The collagen binding integrins: structure and regulation

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

Collagen is the most abundant protein in the body which provides structural support in connective tissue. Collagen binds to many other proteins, including cell-surface receptors. Important collagen receptors include four members of a family of called integrins, which can bind collagen only when the cell is activated, not at rest. The integrins change shape and can then form tight links with specific regions of collagen. We will synthesise and identify those collagen fragments that interact selectively with the four integrins, alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1. This will indicate the role of these different integrins in different tissues, where different collagen types may occur. We will investigate changes that occur in the receptor as it is activated, by making purified receptor and studying its structure crystallographically. Specifically, we will make the fragment of the receptor known as its I-domain in different forms and examine their ability to bind collagen, so identifying the active form needed to bind collagen. We will examine the ability of the different integrin-binding collagen fragments to support cell adhesion in a variety of experimental model systems that will give insight into disease processes including thrombosis, tumour cell invasion and wound healing.

Technical Summary

The integrins comprise a widespread family of adhesive receptors. A subset of these, (alpha1-, alpha2-, alpha10- and alpha11-beta1), recognises GER-sequences within collagen through their alpha subunit I-domain, which is regulated by other parts of the integrin. Our corresponding synthetic triple-helical GER-peptides are recognised by these integrins, and we express mutant and wild type recombinant I-domains and heterodimeric integrins as both soluble and cell-surface forms. These materials, together with platelets and different integrin expressing cell lines, will allow the interface between collagens and integrins to be investigated, with the following objectives in view: to establish the structural properties of the integrins in complex with their ligands, to investigate both the structural changes and physiological processes leading to activation of integrins (affinity regulation). For these aims the following topics will be investigated: co-operation of alpha I-domain and the beta subunit in soluble integrin, role of disulphide-exchange, intermediate affinity states, interaction with peptides. The programme will examine the binding of integrins and cells to collagen substrates, collaborating on the direct measurement of the affinity of the interaction using atomic force microscopy. Integrin preparations will be co-crystallised with triple-helical collagen peptides in collaboration with Dr J Emsley, Nottingham. The ability of peptides specific for Glycoprotein (Gp) VI, VWF/Gp Ib and integrin alpha2beta1 to support platelet adhesion under blood flow conditions will be studied using confocal microscopy, establishing the parameters controlling adhesion and offering insight into the specific role of alpha2beta1 in platelets, where it is thought to serve a primary adhesive function and a secondary signalling role.

Publications

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Alexander SP (2013) The Concise Guide to PHARMACOLOGY 2013/14: overview. in British journal of pharmacology

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

 
Description BBSRC Project Grant (MJD)
Amount £10,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2009 
End 09/2012
 
Description BHF New Horizons (1)
Amount £304,000 (GBP)
Funding ID NH/11/1/28922 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 08/2014
 
Description BHF Programme (RWF3)
Amount £664,000 (GBP)
Funding ID RG/09/003/27122 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2009 
End 09/2014
 
Description BHF Project Grant Scheme (SMJ)
Amount £252,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2014
 
Description MRC Research Grant (GEJ)
Amount £350,000 (GBP)
Funding ID G0601378 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2007 
End 03/2010
 
Description Studentship (EH)
Amount £118,000 (GBP)
Funding ID FS/15/20/31335 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description WT Biomedical Resource Grant
Amount £472,000 (GBP)
Funding ID 094470/Z/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2011 
End 05/2015
 
Title Collagen mimetic peptides 
Description Libraries of triple-helical collagen-mimetic peptides used to map the binding of proteins (receptors etc) to collagen; the collagen III Toolkit was completed with Wellcome support, but the collagen II Toolkit was entirely MRC supported. Specific ligands for collagen receptors etc derived from the above. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Materials supplied to the platelet community have underpinned research worldwide on platelet activation, and integrin ligands have again found application in several laboratories both in UK and abroad. VWF-specific peptides are being developed for diagnostics. 
 
Title Receptor-specific and Integrin-binding peptides 
Description A short collagenous peptide that binds integrin by virtue of containing a Gxx'GEx'' motif. Industry standard for integrin-binding activity for collagen-binding integrins. Funding allowed us to maintain the supply of Glycoprotein VI-specific peptides and VWF-specific peptides for platelet and haemostasis research. This lab is the gold standard for such reagents. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Has the potential to support a wide-range of biocompatible materials; to act as a substrate for cell attachment. 
 
Description Alpha11Beta1 
Organisation University of Bergen
Department Department of Biochemistry and Cell Biology
Country Norway 
Sector Academic/University 
PI Contribution Peptides, collagen homology searches, mapping of sites in-house, etc.
Collaborator Contribution Provision of alpha11-bearing cells; insight, suggestion discussion. (Prof Donald Gulberg)
Impact Diversification into non-fibrillar collagens to map sites for the integrin alpha11beta1. No publications as yet. But we expect to complete the project within the timescale of this grant.
 
Description DDRs 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We supplied Toolkits to Imperial College, to map DDR2, then DDR1 binding to collagen. We used truncated and substituted peptides to pinpoint critical residues within the DDR2-binding motif, and synthesised a peptide for crystallisation of a DDR2-peptide complex.
Collaborator Contribution Publication; networking
Impact Publications: co-ordinates of complex; DDR2-binding motif patent application.
Start Year 2006
 
Description Fibronectin 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Synthesis and mapping of FN binding to collagen fragments. From our side, this arose from observations of indirect but integrin-dependent binding of cells to collagen. Led to link with Prof I. Campbell and Dr John Vakonakis.
Collaborator Contribution Insight into FN-collagen interaction; provision of FN domains for mapping
Impact Publications 19251642, 20739283 Involves mapping of sites and structural studies.
Start Year 2007
 
Description Maastricht 
Organisation Maastricht University (UM)
Country Netherlands 
Sector Academic/University 
PI Contribution Provision of peptides; conducting experiments; analysing data. Hosting of students in Cambridge.
Collaborator Contribution Exchange of personnel and ideas; asssitance in design of prototype apparatus.
Impact Publication 18826391 Understanding role of GpVI and alpha2beta1 in supporting platelet adhesion to collagen under flow; development of flow apparatus in Cambridge. Exchange of personnel (Maastricht students visiting Cambridge during masters programme). Development of synthetic surfaces for bed side analysis of haemostasis defect and anti-platelet therapy. Latter under auspices on Standardization Subcommittee of Int Soc on Thrombosis and Haemostasis.
 
Description Nitric Oxide 
Organisation University of Hull
Department Postgraduate Medical Institute Hull
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of peptides for platelet binding assays
Collaborator Contribution Understanding the regulation of platelet receptor affinity.
Impact Publication 18983487
 
Description VWF and LAIR-1 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution Provision of peptides; analysis interpretation of data. Binding interactions with mutant proteins.
Collaborator Contribution Crystallisation of collagen binding proteins in complex with our peptides, or by themselves. Therefore, detailed understanding of how proteins interact with collagen.
Impact Publications; development of VWF-binding peptides as detailed elsewhere. 20007810 19345263 18363567 17403111 16912226
 
Title Anti-GPVI antibodies 
Description Specific binding members directed to human glycoprotein VI (GPVI), in particular human antibodies, may employ the antibody VH and/or VL domain of the scFv fragment herein termed 10B12 or one or more complementarity determining regions (CDRs) of the 10B12 heavy chain variable (VH) and/or light chain variable (VL) domains, especially VH CDR3 in other antibody framework regions. Antibody molecules are provided with advantageous and unexpected properties, especially ability to inhibit collagen-induced platelet aggregation and the adhesion of platelets to Collagen-Related Peptide (CRP). Domain 1 of human GPVI is a primary target for the 10B12 antibody with these properties, and the epitope includes lysine 59. 
IP Reference WO2003054020 
Protection Patent application published
Year Protection Granted
Licensed No
Impact n/a
 
Title OSCAR-specific ligands 
Description Motifs within collagen that bind OSCAR and mediate osteoclast differentiation from monocytic cells. 
IP Reference US2012028903   
Protection Patent application published
Year Protection Granted 2012
Licensed Yes
Impact None
 
Description Jo Howes SET for Britain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Local MPs and guests, introduced to your researchers activities in the UK. See
http://www.setforbritain.org.uk

Group kept informed of related workshops & presentations.
Year(s) Of Engagement Activity 2012
URL http://www.setforbritain.org.uk
 
Description Schools work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We take one or two school children, typically at GCSE or AS level, into the lab for up to one week, and set them lab tasks that they can achieve.

Students able to make an informed choice about A level selection or university course applied for.
Year(s) Of Engagement Activity 2009,2010,2011,2012