Discoidin domain receptor signalling: from crystal structures to mechanisms

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

Abstract

We want to find out how specialised sensor proteins on the cell surface, called DDRs, transmit a signal into the cell. This research is important because faulty DDR signalling in humans can cause disease, for example arthritis and cancer. In order to develop drugs that target the DDRs, it is necessary to know the structure and understand the signalling mechanism of the DDRs. Our research will contribute towards this aim. The DDRs are composed of three parts: one part sticks out of the cell, a second part is embedded in the cell membrane, and a third part faces the interior of the cell. Previous studies have established that the DDRs are sensing the presence of collagen, which is a major constituent of all connective tissues. This allows cells to react to their environment. When collagen binds to the exterior part of DDRs, the interior part becomes active and informs the cell that collagen has been recognised. We do not understand what happens during this process, but we believe that the DDRs change their shape when they react with collagen. We will use a range of experimental techniques to study how DDRs bind collagen and how they respond to collagen binding. Collagen is difficult to study because it is a very large molecule. In our experiments we will use collagen peptides, which are small synthetic fragments of collagen. We have found that some collagen peptides can stimulate DDR activity, whereas others block DDR activity. Our previous experiments have revealed how DDR recognises a stimulatory collagen peptide. We now want to see how a blocking peptide is recognised. By comparing the DDR structures obtained with stimulatory and blocking peptides, we hope to learn more about the shape changes that occur during signal transmission. Our previous experiments have also revealed that the whole exterior part of DDRs pairs with itself to form what is called a dimer. We will make small, precise changes in the DDR dimer and study their effects on signalling. By combining many such observations, we can construct a detailed map of the regions within the DDR molecule that are involved in transmitting a signal across the cell membrane. Finally, we have created a very useful tool for studying DDR function: a set of antibodies directed against DDR1 (DDR1 is one of the two DDRs in humans). Antibodies are normally made by animals as a defence against pathogens, but by injecting mice with DDR1 protein, we have been able to obtain antibodies that bind to DDR1. Some of these antibodies block DDR1 signalling, whereas others stimulate this activity even in the absence of collagen. In order to understand how these antibodies influence DDR1 activity, we want to determine where on the DDR1 molecule they bind. This information will complement our map of functionally important regions within the DDR1 molecule. We also plan to create antibodies against the second human DDR, DDR2. Our antibodies will be extremely useful research tools that can be shared with other researchers. They could also be used for diagnostic or therapeutic purposes, but the development of such reagents would have to be carried out in a pharmaceutical company.

Technical Summary

The human discoidin domain receptors, DDR1 and DDR2, are unusual receptor tyrosine kinases that are activated by a major constituent of the extracellular matrix, collagen. The DDRs play fundamental roles in cell adhesion, migration and proliferation, and regulate matrix remodelling. With regards to human health, they are implicated in the progression of fibrotic diseases, arthritis and several types of cancer. Understanding DDR function at the molecular and cellular level is of great importance, but many fundamental questions remain unanswered. We previously showed that the DDRs are constitutively dimeric in the absence of ligand, unlike conventional receptor tyrosine kinases, and that specific residues within the DDR transmembrane domain are essential for receptor activation. We also determined crystal structures of the entire DDR1 ectodomain and of the DDR2 discoidin domain bound to an agonistic collagen peptide. The aim of this proposal is to define the mechanism of transmembrane signalling by constitutive DDR dimers. Using protein crystallography and our own function-blocking anti-DDR1 monoclonal antibodies we will reveal the binding modes of antagonistic collagen sequences and the conformation of the DDR1 dimer in its inactive state. Using extensive site-directed mutagenesis we will identify the DDR1 regions and dimer contacts that are associated with receptor activation. Finally, we want to generate functional anti-DDR2 monoclonal antibodies to enable further structure-function studies. The proposed research will provide detailed mechanistic insight into how the dimeric DDRs transmit a signal across the cell membrane. In addition, our research will generate unique tools that may prove valuable in the development of therapies that target unwanted DDR signalling in human diseases.

Planned Impact

The proposed research will significantly advance the knowledge base of academic research into receptor tyrosine kinases and collagens. Researchers in many fields will profit directly from the fundamental insights and reagents that will be generated by our research (for details, see the section on academic beneficiaries). Although the proposed research is basic, it is conceivable that commercially exploitable results will be obtained. Any intellectual property arising from this research grant will be exploited through Imperial College Innovations Ltd, the College's technology transfer company. As the DDRs are new drug targets for a number of human diseases, pharmaceutical companies aiming to develop anti-DDR compounds will be interested in the results of our studies. In this regard, BL has already consulted for Novartis Institute of Biomedical Research, Horsham, UK and Boehringer Ingelheim Pharmaceuticals USA, who are establishing a drug development programme against unwanted DDR signalling. A detailed biochemical understanding of the DDR activation mechanism is likely to be required for any such programme. Other receptor tyrosine kinases (RTKs) are the targets of several drugs, but our recent research has identified fundamental mechanistic differences between DDRs and canonical RTKs. The proposed research will provide the necessary framework for future diagnostic and therapeutic applications. The generation of anti-DDR2 monoclonal antibodies (mAbs) in rabbits may benefit patients in the long term, if these mAbs can be developed into drugs. We already obtained mouse anti-DDR1 mAbs that inhibit DDR1 signalling, and aim to obtain rabbit mAbs against DDR2 with similar characteristics. Like mouse mAbs, rabbit mAbs will need to be humanised before they can be used as drugs in humans, but this process is firmly established. Rabbit mAbs have a potential advantage over mouse mAbs in that they are often cross-reactive with the mouse antigens. Thus rabbit mAbs can be used in mouse models of human diseases, which is extremely useful for pre-clinical evaluations.

Publications

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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

 
Description We mapped the epitopes of our function-blocking anti-DDR1 mAbs to regions near the "bottom" of the DDR1 DS-like domain. Mutagenesis and receptor activation experiments showed that the crystallographic dimer does not represent the signal-transducing state of DDR1. Interestingly, these experiments helped uncover a region in the DDR1 DS domain that is essential for signalling without being involved in ligand binding.

To define functionally important dimer contacts within the DDR1 extracellular domain, we used cysteine- scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerisation did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines further away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signalling thus occurs in the absence of conformational coupling across the plasma membrane, plausibly by ligand-induced receptor clustering.

In addition to the above, the funding in this award also allowed us to complete a study started on another award. Here we showed that DDR1 and DDR2 activation promotes cell adhesion by activating the collagen binding integrins a1ß1 and a1ß1 through increasing their affinity for collagen.

In collaboration with researchers in the US, we used recombinant collagen, based on a bacterial collagen-like protein that was engineered to contain a DDR binding site. We showed that while the reagent bound both DDR1 and DDR2 specifically, it failed to induce receptor phosphorylation. The ability to bind the DDRs without inducing kinase activity suggested it could interfere with interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed both in vitro and in a cell migration assay. Thus, through engineering of a bacterial collagen-like protein, we created a biomaterial which could inhibit DDR1 activity in a biological context.
Exploitation Route The project provides novel insight into the activation mechanism of DDR1, specifically with regards to identifying receptor regions that are required for signalling without being involved in ligand binding. We also showed that the extracellular juxtamembrane region is unusually flexible, underpinning the notion that the DDR activation mechanism is different from that of other receptor tyrosine kinases. These findings may be exploited by the pharmaceutical industry in the drug discovery process. Furthermore, the generation of recombinant collagen-based biomaterials with DDR inhibitory properties could be useful in the study of the pathological roles of DDR signalling in a wide range of diseases.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The findings from the grant have had an impact on the commercial sector. Birgit Leitinger has engaged with industry. In particular, she has been contacted by several pharmaceutical companies who are establishing drug development programmes against unwanted DDR signaling. She has consulted for Merck-Serono (Germany) and Astex Pharmeceuticals in 2012 and was approached by Boehringer Ingelheim (USA), Hoffmann La-Roche (Switzerland) and Servier (France) in 2014 about possible collaborations. She has further consulted for Ono Pharma UK Ltd (two sessions in 2015) and Tizona Therapeutics, USA (phone consultation in 2015). In addition, she engaged in the planning of a collaborative project with Servier, France. Unfortunately, due to company-internal re-organisation, this collaborative project could not be started. Birgit Leitinger has also sought a licence for the distribution of her anti-DDR1 monoclonal antibodies. Imperial Innovations Ltd, Imperial College's technology transfer company, set up a licence agreement with Merck Millipore.
First Year Of Impact 2012
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description BBSRC Research grant responsive mode
Amount £479,586 (GBP)
Funding ID BB/R006245/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2021
 
Description Characterisation of DDR ligands 
Organisation University Hospital of Münster
Country Germany 
Sector Hospitals 
PI Contribution Analyse DDR activation by different types of collagen and different aggregate states of collagen
Collaborator Contribution Provides us with purified collagens in different aggregate states
Impact PhD project
Start Year 2012
 
Description Disease causing DDR2 mutations 
Organisation United Arab Emirates University
Country United Arab Emirates 
Sector Academic/University 
PI Contribution Together with the collaborators, we analysed disease-causing mutants of DDR2, the protein we study in BBSRC-funded research. A manuscript reporting these findings has been published in 2014.
Collaborator Contribution clinical evaluation of patient, identification of mutation in patient,
Impact Manuscript published in 2014 Al-Kindi et al, BMC Genet). Multidisciplinary: clinical medicine, cell biology, genetics
Start Year 2013
 
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 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 shedding 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided reagents (mutant DNA, antibodies that are not available commercially) and advice on receptor activation assays
Collaborator Contribution Biochemical assays of DDR1 shedding, cell migration assays
Impact Manuscript published, Shitomi et al, Mol BiolCell, 201; doi: 10.1091/mbc.E14-10-1463
Start Year 2012
 
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
 
Description 3 minute thesis challenge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact National competition for PhD students, in which participants have 3 minutes to explain their thesis to a lay audience. Anna Cocking won the competition at Imperial College London and was a finalist in Manchester (September 2014).

As a result of her success, Anna is now interested in public engagement activities and has been participating at public engagement events organised by NHLI, Imperial College London
Year(s) Of Engagement Activity 2014
 
Description NHLI outreach activity 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Several pupils visited my lab and I gave an overview of my research. The pupils were very engaged and asked good questions.
Year(s) Of Engagement Activity 2018
 
Description Seminar London Matrix Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Research talk at London Matrix Group autumn 2015 symposium
Year(s) Of Engagement Activity 2015
 
Description Seminar Matrix Biology Europe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited guest lecture at international conference which was very well received.

Some researchers approached me afterwards about possible collaborations.
Year(s) Of Engagement Activity 2014
 
Description Seminar at King's College London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Research talk, Seminar Series, Cardiovascular Division, King's College London
Year(s) Of Engagement Activity 2015
 
Description Seminar at Roche Basel 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Engagement with industry, talk about my research and discussions with lead scientists at Roche Innovation Centre, Basel Switzerland
Year(s) Of Engagement Activity 2015
 
Description Seminar in Berlin 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited guest lecture for Seminar series at Leibniz-Institut für Molekulare Pharmakologie and Max Delbrück Centrum für Molekulare Medizin, Berlin, Germany invited guest lecture held at Leibniz-Institut für Molekulare Pharmakologie, Berlin, Germany

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Seminar in Bordeaux 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited guest lecture; INSERM U1053 & SFR TransBioMed, Université Bordeaux Segalen, Bordeaux, France (January 2012) Invited guest lecture about my science at INSERM U1053 & SFR TransBioMed, Université Bordeaux Segalen, Bordeaux, France

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Seminar in Münster, Germany 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research talk at Seminar Series, Institute of Experimental Musculosceletal Medicine (IEMM), Westfälische Wilhelms University, Münster, Germany
Year(s) Of Engagement Activity 2015
 
Description Seminar in Navarra 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited guest lecture on my research at Center for Applied Medial Research, University of Navarra, Spain

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Talk at St George's hospital 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Discussed own research with clinically oriented audience
Year(s) Of Engagement Activity 2016
 
Description seminar at Merck Serono 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Invited guest lecture at Merck Serono R&D, Darmstadt, Germany invited seminar to researchers at Merck Serono R&D, Darmstadt, Germany

no actual impacts realised to date
Year(s) Of Engagement Activity 2012