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  • Collectively the results presented here provide new insights


    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 MMP-2 Inhibitor II mg non-fibrillar collagen present during the early stages of collagen fibrillogenesis. These DDR1b clusters undergo MMP-2 Inhibitor II mg 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]. Finally, it is important to emphasize the limitations and sensitivity of the techniques and reagents used (e.g., a limited number of anti-phosphotyrosine antibodies) here to follow the spatial–temporal profile of receptor phosphorylation. Another caveat in our studies was that only partial and/or intermittent co-localization of immuno-stained collagen was observed with the clusters and filamentous structures formed by DDRs. We postulate that this could be due to masking of antibody-recognizing epitopes on collagen as a result of DDR binding. Future studies using additional antibodies, fluorescently labeled collagen, submicroscopic high-resolution imaging of molecular complexes, laser capture-microdissection [40] and identification of phosphorylated receptor species by phospho-proteomics approaches at various time points are warranted to elucidate the state of collagen and of these unique collagen receptors upon ligand interactions.
    Acknowledgment This work was supported by NSF CMMI award 1201111 to G.A., AHA predoctoral award 16PRE31160013 to D.Y., and grants from the NIH-NCI (CA1986), Department of Defense (W81XWH-15-1-0226), and the Sky Foundation to R.F. We acknowledge Nabanita Chatterjee at OSU for her assistance with cell-culture experiments.
    Introduction Collagen fibrillogenesis, the assembly of collagen fibers, is a critical process in the development, maturation, and repair of mammalian tissue. Alterations in the structure and amount of deposited collagen fibers can greatly alter the integrity of the whole tissue. Even a single point mutation in collagen type I can severely compromise the strength of cortical bone tissue leading to osteogenesis imperfecta. Further, the interaction between collagen-binding proteins and collagen molecules during fibrillogenesis can promote significant alterations in the resulting collagen fiber structure and subsequent extracellular matrix (ECM) remodeling.2, 3 For example, soluble collagen-binding proteins such as decorin, biglycan, fibronectin, and vitronectin are thought to play a significant role in the process of collagen fibrillogenesis and bone mineralization due to their interaction with collagen molecules. The collagen-binding membrane proteins discoidin domain receptors (DDR1 and DDR2) are transmembrane receptors belonging to the family of receptor tyrosine kinases and have been studied for ECM remodeling in atherosclerosis,5, 6, 7 osteoarthritis,8, 9, 10 and several malignancies. It is well established that activation of the DDR1 and DDR2 kinase domain up-regulates the expression of various matrix metalloproteinases5, 11 and alters the biosynthesis of collagen. The extracellular domain (ECD) of DDRs is known to be necessary and sufficient for its interaction with collagen. Besides the full-length receptor, the DDR1 ECD is also found in five distinct isoforms and as a shed protein in the ECM.13, 14 Several protein and mRNA15, 16, 17 species consisting of the DDR2 ECD have also been observed in vivo. However, the functional roles of these ECDs of DDRs lacking their kinase domain are not well understood. We had previously elucidated that DDR1 ECD and DDR2 ECD inhibit collagen fibrillogenesis in vitro when used as purified proteins. Further, we have recently demonstrated that the DDR2 ECD when anchored on the cell surface preserves the capacity to inhibit collagen fibrillogenesis independent of its kinase activity. It is therefore likely that the expression of soluble ECD of DDRs by cells such as those found in the shedding of DDR1 ECD may play an important role in matrix remodeling.