Collagen IX is the prototype
fibril-associated collagen with interruptions in triple helix. In human cartilage it covers
collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four
collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for
collagen IX. Furthermore primary chondrocytes and
chondrosarcoma cells express the four
integrins simultaneously.
Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1
integrin as their only
collagen receptor, showed fast attachment and spreading on human recombinant
collagen IX indicating that it is an effective cell adhesion
protein. To further study the recognition of
collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains,
collagen IX was among the best collagenous
ligands, making
collagen IX exceptional compared with all other
collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of
collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First,
collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to
collagen I and GFOGER, had very different effects on its binding to
collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that
collagen IX has unique cell adhesion properties when compared with other
collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.