br Materials and Methods br Acknowledgements br
Materials and Methods
Introduction The extracellular matrix (ECM) is a dynamic structure that acts as a scaffold for the mechanical support of multicellular organisms. It is responsible for the organisation of different tissues and regulates critical events in development and disease through interactions with other matrix proteins and cell-surface receptors. The integrins are important cell-surface receptors mediating both cell–cell and cell–ECM contacts, resulting in signal transduction. Another important class of signalling receptors are the receptor tyrosine kinases (RTKs). Most RTKs bind small soluble proteins, but the two receptors of the discoidin domain RTK subfamily, DDR1 and DDR2, act as receptors for a major class of ECM proteins, the collagens.3, 4 Several, mostly fibrillar collagen types have been shown to induce DDR signalling. An unusual feature of the DDRs is that collagen-stimulated receptor autophosphorylation is very slow and sustained (∼hours), compared to the rapid response of typical RTKs to their ligands (∼seconds to minutes). Evidence from in vitro studies and the generation of DDR1 −/− and DDR2 −/− mice suggests that the DDRs regulate cell proliferation, adhesion and motility.5, 6, 7, 8, 9, 10 In addition, the DDRs can control ECM remodelling by influencing the expression and activity of matrix metalloproteinases.3, 6, 9, 11 The highly homologous DDR1 and DDR2 proteins are composed of an N-terminal discoidin homology (DS) domain of ∼150 amino Rosiglitazone residues, followed by a sequence of ∼220 amino acid residues unique to the DDRs, a transmembrane domain, a large cytosolic juxtamembrane domain and a conserved, C-terminal tyrosine kinase domain. In a previous study we showed that DDR activation by collagen is a consequence of direct, high-affinity DDR-collagen interaction and we mapped the collagen I binding site to three spatially adjacent surface loops within the DDR2 DS domain. Collagens constitute a large protein family of more than 20 members. Within this family, the fibrillar collagens (types I, II, III, V and XI), characterised by the presence of a 300nm long triple-helical domain, play a key architectural role as the most abundant proteins in the body of complex organisms. The self-assembly of fibrillar collagen monomers into highly organised fibrils provides mechanical strength to connective tissues. The triple-helical structure of fibrillar collagens consists of three collagen α chains, each of ∼1000 amino acid residues. Fibrillar collagens are secreted by cells as a soluble procollagen form, in which the N and C termini of the triple helix are joined to globular N and C-terminal propeptides. Upon secretion from the cell, the propeptides are cleaved by specific proteinases, and the monomers self-assemble spontaneously into characteristic collagen fibrils with molecules packed in a quarter-staggered array. In this packing arrangement, adjacent monomers overlap each other by 234 amino acid residues, giving rise to a 67nm wide repeat structure, termed D period, visible by electron microscopy. Collagen II is the major collagen found in cartilage. The collagen II triple helix is a homotrimer of three α1 chains. We have been interested in mapping the functional domains of the collagen II triple helix and developed a cDNA cassette system for recombinant production of collagen II variants. Using this system, we engineered deletion mutants that lack one of the four repeating D periods of the collagen triple helix and procollagen II-like proteins that are predominantly composed of a tandem repeat of one of the four D domains.16, 17
Discussion The present study demonstrates that DDR2 interacts with collagen II in a highly specific manner. Our solid-phase binding data (Figure 1) and receptor autophosphorylation assays (Figure 3) show that collagen II is primarily a ligand for DDR2. These results differ somewhat from two previous studies. In one study, which examined autophosphorylation of the DDRs in a similar set-up to the present study, using transiently transfected 293 cells, collagen II induced DDR1 phosphorylation preferentially. In a different study, in agreement with the present results, collagen II seemed to bind more strongly to the DDR2 ECD than to the DDR1 ECD. However, DDR1, expressed transiently in COS cells, was found to respond to collagen II with robust autophosphorylation. The phosphorylation response of DDR2 was not tested. We cannot offer any satisfactory explanation for the discrepancy between our results and those of the other studies.