535 2 It is interesting to note that our results show a

It is interesting to note that 535 2 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 [6], [33]. 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 [34]. 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 [22]. 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 [18], 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 [16], 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 [16], Cos-7 [21], and GD25 [20], by us and others, suggesting that DDR1 clustering is likely a ubiquitous phenomenon. Our results suggest that formation of DDR1b clusters may be important for and precede receptor phosphorylation. Indeed, while DDR1b clustering was readily detected (by YFP signal) 30 min after collagen administration, phosphorylated DDR1 species at Y513 (present in the IJXM) were evident after 4 h of collagen stimulation. Furthermore, the observation that not all DDR1b clusters were positive for Y513 signal lends support to the hypothesis that receptor clustering may be a prerequisite for receptor phosphorylation. Moreover, our findings that DDR1b clusters were positive for Y513 and not Y792 (present in the KD) suggest that differentially phosphorylated DDR1b receptor subpopulations may be segregated to various subcellular sites. However, whether the stronger detection of DDR1b/c-Y513 versus DDR1-Y792 signals in DDR1b clusters is due to differences in antibody affinity or differences in epitope availability and/or phosphorylation/dephosphorylation kinetics needs to be determined.