Synthetic peptides activating discoidin domain receptor 2 and collagen-binding integrins cooperate to stimulate osteoblast differentiation of skeletal progenitor cells.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.

Development of a Selective Dual Discoidin Domain Receptor (DDR)/p38 Kinase Chemical Probe.

Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.

The Journey of DDR1 and DDR2 Kinase Inhibitors as Rising Stars in the Fight Against Cancer.

Discoidin domain receptor (DDR) is a collagen-activated receptor tyrosine kinase that plays critical roles in regulating essential cellular processes such as morphogenesis, differentiation, proliferation, adhesion, migration, invasion, and matrix remodeling. As a result, DDR dysregulation has been attributed to a variety of human cancer disorders, for instance, non-small-cell lung carcinoma (NSCLC), ovarian cancer, glioblastoma, and breast cancer, in addition to some inflammatory and neurodegenerative disorders. Since the target identification in the early 1990s to date, a lot of efforts have been devoted to the development of DDR inhibitors. From a medicinal chemistry perspective, we attempted to reveal the progress in the development of the most promising DDR1 and DDR2 small molecule inhibitors covering their design approaches, structure-activity relationship (SAR), biological activity, and selectivity.

Active MT1-MMP is tethered to collagen fibers in DDR2-containing remnants.

Type I collagen is a major extracellular matrix (ECM) component in the interstitial stroma of solid tumors, and it represents the first barrier against tumor cell invasion after basement-membrane degradation. The collagen receptors that convey molecular signals into the cells are collagen-binding discoidin domain receptors (DDRs) and integrins. Collagen-activated DDR2 clusters form DDR2-containing remnants in an integrin-dependent manner in three-dimensional (3D) collagen matrix. Although DDR2-containing remnants in the collagen matrix may generate sustained perturbation to ECM remodeling, the molecular components and function of the remnants are largely unknown. Here we determined the interaction and co-localization between DDR2 and membrane type I-matrix metalloproteinase (MT1-MMP) in the cells and the DDR2-containing remnants on collagen fibers, and we found that MT1-MMP was co-tethered to collagen fibers in the remnants. These collagen fiber-associated MT1-MMP remained active. Furthermore, DDR2 enhanced MT1-MMP proteolytic activity. These results demonstrate that DDR2 ensures the remnant-associated MT1-MMP to continue the degradation of ECM in addition to pericellular ECM degradation mediated by cell surface tethered MT1-MMP. Thus, our findings reveal a new alternative ECM degradation mechanism mediated by MT1-MMP in the DDR2-containing remnants.

Complex roles of discoidin domain receptor tyrosine kinases in cancer.

Discoidin domain receptors, DDR1 and DDR2 are members of the receptor tyrosine kinase (RTK) family that serves as a non-integrin collagen receptor and were initially identified as critical regulators of embryonic development and cellular homeostasis. In recent years, numerous studies have focused on the role of these receptors in disease development, in particular, cancer where they have been reported to augment ECM remodeling, invasion, drug resistance to facilitate tumor progression and metastasis. Interestingly, accumulating evidence also suggests that DDRs promote apoptosis and suppress tumor progression in various human cancers due to which their functions in cancer remain ill-defined and presents a case of an interesting therapeutic target. The present review has discussed the role of DDRs in tumorigenesis and the metastasis.

Modulation of biomimetic mineralization of collagen by soluble ectodomain of discoidin domain receptor 2.

Collagen fibrils serve as the major template for mineral deposits in both biologically derived and engineered tissues. In recent years certain non-collagenous proteins have been elucidated as important players in differentially modulating intra vs. extra-fibrillar mineralization of collagen. We and others have previously shown that the expression of the collagen receptor, discoidin domain receptor 2 (DDR2) positively correlates with matrix mineralization. The objective of this study was to examine if the ectodomain (ECD) of DDR2 modulates intra versus extra-fibrillar mineralization of collagen independent of cell-signaling. For this purpose, a decellularized collagenous substrate, namely glutaraldehyde fixed porcine pericardium (GFPP) was subjected to biomimetic mineralization protocols. GFPP was incubated in modified simulated body fluid (mSBF) or polymer-induced liquid precursor (PILP) solutions in the presence of recombinant DDR2 ECD (DDR2-Fc) to mediate extra or intra-fibrillar mineralization of collagen. Thermogravimetric analysis revealed that DDR2-Fc increased mineral content in GFPP calcified in mSBF while no significant differences were observed in PILP mediated mineralization. Electron microscopy approaches were used to evaluate the quality and quantity mineral deposits. An increase in the matrix to mineral ratio, frequency of particles and size of mineral deposits was observed in the presence of DDR2-Fc in mSBF. Von Kossa staining and immunohistochemistry analysis of adjacent sections indicated that DDR2-Fc bound to both the matrix and mineral phase of GFPP. Further, DDR2-Fc was found to bind to hydroxyapatite (HAP) particles and enhance the nucleation of mineral deposits in mSBF solutions independent of collagen. Taken together, our results elucidate DDR2 ECD as a novel player in the modulation of extra-fibrillar mineralization of collagen.

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.

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.

Spatial localisation of Discoidin Domain Receptor 2 (DDR2) signalling is dependent on its collagen binding and kinase activity.

Discoidin Domain Receptor 2 (DDR2) is a collagen-binding receptor tyrosine kinase that initiates delayed and sustained tyrosine phosphorylation signalling. To understand the molecular basis of this unique phosphorylation profile, here we utilise fluorescence microscopy to map the spatiotemporal localisation of DDR2 and tyrosine phosphorylated proteins upon stimulation with collagen. We show that cellular phosphorylated proteins are localised to the interface where DDR2 is in contact with collagen and not in the early endosomes or lysosomes. We find that DDR2 localisation is independent of integrin activation and the key DDR2 signalling effector SHC1. Structure-function analysis reveals that DDR2 mutants defective for collagen binding or kinase activity are unable to localise to the cell surface, demonstrating for the first time that both collagen binding and kinase functions are required for spatial localisation of DDR2. This study provides new insights into the underlying structural features that control DDR2 activation in space and time.

DDR1 and DDR2 physical interaction leads to signaling interconnection but with possible distinct functions.

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are members of the tyrosine kinase receptors activated after binding with collagen. DDRs are implicated in numerous physiological and pathological functions such as proliferation, adhesion and migration. Little is known about the expression of the two receptors in normal and cancer cells and most of studies focus only on one receptor. Western blot analysis of DDR1 and DDR2 expression in different tumor cell lines shows an absence of high co-expression of the two receptors suggesting a deleterious effect of their presence at high amount. To study the consequences of high DDR1 and DDR2 co-expression in cells, we over-express the two receptors in HEK 293T cells and compare biological effects to HEK cells over-expressing DDR1 or DDR2. To distinguish between the intracellular dependent and independent activities of the two receptors we over-express an intracellular truncated dominant-negative DDR1 or DDR2 protein (DDR1DN and DDR2DN). No major differences of Erk or Jak2 activation are found after collagen I stimulation, nevertheless Erk activation is higher in cells co-expressing DDR1 and DDR2. DDR1 increases cell proliferation but co-expression of DDR1 and DDR2 is inhibitory. DDR1 but not DDR2 is implicated in cell adhesion to a collagen I matrix. DDR1, and DDR1 and DDR2 co-expression inhibit cell migration. Moreover a DDR1/DDR2 physical interaction is found by co-immunoprecipitation assays. Taken together, our results show a deleterious effect of high co-expression of DDR1 and DDR2 and a physical interaction between the two receptors.