The discoidin domain receptors: collagen binding specificity and cooperation with integrins in cell adhesion / migration

Lead Research Organisation: Imperial College London
Department Name: National Heart and Lung Institute

Abstract

We aim to study how cells use specialised proteins, called DDRs, to interact with collagen. When DDRs are faulty, they can cause disease. It is hoped that our work will benefit human health by creating reagents that may be useful in anti-DDR drug discovery programmes.

The DDRs are novel drug targets for a number of human diseases, including arthritis and certain cancers. The DDRs recognise collagen, but we know very little about this interaction. When the DDRs bind collagen, they transmit a signal into the cell. We do not understand how the DDRs transmit such information.

Collagen is the most abundant protein in our body and has a very complex structure. It is built like a rope, composed of three very long strands of amino acids that wind around each other. We will characterise the parts of collagen that are recognised by the DDRs. We will produce synthetic collagen fragments with the same rope-like structure as collagen and test which of these the DDRs use to transmit signals into cells.

The DDRs are not the only proteins that interact with collagen. Another type of protein called integrin also binds collagen. Because the DDRs and integrins both recognise collagen, their relative contribution to biological function has been difficult to study. We have recently discovered that DDRs recognise a different part of collagen than integrins. We can thus make synthetic collagen fragments that will bind only DDRs, but not integrins, and use these to understand how the DDRs regulate the cell’s function.

Technical Summary

The human discoidin domain receptors, DDR1 and DDR2, are highly unusual receptor tyrosine kinases that are activated by a major constituent of the extracellular matrix, collagen. Next to the collagen-binding integrins, the DDRs are the most widely distributed collagen receptors in mammals. The DDRs are important in development and aberrant DDR function leads to human disease. In particular, DDRs are responsible for disease progression in fibrotic diseases, arthritis and several types of cancer. In contrast to the situation with integrins, not much is known about the DDR-collagen interaction. Both DDRs are activated by a number of different collagen types, and activation is strictly dependent on the native, triple-helical conformation of collagen.

Our previous work has shown that the DDRs bind collagen with high affinity and in a highly specific manner. Very recently, using synthetic triple-helical peptides derived from collagen, we identified a specific sequence in collagen II recognised by DDR2 (unpublished results). This amino acid motif is distinct from that recognised by integrins and the platelet collagen receptor GPVI. Importantly, triple-helical peptides comprising the DDR2 binding site not only inhibit DDR2 binding to collagen II, but activate DDR2 autophosphorylation in a specific manner. Thus, we are now in a unique position to use high-affinity DDR2-specific collagen peptides as a research tool to investigate the cellular function of DDR2.

The aims of this proposal are twofold: 1) to define DDR1 and DDR2 binding sites in other collagens, and 2) to use our DDR-selective ligands to probe the contribution of the DDRs to cell adhesion and migration. Although the two DDRs are homologous, their collagen binding specificities are distinct; our studies will establish the molecular basis for these differences. The DDRs play important roles in cell adhesion and migration, but integrins are thought to be the primary mediators of these processes. The fact that both types of receptor recognise the same ligand (collagen) has complicated the analysis of their relative contributions. Using our new DDR-specific peptides, together with previously developed integrin-specific peptides, we are now able to discriminate between integrin-initiated and DDR-initiated events in cell adhesion and migration. We will study the possible cooperation between the DDRs and integrins. The results obtained from this research programme will establish the collagen binding specificities of the DDRs and give important novel insight into the roles of the DDRs in cell adhesion and migration.

Publications

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Borza CM (2017) Discoidin domain receptor 1 kinase activity is required for regulating collagen IV synthesis. in Matrix biology : journal of the International Society for Matrix Biology

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Carafoli F (2009) Crystallographic insight into collagen recognition by discoidin domain receptor 2. in Structure (London, England : 1993)

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Leitinger B (2011) Transmembrane collagen receptors. in Annual review of cell and developmental biology

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Leitinger B (2014) Discoidin domain receptor functions in physiological and pathological conditions. in International review of cell and molecular biology

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Multhaupt HA (2016) Extracellular matrix component signaling in cancer. in Advanced drug delivery reviews

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Valencia K (2012) Inhibition of collagen receptor discoidin domain receptor-1 (DDR1) reduces cell survival, homing, and colonization in lung cancer bone metastasis. in Clinical cancer research : an official journal of the American Association for Cancer Research

 
Description Project grant; Discoidin domain receptor signalling: from crystal structures to mechanisms
Amount £439,000 (GBP)
Funding ID BB/I011226/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2011 
End 11/2014
 
Title anti-DDR1 antibodies 
Description Although the anti-DDR1 antibodies were created without MRC funds, MRC funds helped to characterise these antibodies. 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact The antibodies used by my collaborators led to two high impact publications: Hidalgo-Carcedo, C., Hooper, S., Chaudhry, S.I., Williamson, P., Harrington, K., Leitinger, B., & Sahai, E. (2011). Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6. Nat. Cell Biol., 13, 49-58. Juin A, Di Martino, J, Leitinger, B, Henriet, E, Gary, AS, Paysan, L, Bomo, J, Baffet, G, Gauthier Rouviere C, Rosenbaum, J, Moreau, V & Saltel, F (2014). Discoidin domain receptor 1 controls linear invadosome formation via Cdc42/Tuba pathway, J Cell Biol, in press The antibodies were described in Carafoli et al, Structure (2012) and have led to new collaborations. The antibodies are also the basis for a patent. 
 
Description Characterisation of DDR2 SMED mutants 
Organisation United Arab Emirates University
Country United Arab Emirates 
Sector Academic/University 
PI Contribution We performed experiments that characterised biochemically the DDR2 missense mutations identified by our collaborators
Collaborator Contribution The collaborators have identified disease-causing mutants of DDR2, the protein we study in MRC-funded research. Together with the collaborators we have identified the underlying causes for a growth defect caused by DDR2 missense mutations. A manuscript reporting these findings has been published in 2010.
Impact A manuscript reporting the collaborative findings has been published in 2010 (see Publications, Ali et al, 2010)
Start Year 2009
 
Description Proteomics of DDR2 
Organisation Institute of Cancer Research UK
Department Division of Cancer Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution provided reagents, did flow cytometry assays, provided guidance on research and manuscript writing
Collaborator Contribution performed research on DDR2 phosphoproteomics
Impact Manuscript published: Iwai et al, Biochem J, 2013.
Start Year 2010
 
Description Role of DDR1 in cancer dormancy 
Organisation Memorial Sloan Kettering Cancer Center
Department Weill Graduate School of Biomedical Sciences
Country United States 
Sector Academic/University 
PI Contribution Provide antibodies made in my lab that are not commercially available; provide hybridoma cells to allow Ab production
Collaborator Contribution research on role of DDR1 in cancer dormancy
Impact Publlcation, Gao et al, Cell, 2016. doi: 10.1016/j.cell.2016.06.009
Start Year 2012
 
Description Role of DDR1 in cell migration 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Provide essential reagents for the study
Collaborator Contribution The collaborators have identified a novel role for DDR1, the protein of interest in the MRC-funded grant, in cell migration.
Impact A manuscript describing the findings has been published (Hidalgo-Carcedo, C., Hooper, S., Chaudhry, S.I., Williamson, P., Harrington, K., Leitinger, B., & Sahai, E. (2011). Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6. Nat. Cell Biol., 13, 49-58.)
Start Year 2009
 
Description Role of DDR1 in invadosomes 
Organisation National Institute of Health and Medical Research (INSERM)
Department INSERN (1053) (Université Bordeaux Segalen)
Country France 
Sector Public 
PI Contribution Advice on how to do receptor activation assays; provided antibodies made in my lab
Collaborator Contribution Research on role of DDR1 in invadosome formation
Impact Invitation to give seminar in Bordeaux, manuscript published (Juin et al, J Cell Biol, 2014); doi: 10.1083/jcb.201404079
Start Year 2012
 
Description Role of DDR1 in lung cancer 
Organisation University of Navarra
Department Center for Applied Medical Research
Country Spain 
Sector Academic/University 
PI Contribution Provided training in receptor activation assays, provided antibody that was made in my lab, will analyse the function of DDR1 in lung cancer cell lines
Collaborator Contribution For new experiments, the partner will provide us with cell lines
Impact Publication in Clin Cancer Research, doi: 10.1158/1078-0432.CCR-11-1686; invitation to give seminar in Pamplona; invitation to be external examiner for thesis in Spain and invitation to give research seminar in Spain
Start Year 2011
 
Description Study with engineered bacterial collagen 
Organisation Tufts University
Department Department of Biomedical Engineering
Country United States 
Sector Academic/University 
PI Contribution MRC and BBSRC employed post doc and PhD student performed experiments with material obtained from collaborators at Tufts university
Collaborator Contribution Provided engineered recombinant collagen for experiments
Impact Published paper (An et al, JBC, 2015)
Start Year 2010
 
Title Anti-DDR1 mAb licensed 
Description Anti-DDR1 mAb 5D5, which was characterised with MRC funds, was licensed to Millipore for sale 
IP Reference  
Protection Protection not required
Year Protection Granted 2015
Licensed Yes
Impact no other impacts so far
 
Title Blocking antibodies against DDR1 
Description DDR1 is a novel drug target for a number of human diseases. Function-blocking antibodies against DDR1, which were made prior to the MRC-funded research were characterised with MRC funds. These antibodies have the potential to be developed into therapeutic antibodies that could be used in a number of human diseases where DDR1 is involved in disease progression. 
IP Reference WO2013034933 
Protection Patent application published
Year Protection Granted 2011
Licensed No
Impact no impacts yet