The collagen binding integrins: structure and regulation

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.

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.