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The discoidin domain receptor DDR2 is a collagen-binding receptor tyrosine kinase whose dysregulation is associated with a wide range of diseases. Missense mutations in the DDR2 kinase domain cause Warburg-Cinotti syndrome in an autosomal dominant manner. Warburg-Cinotti syndrome is a severe connective tissue disorder, characterised by a range of manifestations including joint contractures of the hand, corneal vascularisation and pannus, skin fusion and infection, keloid plaques and acro-osteolysis. The Warburg-Cinotti variants, p.Leu610Pro and p.Tyr740Cys, were previously hypothesised to cause disease through a gain-of-function mechanism but mechanistic studies addressing this notion have been lacking. Here we show that both disease variants exhibit ligand-independent constitutive autophosphorylation when expressed as full-length proteins in mammalian cells. We also characterised the enzyme kinetics of soluble WT and DDR2-Y740C kinase constructs. WT DDR2 kinase was found to follow the same two-step activation mechanism previously characterised for DDR1 kinase but with enhanced autophosphorylation and substrate phosphorylation rates. Compared with WT DDR2, DDR2-Y740C displayed further enhanced autophosphorylation and substrate phosphorylation rates, but no effect on ATP binding affinity. The increased catalytic rates of unphosphorylated DDR2-Y740C kinase were similar to those of fully phosphorylated WT DDR2, indicating that the missense variant bypasses all autoinhibitory constraints and adopts the fully active kinase conformation. Tyrosine-740 is a residue in the A-loop of DDR2 kinase that forms autoinhibitory hydrogen bonds with key catalytic residues. These hydrogen bonds cannot form in the cysteine-substituted variant, providing a structural explanation for the release of the A-loop from its autoinhibitory conformation.

The collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants (‘receiver’) with functional DDR1 (‘donor’) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers. The membrane surrounding each living cell contains a variety of proteins that carry out different roles. For example, proteins called receptor tyrosine kinases help a cell to receive signals from its external environment. Receptor tyrosine kinases span the membrane so that one part of the protein known as the ectodomain sticks out from the surface of the cell, while another part (called the kinase domain) sits inside the cell. When a signalling molecule binds to the ectodomain, the kinase domain becomes active and starts to add chemical groups called phosphates to other proteins. This process, known as phosphorylation, changes the protein’s activity, which in turn influences the cell’s behaviour. In most cases, the signalling molecule causes two receptor tyrosine kinase proteins to bind to each other and form a “dimer” in which the kinase domains are able to phosphorylate, and thus activate, each other. Female mammals need a receptor tyrosine kinase called DDR1 to develop mammary glands (the glands that produce milk). DDR1 is activated when a signalling molecule called collagen binds to its ectodomain. Unlike many other receptor tyrosine kinases, DDR1 exists as a dimer even before it binds to collagen, so it is not clear how collagen activates DDR1. One possibility is that collagen causes several DDR1 dimers to form clusters on the membrane so that kinases on neighbouring dimers can phosphorylate each other. Juskaite et al. explored this idea by pairing up normal DDR1 proteins with mutant versions that are unable to bind to collagen. The experiments show that when collagen binds to the normal DDR1 molecules, DDR1 dimers do indeed form clusters. This enables the normal protein molecules in neighbouring dimers to phosphorylate each other as well as the mutant proteins. In this way, the clustered DDR1 dimers can become active even if the clusters contain one or more mutant versions that are unable to detect collagen. Further experiments show that specific contacts need to form between neighbouring dimers for this phosphorylation to occur. Abnormal DDR1 activity is associated with several diseases including cancer, inflammation and fibrosis. The findings of Juskaite et al. suggest that developing new drugs that can prevent DDR1 from forming clusters may help to treat people with these conditions. Further work is also needed to analyse the size and structure of DDR1 clusters and investigate if other proteins also associate with the clusters.

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register. Designed heterotrimers of collagen I, locked in three possible chain registers, enable structural and functional characterization of each permutation, leading to identification of the AAB heterotrimer as the most active and therefore likely to occur biologically.

Almost all human cancers display dysregulated expression and/or function of one or more receptor tyrosine kinases (RTKs). The strong causative association between altered RTK function and cancer progression has been translated into novel therapeutic strategies that target these cell surface receptors in cancer. Yet, the full spectrum of RTKs that may alter the oncogenic process is not completely understood. Accumulating evidence suggests that a unique set of RTKs known as the discoidin domain receptors (DDRs) play a key role in cancer progression by regulating the interactions of tumor cells with their surrounding collagen matrix. The DDRs are the only RTKs that specifically bind to and are activated by collagen. DDRs control cell and tissue homeostasis by acting as collagen sensors, transducing signals that regulate cell polarity, tissue morphogenesis, and cell differentiation. In cancer, DDRs are hijacked by tumor cells to disrupt normal cell–matrix communication and initiate pro-migratory and pro-invasive programs. Importantly, several cancer types exhibit DDR mutations, which are thought to alter receptor function and contribute to cancer progression. Other evidence suggests that the actions of DDRs in cancer are complex, either promoting or suppressing tumor cell behavior in a DDR type/isoform specific- and context-dependent manner. Thus, there is still a considerable gap in our knowledge of DDR actions in cancer tissues. This review summarizes and discusses the current knowledge on DDR expression and function in cancer. It is hoped that this effort will encourage more research into these poorly understood but unique RTKs, which have the potential of becoming novel therapeutic targets in cancer.

A complex containing the Par3/6 polarity proteins and the matrix receptor DDR1 modulates migration by downregulating cortical actomyosin contractility. Collective cell migration occurs in a range of contexts: cancer cells frequently invade in cohorts while retaining cell–cell junctions. Here we show that collective invasion by cancer cells depends on decreasing actomyosin contractility at sites of cell–cell contact. When actomyosin is not downregulated at cell–cell contacts, migrating cells lose cohesion. We provide a molecular mechanism for this downregulation. Depletion of discoidin domain receptor 1 (DDR1) blocks collective cancer-cell invasion in a range of two-dimensional, three-dimensional and 'organotypic' models. DDR1 coordinates the Par3/Par6 cell-polarity complex through its carboxy terminus, binding PDZ domains in Par3 and Par6. The DDR1–Par3/Par6 complex controls the localization of RhoE to cell–cell contacts, where it antagonizes ROCK-driven actomyosin contractility. Depletion of DDR1, Par3, Par6 or RhoE leads to increased actomyosin contactility at cell–cell contacts, a loss of cell–cell cohesion and defective collective cell invasion.

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.

The architecture of tumor collagen greatly influences tumor biology and therapeutic response. Two new studies identify tumor DDR1 as a central player in stromal collagen deposition and organization in the primary tumor and in disseminated tumor cells, resulting in immune exclusion or sustained dormancy, respectively.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro . In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of β integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin β1 to focal adhesions and enhances integrin β1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis. Aberrant DDR1 activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity. Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps: rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells. The insights may be used to develop allosteric, DDR1-specific, kinase inhibitors.

We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.