The monomeric oligomeric states of
The monomeric/oligomeric states of DDR2-V5-His and DDR2-Fc were confirmed by Western blotting under reducing and non-reducing conditions. As shown in Fig. 1b, DDR2-V5-His exhibited a relative molecular mass of ~60 kDa under both reducing and non-reducing conditions, consistent with this protein being in a monomeric state. In contrast, when resolved under non-reducing conditions, DDR2-Fc displayed a molecular mass of ~190 kDa, consistent with being an Fc-tagged dimer . Under reducing conditions, DDR2-Fc displayed a mass of ~90 kDa. The oligomeric state of DDR2-Fc oligomers, induced by the presence of anti-Fc antibodies, was determined earlier using size-exclusion chromatography .
Discussion DDRs are type I transmembrane proteins in which their ECD is exposed to the extracellular milieu, ready to interact with collagens. On the ascorbic acid membrane, DDRs are displayed as a mixture of monomeric and homodimeric forms, and thus, they also exist as inactive preformed non-covalent homodimers . Here we focused on the specific contribution of isolated ECDs of DDR2, capable of displaying monomeric, dimeric, or oligomeric forms, on collagen binding and collagen fibrillogenesis in vitro. We found that oligomeric and dimeric DDR2 ECD species showed the highest affinity toward immobilized collagen I, when compared to the monomeric form. These results are in agreement with earlier reports showing that a dimeric state of DDR2 ECD is required for high-affinity binding to collagen I  and oligomeric state of DDR2 ECD enhanced its binding to collagen . Our results also help resolve the discrepancies arising from the different binding assays utilized in these earlier reports. Furthermore, pre-oligomerization of DDR2-Fc enhanced collagen binding in solid-phase binding assays in a manner similar to that observed for DDR1-Fc . Conversely, monomeric DDR2-V5-His exhibited reduced binding, consistent with earlier reports using monomeric His-DS2, which comprised only the DDR2 discoidin domain , . It is important to note that in an earlier study, amino-terminal tagged His-DDR2 ECD was characterized to be a non-covalently linked dimer . This difference in the oligomeric state of His-tagged DDR2 ECD from this earlier work versus our current study could be due to the different sites for epitope tagging and/or differences in protein purification protocols. One possible explanation for differences in the binding ability of monomeric versus dimeric forms of DDR2 ECD to collagen could be that the monomeric form only binds to the primary GVMGFO site, whereas dimeric (and oligomeric) DDR2 ECD binds to additional sites on the collagen triple-helical molecule. Our results from AFM imaging support this hypothesis because the binding events for dimeric DDR2-Fc were observed to be more frequent than those for monomeric DDR2-V5-His on the collagen triple-helical molecule, under identical experimental conditions. Our earlier AFM studies had showed the existence of three possible binding sites for DDR2-Fc oligomers on the collagen I triple helix . Recent studies using col II and col III toolkit peptides have identified that besides the primary GVMGFO site, four additional DDR2 binding sites exist on the collagen triple helix , . Molecular modeling  and X-ray crystallographic studies  have provided insight into how the monomeric and dimeric forms of the DDR2 ECD can bind to the GVMGFO site, but no such studies exist for the remaining DDR2 binding sites. Although not discussed by the authors of these studies, it is interesting to note that in their toolkit studies, the various DDR2 ECD variants (including monomeric and dimeric DDR2 ECD) showed different relative affinities to these additional binding sites , . For instance, while dimeric DDR2-Fc recognized the toolkit peptide II-5 , this site was not recognized by DDR2-His . Thus, it is likely that binding of dimeric (and oligomeric) DDR2 ECD to additional sites on the collagen triple-helix could account for their stronger binding and inhibition of collagen fibrillogenesis when compared to the monomeric DDR2 ECD form.