One possible explanation for differences in the binding abil
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 Neuregulin/Heregulin-1β (NRG-1β/HRG-1β), human recombinant protein 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. It is interesting to note that our results show a very similar behavior in binding of DDR2 ECD to immobilized a-telo (bovine-dermal) versus telo- (rat-tail) collagen I as a function of its oligomeric state. Thus, the telopeptide region of tropocollagen exerts little influence on DDR2-collagen binding, consistent with earlier findings that DDRs bind to the collagen triple-helix , . However, the presence of telopeptides did influence the modulation of collagen fibrillogenesis by DDR2 ECDs. Oligomeric and dimeric DDR2 ECD inhibited fibrillogenesis of the rat-tail collagen to a much lesser extent as compared to the bovine-dermal collagen. One possible explanation for the reduced effect of DDR2 ECD on fibrillogenesis of telo- collagen could be the differences in the rate of fibrillogenesis versus that of binding of DDR2 ECD to the collagen triple helix. As shown in our studies, the telo- rat-tail collagen exhibited a faster rate of fibrillogenesis and an overall higher turbidity when compared to the a-telo bovine-dermal collagen, consistent with the important role of telopeptides in promoting collagen fibrillogenesis . Our earlier studies using surface plasmon resonance have shown that binding of DDR2-Fc oligomers to immobilized collagen did not reach a saturation even after ~10 min . These observations suggest that the binding of DDR2 ECD to the collagen triple helix may be slow in comparison to the rate of fibrillogenesis of telo-collagen. A notable feature in our findings was the dissimilarity in the clustering ability of dimeric DDR1 ECD (DDR1-Fc) versus that of DDR2-Fc post-ligand binding, which was evident in the results from our AFM experiments. While the recombinant DDR1-Fc ECD spontaneously clustered to form high-order structures upon collagen binding in vitro , no such feature was observed for DDR2-Fc. Measurement of particle sizes from AFM images revealed that DDR2-Fc preserved its globular morphology and size post-collagen binding. Consistent with these in vitro observations, live-cell imaging showed that while the full-length DDR1b-YFP underwent a spatial re-distribution and cluster formation within minutes after collagen stimulation , DDR2-GFP maintained a homogenous distribution on the cell surface with no clustering at similar time points. While we cannot completely rule out potential contributions of the TMD or ICD domains of DDR2 in small cluster formation, which could not be resolved by wide-field light microscopy of cells, our results suggest that, unlike DDR1b, DDR2 is not able to organize into large clusters upon ligand binding. Interestingly, while clustering of DDR1b-YFP was observed in the MC3T3-E1 cells utilized in this study, clustering of DDR1 upon collagen stimulation has also been reported in other cell types, for example, HEK293 , Cos-7 , and GD25 , by us and others, suggesting that DDR1 clustering is likely a ubiquitous phenomenon.