Collagen recognition and transmembrane signalling by discoidin domain receptors

The discoidin domain receptors, DDR1 and DDR2, are two closely related receptor tyrosine kinases that are activated by triple-helical collagen in a slow and sustained manner. The DDRs have important roles in embryo development and their dysregulation is associated with human diseases, such as fibrosis, arthritis and cancer. The extracellular region of DDRs consists of a collagen-binding discoidin (DS) domain and a DS-like domain. The transmembrane region mediates the ligand-independent dimerisation of DDRs and is connected to the tyrosine kinase domain by an unusually long juxtamembrane domain. The major DDR binding site in fibrillar collagens is a GVMGFO motif (O is hydroxyproline), which is recognised by an amphiphilic trench at the top of the DS domain. How collagen binding leads to DDR activation is not understood. GVMGFO-containing triple-helical peptides activate DDRs with the characteristic slow kinetics, suggesting that the supramolecular structure of collagen is not required. Activation can be blocked allosterically by monoclonal antibodies that bind to the DS-like domain. Thus, collagen most likely causes a conformational change within the DDR dimer, which may lead to the formation of larger DDR clusters. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Structure of the discoidin domain receptor 1 extracellular region bound to an inhibitory Fab fragment reveals features important for signaling

The discoidin domain receptors, DDR1 and DDR2, are constitutively dimeric receptor tyrosine kinases that are activated by triple-helical collagen. Aberrant DDR signaling contributes to several human pathologies, including many cancers. We have generated monoclonal antibodies (mAbs) that inhibit DDR1 signaling without interfering with collagen binding. The crystal structure of the monomeric DDR1 extracellular region bound to the Fab fragment of mAb 3E3 reveals that the collagen-binding discoidin (DS) domain is tightly associated with the following DS-like domain, which contains the epitopes of all mAbs. A conserved surface patch in the DS domain outside the collagen-binding site is shown to be required for signaling. Thus, the active conformation of the DDR1 dimer involves collagen-induced contacts between the DS domains, in addition to the previously identified association of transmembrane helices. The mAbs likely inhibit signaling by sterically blocking the extracellular association of DDR1 subunits.     

Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.     

Identification of the collagen-binding site of the von Willebrand factor A3-domain

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.     

Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

Inhibition of Collagen Fibrillogenesis by Cells Expressing Soluble Extracellular Domains of DDR1 and DDR2

Collagen fiber assembly affects many physiological processes and is tightly controlled by collagen-binding proteins. However, to what extent membrane-bound versus cell-secreted collagen-binding proteins affect collagen fibrillogenesis is not well understood. In our previous studies, we had demonstrated that the membrane-anchored extracellular domain (ECD) of the collagen receptor discoidin domain receptor 2 (DDR2) inhibits fibrillogenesis of collagen endogenously secreted by the cells. These results led to a novel functional role of the DDR2 ECD. However, since soluble forms of DDR1 and DDR2 containing its ECD are known to naturally exist in the extracellular matrix, in this work we investigated if these soluble DDR ECDs may have a functional role in modulating collagen fibrillogenesis. For this purpose, we created mouse osteoblast cell lines stably secreting DDR1 or DDR2 ECD as soluble proteins. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays were used to demonstrate that DDR ECD expression reduced the rate and quantity of collagen deposition and induced significant changes in fiber morphology and matrix mineralization. Collectively, our studies advance our understanding of DDR receptors as powerful regulators of collagen deposition in the ECM and elucidate their multifaceted role in ECM remodeling.     

Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.     

The D2 period of collagen II contains a specific binding site for the human discoidin domain receptor, DDR2

The human discoidin domain receptors (DDRs), DDR1 and DDR2, are expressed widely and, uniquely among receptor tyrosine kinases, activated by the extracellular matrix protein collagen. This activation is due to a direct interaction of collagen with the DDR discoidin domain. Here, we localised a specific DDR2 binding site on the triple-helical region of collagen II. Collagen II was found to be a much better ligand for DDR2 than for DDR1. As expected, DDR2 binding to collagen II was dependent on triple-helical collagen and was mediated by the DDR2 discoidin domain. Collagen II served as a potent stimulator of DDR2 autophosphorylation, the first step in transmembrane signalling. To map the DDR2 binding site(s) on collagen II, we used recombinant collagen II variants with specific deletions of one of the four repeating D periods. We found that the D2 period of collagen II was essential for DDR2 binding and receptor autophosphorylation, whereas the D3 and D4 periods were dispensable. The DDR2 binding site on collagen II was further defined by recombinant collagen II-like proteins consisting predominantly of tandem repeats of the D2 or D4 period. The D2 construct, but not the D4 construct, mediated DDR2 binding and receptor autophosphorylation, demonstrating that the D2 period of collagen II harbours a specific DDR2 recognition site. The discovery of a site-specific interaction of DDR2 with collagen II gives novel insight into the nature of the interaction of collagen II with matrix receptors.     

Interaction of discoidin domain receptor 1 with collagen type 1

Discoidin domain receptor 1 (DDR1) is a widely expressed tyrosine kinase receptor which binds to and gets activated by collagens including collagen type 1. Little is understood about the interaction of DDR1 with collagen and its possible functional implications. Here, we elucidate the binding pattern of the DDR1 extracellular domain (ECD) to collagen type 1 and its impact on collagen fibrillogenesis. Our in vitro assays utilized DDR1-Fc fusion proteins, which contain only the ECD of DDR1. Using surface plasmon resonance, we confirmed that further oligomerization of DDR1-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Single-molecule imaging by means of atomic force microscopy revealed that DDR1 oligomers bound at overlapping or adjacent collagen molecules and were nearly absent on isolated collagen molecules. Interaction of DDR1 oligomers with collagen was found to modulate collagen fibrillogenesis both in vitro and in cell-based assays. Collagen fibers formed in the presence of DDR1 had a larger average diameter, were more cross-linked and lacked the native banded structure. The presence of DDR1 ECD resulted in "locking" of collagen molecules in an incomplete fibrillar state both in vitro and on surfaces of cells overexpressing DDR1. Our results signify an important functional role of the DDR1 ECD, which occurs naturally in kinase-dead isoforms of DDR1 and as a shedded soluble protein. The modulation of collagen fibrillogenesis by the DDR1 ECD elucidates a novel mechanism of collagen regulation by DDR1.     

Regulation of collagen fibrillogenesis by cell-surface expression of kinase dead DDR2

The assembly of collagen fibers, the major component of the extracellular matrix (ECM), governs a variety of physiological processes. Collagen fibrillogenesis is a tightly controlled process in which several factors, including collagen binding proteins, have a crucial role. Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that bind to and are phosphorylated upon collagen binding. The phosphorylation of DDRs is known to activate matrix metalloproteases, which in turn cleave the ECM. In our earlier studies, we established a novel mechanism of collagen regulation by DDRs; that is, the extracellular domain (ECD) of DDR2, when used as a purified, soluble protein, inhibits collagen fibrillogenesis in-vitro. To extend this novel observation, the current study investigates how the DDR2-ECD, when expressed as a membrane-anchored, cell-surface protein, affects collagen fibrillogenesis by cells. We generated a mouse osteoblast cell line that stably expresses a kinase-deficient form of DDR2, termed DDR2/-KD, on its cell surface. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays demonstrated that the expression of DDR2/-KD reduced the rate and abundance of collagen deposition and induced significant morphological changes in the resulting fibers. Taken together, our observations extend the functional roles that DDR2 and possibly other membrane-anchored, collagen-binding proteins can play in the regulation of cell adhesion, migration, proliferation and in the remodeling of the extracellular matrix.     

The discoidin domain receptor DDR2 is a receptor for type X collagen.

During endochondral ossification, collagen X is deposited in the hypertrophic zone of the growth plate. Our previous results have shown that collagen X is capable of interacting directly with chondrocytes, primarily via integrin alpha2beta1. In this study, we determined whether collagen X could also interact with the non-integrin collagen receptors, discoidin domain receptors (DDRs), DDR1 or DDR2. The widely expressed DDRs are receptor tyrosine kinases that are activated by a number of different collagen types. Collagen X was found to be a much better ligand for DDR2 than for DDR1. Collagen X bound to the DDR2 extracellular domain with high affinity and stimulated DDR2 autophosphorylation, the first step in transmembrane signalling. Expression of DDR2 in the epiphyseal plate was confirmed by RT-PCR and immunohistochemistry. The spatial expression of DDR2 in the hypertrophic zone of the growth plate is consistent with a physiological interaction of DDR2 with collagen X. Surprisingly, the discoidin domain of DDR2, which fully contains the binding sites for the fibrillar collagens I and II, was not sufficient for collagen X binding. The nature of the DDR2 binding site(s) within collagen X was further analysed. In addition to a collagenous domain, collagen X contains a C-terminal NC1 domain. DDR2 was found to recognise the triple-helical region of collagen X as well as the NC1 domain. Binding to the collagenous region was dependent on the triple-helical conformation. DDR2 autophosphorylation was induced by the collagen X triple-helical region but not the NC1 domain, indicating that the triple-helical region of collagen X contains a specific DDR2 binding site that is capable of receptor activation. Our study is the first to describe a non-fibrillar collagen ligand for DDR2 and will form the basis for further studies into the biological function of collagen X during endochondral ossification.     

Using synthetic peptides and recombinant collagen to understand DDR–collagen interactions

The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR–collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein–collagen interactions. Herein we review how these techniques have been used to understand DDR–collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR–collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.     

Collagen-binding mode of vWF-A3 domain determined by a transferred cross-saturation experiment

The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the a2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.     

Mapping the collagen-binding site in the von Willebrand factor-A3 domain

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.     

Molecular analysis of collagen binding by the human discoidin domain receptors,DDR1 and DDR2. Identification of collagen binding sites in DDR2

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.     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.     

Evolution of collagen-based adhesion systems

Collagens are large, triple-helical proteins that form fibrils and network-like structures in the extracellular matrix. The collagens may have participated in the evolution of the metazoans from their very earliest origins. Cell adhesion receptors, such as the integrins, are at least as old as the collagens. Still, the early metazoan cells might not have been able to anchor directly to collagen fibrils, since the integrin-type collagen receptors have only been identified in vertebrates. Instead, the early metazoans may have used integrin-type receptors in the recognition of collagen-binding glycoproteins. It is possible that specialized, high-avidity collagen-receptor integrins have become instrumental for the evolution of bone, cartilage, circulatory and immune systems in the chordates. In vertebrates, specific collagen-binding receptor tyrosine kinases send signals into cells after adhesion to collagen. These receptors are members of the discoidin domain receptor (DDR) group. The evolutionary history of DDRs is poorly known at this time. DDR orthologs have been found in many invertebrates, but their ability to function as collagen receptors has not yet been tested. The two main categories of collagens, fibrillar and non-fibrillar, already exist in the most primitive metazoans, such as the sponges. Interestingly, both integrin and DDR families seem to have members that favor either one or the other of these two groups of collagens.     

Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.     

Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction

Bone sialoprotein (BSP) is an anionic phosphorylated glycoprotein that is expressed almost exclusively in mineralized tissues and has been shown to be a potent nucleator of hydroxyapatite formation. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and in the adhesion of bone cells to the mineralized matrix. Using a solid phase assay, we have investigated the interaction between BSP and collagen. Initial studies showed that raising the ionic strength, decreasing the pH below 7, or introducing divalent cations diminishes but does not abolish the binding of BSP to collagen, indicating that the interaction is only partly electrostatic in nature. Both bone-extracted and recombinant (r)BSP exhibited similar binding affinities, indicating that post-translational modifications are not critical for binding. To identify the collagen-binding domain, recombinant peptides of BSP were studied. Peptide rBSP-(1-100) binds to type I collagen with an affinity similar to that of full-length rBSP, whereas peptides containing the sequences 99-201 or 200-301 do not bind. Further studies showed that rBSP-(1-75) competitively inhibits the binding of rBSP-(1-100), whereas rBSP-(21-100) inhibits binding to a lesser extent, and rBSP-(43-100) does not inhibit binding. These results suggest that the collagen-binding site of rat BSP is within the sequence 21-42, with residues N-terminal of this region likely also involved. This site was confirmed by the demonstration of collagen-binding activity of a synthetic peptide corresponding to residues 19-46. The collagen-binding domain, which is highly conserved among species, is enriched in hydrophobic residues and lacks acidic residues. We conclude that residues 19-46 of BSP represent a novel collagen-binding site.