Besides collagen other cytoskeletal and or intracellular pro

Besides collagen, other cytoskeletal and/or intracellular proteins could also have a putative role in modulating the spatial distribution and assembly of DDRs into clusters or filamentous structures. Our initial investigations to identify such proteins yielded limited success. Both DDR1b clusters as well as DDR1b and DDR2 filamentous structures did not co-localize with either vimentin, vinculin (Fig. S8), or f-actin (Fig. S9). This is consistent with an earlier report showing the lack of co-localization between DDR1 with these cytoskeletal proteins in voltage gated sodium channel cultured on immobilized collagen fibrils [20]. However, DDR1 has been reported to co-localize with non-muscle myosin II [20]. Along similar lines, we found that the filamentous structures formed by DDR1b-YFP and DDR2-GFP were enriched with the actin-binding protein cortactin (Fig. S10). It is tempting to speculate that the higher-order assembly of DDRs into filamentous structures may serve as a scaffold for recruiting proteins like cortactin and myosin II to communicate with the cell cytoskeleton. In this regard, it is important to note that DDRs have reported to play a role in cell-mediated traction forces and mechano-transduction [20] and in formation of linear invadosomes [36]. Further studies are required to understand how the DDR–collagen assembly and ensuing receptor phosphorylation may be involved in these processes. Collectively, the results presented here provide new insights into ligand binding, clustering, spatial distribution, and phosphorylation of DDR1b and DDR2 in response to soluble collagen I. As depicted in the cartoon of Scheme 1, we postulate a model in which the spatial distribution and assembly of DDRs is dependent on the morphological state of collagen and precedes receptor phosphorylation. In this model, we propose that DDR1b cluster formation is promoted by the presence of non-fibrillar collagen present during the early stages of collagen fibrillogenesis. These DDR1b clusters undergo endocytosis to early endosomes, within a few minutes of collagen stimulation, as shown in our earlier studies [16]. At later time points, a fraction of DDR1b receptor clusters may recycle back to the plasma membrane with their cargo. During this process of recycling, a sub-population of DDR1b clusters is enriched with phosphorylated receptor species at Y513. Whether these phosphorylated DDR1b clusters localize in the endosome or the cell-membrane cannot be presently deciphered. Further studies are required to dissect the molecular composition and sub-cellular location of DDR1b clusters, which may be responsible for specific cell-signaling pathways, as has been defined for other members of the receptor tyrosine kinase family [37], [38]. In this regard, it is interesting to note that DDR1 has also been reported to co-internalize with and phosphorylate upon stimulation of insulin-like-growth factor I (IGF-IR) receptor, and the collagen-dependent phosphorylation of DDR1 was impaired in the absence of IGF-IR [39].