CDB15:0000338 COL2A1 — ITGA2

ABSTRACT: Previously identified high affinity integrin-binding motifs in collagens, GFOGER and GLOGER, are not present in type III collagen. Here, we first characterized the binding of recombinant I domains from integrins alpha(1) and alpha(2) (alpha(1)I and alpha(2)I) to fibrillar collagen types I-III and showed that each I domain bound to the three types of collagens with similar affinities. Using rotary shadowing followed by electron microscopy, we identified a high affinity binding region in human type III collagen recognized by alpha(1)I and alpha(2)I. Examination of the region revealed the presence of two sequences that contain the critical GER motif, GROGER and GAOGER. Collagen-like peptides containing these two motifs were synthesized, and their triple helical nature was confirmed by circular dichroism spectroscopy. Experiments show that the GROGER-containing peptide was able to bind both alpha(1)I and alpha(2)I with high affinity and effectively inhibit the binding of alpha(1)I and alpha(2)I to type III and I collagens, whereas the GAOGER-containing peptide was considerably less effective. Furthermore, the GROGER-containing peptide supported adhesion of human lung fibroblast cells when coated on a culture dish. Thus, we have identified a novel high affinity binding sequence for the collagen-binding integrin I domains.

ABSTRACT: The mechanism of interaction of chondrocytic cells with cartilage-specific type II collagen has been examined using HCS-2/8 human chondrosarcoma cells as a model system. By the criteria of specific collagen secretion and integrin expression profile, HCS-2/8 have a similar differentiated phenotype to normal chondrocytes and are therefore a good model system. HCS-2/8 cells were able to attach and spread on both native and heat-denatured pepsinised type II collagen, and assays using denatured cyanogen bromide fragments apparently localised the major cell binding site to the CB10 fragment. However, when they were used as soluble inhibitors, cyanogen bromide fragments were found to block adhesion to denatured collagen, but had no effect on either attachment or spreading on the native molecule. The inability of cyanogen bromide fragments to reproduce the cell binding site of native collagen demonstrated a strict dependence on collagen conformation. This was also reflected in the receptors that were employed by HCS-2/8 cells for binding to type II collagen: binding to native collagen was mediated by the integrin alpha 2 beta 1 while binding to denatured collagen was mediated by a novel alpha 5 beta 1-fibronectin bridge. The identification of this bridge adds to the mechanisms by which cells can bind to denatured collagens. The previously characterised KDGEA active site peptide from type I collagen was found to be inactive as an inhibitor of type II collagen-mediated adhesion. The implications of these findings for the strategies used to identify adhesive active sites within collagens are discussed. In particular, these data suggest that, unlike other integrin ligands, a synthetic peptide-based approach is not suitable for the identification of collagen active sites.

ABSTRACT: In the past, proteins have been described that may be involved in chondrocyte interactions with extracellular collagen, but little is known about the role of integrins in chondrocyte-collagen interactions. Here we report on the analysis of beta 1-integrin distribution in human fetal cartilage and on the expression of integrins on fetal chondrocytes, using monoclonal and polyclonal antibodies to integrin alpha- and beta-chains. We show the presence of alpha 2-, alpha 5-, alpha 6-, alpha v-, and beta 1-chains on freshly isolated chondrocytes by surface immunofluorescence in the fluorescence-activated cell sorter and by surface iodination followed by immunoprecipitation. Affinity chromatography of bovine chondrocyte membrane proteins on a collagen-Sepharose column followed by immunoprecipitation confirmed the presence of the collagen-binding alpha 2 beta 1-integrin on chondrocytes. Chondrocyte adhesion on native collagens I and II, on fibronectin, and on laminin was completely blocked by anti-beta 1; anti-alpha 2 reduced chondrocyte binding to collagen by only 40-50%; similarly, anti-alpha 1-antibodies were also able to reduce chondrocyte binding to collagen, although alpha 1 could not be unequivocally identified on chondrocytes. Chondrocyte adhesion to fibronectin was Mg(2+)- and Ca(2+)-dependent and could be inhibited by anti-alpha 5 and by RGD peptides. Chondrocyte adhesion to native collagens is Mg(2+)-, but not Ca(2+)-dependent and RGD-independent. Interestingly, although these data point to a role of alpha 2 beta 1 in chondrocyte-collagen interactions in vitro, alpha 2 could not be visualized in sections of human fetal cartilage, in contrast to the beta 1-, alpha v-, and alpha 5-chains which were present. This suggests that alpha 2 beta 1-integrin may be involved in the assembly of a pericellular collagen matrix in vitro, but may not be required for chondrocyte-collagen interactions in intact cartilage.