Discoidin domain receptors: Micro insights into macro assemblies.

Assembly of cell-surface receptors into specific oligomeric states and/or clusters before and after ligand binding is an important feature governing their biological function. Receptor oligomerization can be mediated by specific domains of the receptor, ligand binding, configurational changes or other interacting molecules. In this review we summarize our understanding of the oligomeric state of discoidin domain receptors (DDR1 and DDR2), which belong to the receptor tyrosine kinase family (RTK). DDRs form an interesting system from an oligomerization perspective as their ligand collagen(s) can also undergo supramolecular assembly to form fibrils. Even though DDR1 and DDR2 differ in the domains responsible to form ligand-free dimers they share similarities in binding to soluble, monomeric collagen. However, only DDR1b forms globular clusters in response to monomeric collagen and not DDR2. Interestingly, both DDR1 and DDR2 are assembled into linear clusters by the collagen fibril. Formation of these clusters is important for receptor phosphorylation and is mediated in part by other membrane components. We summarize how the oligomeric status of DDRs shares similarities with other members of the RTK family and with collagen receptors. Unraveling the multiple macro-molecular configurations adopted by this receptor-ligand pair can provide novel insights into the intricacies of cell-matrix interactions.

A structural prospective for collagen receptors such as DDR and their binding of the collagen fibril.

The structure of the collagen fibril surface directly effects and possibly assists the management of collagen receptor interactions. An important class of collagen receptors, the receptor tyrosine kinases of the Discoidin Domain Receptor family (DDR1 and DDR2), are differentially activated by specific collagen types and play important roles in cell adhesion, migration, proliferation, and matrix remodeling. This review discusses their structure and function as it pertains directly to the fibrillar collagen structure with which they interact far more readily than they do with isolated molecular collagen. This prospective provides further insight into the mechanisms of activation and rational cellular control of this important class of receptors while also providing a comparison of DDR-collagen interactions with other receptors such as integrin and GPVI. When improperly regulated, DDR activation can lead to abnormal cellular proliferation activities such as in cancer. Hence how and when the DDRs associate with the major basis of mammalian tissue infrastructure, fibrillar collagen, should be of keen interest.

Expression and crystallographic studies of the ligand-binding region of the human endocytic collagen receptor uPARAP.

Urokinase plasminogen activator receptor-associated protein (uPARAP) is an endocytic receptor that internalizes collagen for lysosomal degradation and plays an important role in matrix remodelling. Previous recombinant protein production of uPARAP in Pichia pastoris generated protein with highly heterogeneous glycans that was prone to proteolytic degradation, resulting in highly twinned crystals. In this study, the uPARAP ligand-binding region was expressed in stably transfected Drosophila S2 insect cells. The recombinant protein was homogeneous after purification by metal-affinity and anion-exchange chromatography. Crystals were obtained at two different pH values (5.3 and 7.4) and diffracted to 2.44 and 3.13 Å resolution, respectively. A model of the ligand-binding region of uPARAP was obtained by molecular replacement combined with autobuilding. As the first multidomain crystal structure of the mannose receptor family, structural characterization of the uPARAP ligand-binding region will provide insight into the pH-induced conformational rearrangements of the mannose receptor family.

Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors.

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a ß-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3.

Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)ß(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.

G6f-like is an ITAM-containing collagen receptor in thrombocytes.

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the ß(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Transmembrane collagen receptors.

Collagen, the most abundant protein in animals, is a key component of extracellular matrices. Not only do collagens provide essential structural support for connective tissues, but they are also intimately involved in controlling a spectrum of cellular functions such as growth, differentiation, and morphogenesis. All collagens possess triple-helical regions through which they interact with a host of other proteins including cell surface receptors. A structurally diverse group of transmembrane receptors mediates the recognition of the collagen triple helix: integrins, discoidin domain receptors, glycoprotein VI, and leukocyte-associated immunoglobulin-like receptor-1. These collagen receptors regulate a wide range of behaviors including cell adhesion and migration, hemostasis, and immune function. Here these collagen receptors are discussed in terms of their molecular basis of collagen recognition, their signaling and developmental functions, and their roles in disease.

Structural insights into the interactions between platelet receptors and fibrillar collagen.

Collagen peptides have been used to identify binding sites for several important collagen receptors, including integrin alpha(2)beta(1), glycoprotein VI, and von Willebrand factor. In parallel, the structures of these collagen receptors have been reported, and their interactions with collagen peptides have been studied. Recently, the three-dimensional structure of the intact type I collagen fiber from rat tail tendon has been resolved by fiber diffraction. It is now possible to map the binding sites of platelet collagen receptors onto the intact collagen fiber in three dimensions. This minireview will discuss these recent findings and their implications for platelet activation by collagen.

Integrins during evolution: evolutionary trees and model organisms.

The integrins form a large family of cell adhesion receptors. All multicellular animals express integrins, indicating that the family evolved relatively early in the history of metazoans, and homologous sequences of the component domains of integrin alpha and beta subunits are seen in prokaryotes. Some integrins, however, seem to be much younger. For example, the alphaI domain containing integrins, including collagen receptors and leukocyte integrins, have been found in chordates only. Here, we will discuss what conclusions can be drawn about integrin function by studying the evolutionary conservation of integrins. We will also look at how studying integrins in organisms such as the fruit fly and mouse has helped our understanding of integrin evolution-function relationships. As an illustration of this, we will summarize the current understanding of integrin involvement in skeletal muscle formation.

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.

Identification of the first prokaryotic collagen sequence motif that mediates binding to human collagen receptors, integrins alpha2beta1 and alpha11beta1.

Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin alpha2beta1. The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the alpha2beta1 and the alpha11beta1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant alpha2-integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-alpha2+myoblasts expressing the alpha2beta1 integrin as the sole collagen receptor. The C2C12-alpha11+myoblasts expressing the alpha11beta1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-alpha11+cells attached to rScl1 more efficiently than C2C12-alpha2+cells, suggesting that the alpha11beta1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria.

Snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of the platelet collagen receptor, glycoprotein VI.

Glycoprotein (GP)VI, that binds collagen, together with GPIb-IX-V which binds vonWillebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases. Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus ( approximately 60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, approximately 0.3 microg/ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming crotarhagin targeted GPVI. Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation. Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.

The collagen family members as cell adhesion proteins.

The collagen family of extracellular matrix proteins has played a fundamental role in the evolution of multicellular animals. At the present, 28 triple helical proteins have been named as collagens and they can be divided into several subgroups based on their structural and functional properties. In tissues, the cells are anchored to collagenous structures. Often the interaction is indirect and mediated by matrix glycoproteins, but cells also express receptors, which have the ability to directly bind to the triple helical domains in collagens. Some receptors bind to sites that are abundant in all collagens. However, increasing evidence indicates that the coevolution of collagens and cell adhesion mechanisms has given rise to receptors that bind to specific motifs in collagens. These receptors may also recognize the different members of the large collagen family in a selective manner. This review summarizes the present knowledge about the properties of collagen subtypes as cell adhesion proteins.

A recombinant protein and a chemically synthesized peptide containing the active peptides of the platelet collagen receptors inhibit ferric chloride-induced thrombosis in a rat model.

We have previously reported that a recombinant protein (M(r) 47 kDa), which contains both active peptide of platelet receptors for types I and III collagen inhibits both types I and III collagen-induced platelet aggregation. In order to eliminate non-reactive portion of the protein, we have constructed a recombinant of rHyB (M r 6 kDa). In addition, we chemically synthesized a hybrid peptide with 30 amino acid residues (cHyB, M r 3 kDa) that contains each of the active peptide derived from platelet receptors for types I and III collagen and a linker of 12 amino acid residues. In the present investigation, we report that both rHyB and cHyB inhibit type I and type III collagen-induced platelet aggregation, and the adhesion of radiolabeled platelets onto rabbit aortic segments in a dose-dependent manner. We have used an animal model, which employs FeCl3 to induce thrombi formation to study the effectiveness of both rHyb and cHyB on preventing thrombi formation. We obtained results that show that both rHyB and cHyB can inhibit thrombi formation in a dose-dependent manner. These results suggest that either rHyB or cHyB may be a possible therapeutic agent in preventing thrombi formation.

Peptides derived from platelet non-integrin collagen-receptors or types I and III collagen inhibit collagen-platelet interaction.

Platelet-collagen interaction plays an important role in hemostasis and pathological thrombosis. Upon an injury to the subendothelium of a blood vessel wall, platelets adhere to the denuded substrate, aggregate, and release biological substances. Many investigators have explored the use of blocking agents to interrupt the final step of binding fibrinogen on glycoprotein (GP) IIb/IIIa of activated platelets. A potent peptide is Arg-Gly-Asp-Ser (RGDS) and its derivatives in various forms. Results from many clinical trials show that the efficacy of these antagonists does not lie in blocking the adhesion of platelets to the distal site(s) of the injury as expected. Because type I and type III collagens are predominant components of blood vessel walls, other laboratories and ours have defined various active peptides from either collagen molecules or platelets as useful for blocking collagen-platelet interaction. An active hybrid peptide derived from both platelet types I and type III collagen receptors that abolishes type I and type III collagen-induced platelet aggregation has been obtained. The hybrid peptide inhibits the binding of type I and type III collagens to washed human platelets, platelet aggregation, and the adhesion of washed platelets to rabbit aortic segments. However, the usefulness of the defined hybrid peptide in preventing thrombi formation in vivo requires further investigation.

Mammalian collagen receptors.

Collagen-rich extracellular matrices are abundant and ubiquitous in the mammalian body. Collagens are not only essential for the mechanical stability of tissues, but are also intimately involved in controlling cell behaviour. The hallmark of collagens is a triple helix made up of polypeptide chains containing glycine-X-Y repeats. A structurally and functionally diverse group of cell surface receptors mediates the recognition of triple-helical collagen: integrins, discoidin domain receptors, glycoprotein VI, leukocyte-associated IG-like receptor-1, and members of the mannose receptor family. In this review, we discuss the structure and function of these receptors, focussing on the principles involved in collagen recognition.

Integrin evolution: insights from ascidian and teleost fish genomes.

Integrins are a family of alphabeta heterodimeric receptors essential to cell adhesion in all metazoans. In humans, the family consists of 18 alpha and 8 beta subunits that combine to form 24 dimers. Here, we present phylogenetic reconstructions for the alpha and beta integrin subunits based on sequences from 24 invertebrate and vertebrate species, including the fully sequenced genomes of the ascidian Ciona intestinalis (a urochordate) and the pufferfish Takifugu rubripes (a teleost). Both genomes contain integrin alpha subunits that have the inserted alphaI domain. As for the one alphaI domain containing integrin alpha subunit discovered earlier from the ascidian Halocynthia roretzi, the Ciona alphaI domains are missing the distinctive characteristics of mammalian collagen receptors and segregate from all vertebrate alphaI domain integrins in a phylogenetic tree, forming a new subgroup of alpha subunits with alphaI domains. Each of the pufferfish alphaI domain sequences does have characteristics of the collagen receptor alphaI domains, but no leukocyte-specific alphaI domains were found in pufferfish. Comparative protein modeling suggests that several of these fish alphaI domains are structurally compatible with binding to a GFOGER sequence in a collagen triple helix.