant receptors showed increased phosphorylation (data not shown), indicating that mutagenesis of the DS domain did not affect the function of the kinase domain in the cytoplasmic tail. Moreover, this suggests proper localization of the mutant receptors within the cell.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study has given important new insight into several aspects of collagen binding and signaling by DDRs. I have shown, for the first time, 1) that the ECDs of DDRs bind collagen directly and with high affinity, 2) that the DDR-collagen interaction requires the receptors to be dimerized, 3) that the DS domain of DDR2 fully contains the collagen binding site, and 4) that three spatially adjacent surface loops within the DS domain are critically involved in collagen binding and DDR2 signaling. The study has also revealed unexpected functional differences between DDR1 and DDR2.

Using properly folded and processed DDR fragments, obtained by expression in human embryonic kidney (293) cells, I have established a robust solid phase assay measuring DDR-collagen binding (Fig. 2). Previous studies used DDR autophosphorylation as an indirect read-out for collagen binding (3, 4), employed slot blot assays on nitrocellulose membranes (4), or relied on cell lysates, rather than purified DDR proteins, to assess collagen binding (22). Hence, none of these studies could formally exclude the possibility that co-receptors, as suggested by L'Hote et al. (27), or other putative accessory proteins are needed for the DDR-collagen interaction. Collagen-induced DDR1 autophosphorylation is cell type-specific (27) and requires the secreted protein Wnt5A (28). My results demonstrate a direct binding of DDR to collagen and suggest that Wnt5A is not required for the DDR1-collagen binding interaction per se. Wnt5A may be required to render DDR1 competent for ligand binding or may be involved in signal transduction events following ligand binding.

The observation that DDR dimerization is a prerequisite for collagen recognition is in contrast to the "classical" model of RTK activation, in which ligand binding leads to receptor dimerization (1). A likely scenario is that collagen engagement by dimeric DDRs triggers conformational changes that are transmitted across the plasma membrane and lead to kinase activation. It may be relevant in this context that the ECD constructs his-DDR1 and his-DDR2 were found to differ in their oligomerization state (Fig. 3), although the present data do not define the regions responsible for this difference. In any case, the oligomeric state of the full-length DDRs on the cell surface is likely to be influenced by the TM and/or cytoplasmic domains. While this manuscript was in preparation, Agarwal et al. (29) reported the binding of a DDR2-Fc fusion protein to collagen I. The presumably dimeric DDR2-Fc fusion protein did not bind to collagen I unless it was clustered (multimerized) with Abs. My attempts to produce the entire ECD of DDR2 as an Fc fusion protein were unsuccessful, but two related constructs, DDR1-Fc and DS2-Fc, bound collagen without prior clustering with Abs. Since the DDR2-Fc sequence used by Agarwal et al. (29) is not disclosed, it remains unclear whether the discrepancy is due to differences in protein constructs or binding assays.

Binding experiments with the DS2-Fc construct demonstrate that the DS domain of DDR2 is sufficient for specific, high affinity collagen binding (Fig. 4). This finding is in agreement with Curat et al. (22), who reported collagen binding of a DDR1 TM receptor construct containing only the DS domain extracellularly. Although both DDRs use the DS domain for collagen binding, the two receptors differ significantly in the contribution of other protein domains to receptor activation. In DDR2, the DS domain (potentially in combination with a short membrane-proximal sequence) is sufficient for both collagen binding and receptor activation, with a large part of the ECD being dispensable for these activities. In contrast, DDR1 signaling requires the entire ECD, since the DS domain, either on its own or in combination with the membrane-proximal Pro/Gly-rich sequence, was not sufficient for collagen-induced autophosphorylation. The DDR ECDs up to their membrane-proximal regions are highly homologous, with 53% sequence identity. Despite this close similarity, DDR1 and DDR2 appear to use subtly different mechanisms for receptor dimerization and/or the propagation of conformational changes leading to kinase activation.

Loop replacement mutagenesis identified three regions in the DS domain of DDR2 as critical for collagen binding and collagen-induced receptor activation (Figs. 7 and 8). The loop replacements arguably could corrupt the overall protein conformation. However, this is unlikely for the following reasons: 1) all chimeric DDR2 ECD fragments were secreted by the 293 cells as soluble proteins with similar yields to the wild-type protein, and 2) the TM receptor chimeras showed the same pattern of (glycosylated) higher molecular weight forms as wild-type DDR2. These observations strongly argue for properly folded structures, since defects in protein folding are known to lead to intracellular protein degradation prior to secretion (30). The proposed collagen binding epitopes in DDR2 also conform to typical protein interaction sites. Crystal structures of three different DS domains have revealed a highly conserved beta -sandwich core and more variable surface-exposed loops (19-21). Residues that are strictly conserved among superfamily members constitute the core of the beta -sandwich and are expected to be crucial for the stability of the domain fold. Regions that are important for the functions of the individual members, on the other hand, are expected to be solvent-exposed and among those of high variability within the protein family, as is the case with the putative collagen binding loops in DDR2. A previous report claims to have mapped DDR1 residues critical for collagen binding (22), but it is unlikely that a collagen binding epitope was identified, since only highly conserved, buried residues were targeted by point mutations (denoted by asterisks in Fig. 6B). Inspection of the factor V C2 domain crystal structure suggests that eight of the nine mutations are very likely to severely destabilize parts of the beta -sandwich core. Two of the targeted residues are, in fact, equivalent to residues of the C2 domain critical for protein folding (19, 26).

Sequence analysis further supports the identification of collagen binding loops in DDR2. The DS domains of DDR1 and DDR2 have an overall sequence identity of 59%, but the three loops defined here as critical for collagen binding are strikingly conserved: five of six residues are identical in L1; five of seven in L2; and seven of nine in L4. In contrast, L3, which does not play an important role in collagen binding, is much less conserved. Since DDR1 and DDR2 both bind collagen I, I propose that the strict conservation of loops L1, L2, and L4 is functionally important and that DDR1 employs the same loops for collagen binding. L1, L2, and L4 of the DDR2 DS domain are equivalent to the three prominent spikes defined in the crystal structures of blood coagulation factors V and VIII (Fig. 6A). In factor V, phospholipid binding is mediated mainly by the hydrophobic L1 (26); in factor VIII, both phospholipids and von Willebrand factor are bound by L1 and L4 (31). The three DDR2 loops defined here also correspond to putative ligand binding regions in the first DS domain of neuropilin-1 (21).

To date, only a few collagen binding sites in other proteins have been defined structurally, and there is only one experimentally determined structure of a receptor-collagen complex. In the crystal structure of the alpha 2beta 1 integrin I domain bound to a triple-helical collagen model peptide (32), the collagen binding epitope is located opposite to the polypeptide chain termini of the I domain, as proposed here for the DDR2 DS domain. Three I domain loops that coordinate a metal ion also engage the collagen, with a Glu residue of the collagen providing extra metal coordination. In the structurally related von Willebrand factor A3 domain, the collagen binding site defined by mutagenesis is on a different face of the domain and comprises residues from two loops and one turn, without the participation of metal ions (33). The collagen binding site of the extracellular matrix protein SPARC was mapped to a rather flat region containing a helix and a loop (34). Finally, in the collagen binding domain of Staphylococcus aureus adhesin, the collagen triple helix is believed to bind in a prominent and narrow groove (35). The dimensions of triple-helical collagen (10-15-Å diameter) are consistent with a direct engagement of all three DDR2 loops, L1, L2, and L4, in collagen binding, but a more detailed analysis will have to await a structure determination of a DDR DS domain.

    ACKNOWLEDGEMENTS

I thank Drs. Michele de Luca and Michel Faure for providing cDNA plasmids. I especially thank Erhard Hohenester for support throughout this project, help with the structural aspect of this work, and helpful comments on the manuscript. I am grateful to Nancy Hogg for critical reading of the manuscript. I thank Mike Horton for continuous support and encouragement.

    FOOTNOTES

* This work was supported by an endowment of the Dr. Mortimer and Mrs. Theresa Sackler Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-20-7679-6167; Fax: 44-20-7679-6219; E-mail: b.leitinger@ucl.ac.uk.

Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M301370200

    ABBREVIATIONS

The abbreviations used are: RTK, receptor tyrosine kinase; DDR, discoidin domain receptor; DS, discoidin homology; TM, transmembrane; ECD, extracellular domain; Ab, antibody; mAb, monoclonal antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; BS3, bis(sulfosuccinimidyl)suberate.

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