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  • Biapenem mg Various domains of DDR have been shown to be imp


    Various domains of DDR1 have been shown to be important for receptor clustering and its oligomeric status. It is now understood that (i) dimerization [7] and higher-order oligomerization [12], [18] of the DDR1 extracellular domain (ECD) enhance its binding to collagen; (ii) DDR1 exists as non-covalent homodimer on the cell surface, which is mediated by critical residues within its ECD [17] and transmembrane domain (TMD) [15]; (iii) both full-length and kinase-dead DDR1 expressed on the cell surface undergo clustering upon collagen binding [16], [17], [18], [20], [21]; and (iv) clustering of DDR1 post-ligand binding is mediated in part by its ECD [18] and by its intracellular domain (ICD) [20], [21]. DDR1 clustering has been postulated to be a mechanism required for receptor activation based on our earlier microscopy-based studies [16], [18], X-ray crystallographic insights by Carafoli et al. [10] and recent cell-based studies [20]. In this regard, mutation of an N-glycosylation site in the DDR1 ECD (which results in a higher population of dimers) has been shown to induce ligand-independent activation of DDR1 [17]. In another study, a function-blocking monoclonal antibody, which binds to DDR1 ECD and inhibits collagen-induced receptor phosphorylation [10], also inhibited DDR1 clustering [21]. Thus, understanding the structural constrains and molecular mechanisms that promote the clustering and/or the oligomeric state of DDR1 could be exploited as a therapeutic avenue to modulate receptor function in diseases involving DDR1 activity. In Biapenem mg to DDR1, the role of oligomerization and/or clustering of DDR2 in mediating its interactions with collagen is less understood. Current data show that in DDR2, like in DDR1, (i) dimerization [7] and higher-order oligomerization of its ECD [11], [22] enhance its binding to collagen, and (ii) in cells, DDR2 exists as a constitutive non-covalent homodimer [15], which is partly promoted by high propensity of the TMD of DDR2 to self-interact [23]. A juxtamembrane segment in the ICD of DDR2 has also been shown to control receptor dimerization and thereby regulate collagen-dependent activation [24]. However, studies on DDR2 clustering, spatial distribution and its correlation with receptor phosphorylation post-ligand binding are lacking.
    Materials and Methods
    Results Using surface plasmon resonance, we previously demonstrated that oligomeric forms of DDR2-Fc displayed enhanced ability to bind bovine-dermal collagen I when compared to dimeric forms of DDR2-Fc [22]. In addition, in separate studies, we showed that monomeric DDR2-V5-His [2], dimeric DDR2-Fc [25], and oligomeric DDR2-Fc [22] forms could all inhibit collagen I fibrillogenesis. However, in these earlier studies, the relative abilities of these structural Biapenem mg states of the DDR2 ECD could not be compared side by side because of the different experimental conditions used. Here, under identical experimental conditions, we examined the relative abilities of the DDR2 ECD, in its various oligomeric states, to bind to soluble collagen I and modulate its fibrillogenesis. Studies were conducted using telopeptide-lacking bovine-dermal collagen as well as telopeptide-containing rat-tail collagen I. 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 [22]. 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 [22].
    Discussion DDRs are type I transmembrane proteins in which their ECD is exposed to the extracellular milieu, ready to interact with collagens. On the plasma membrane, DDRs are displayed as a mixture of monomeric and homodimeric forms, and thus, they also exist as inactive preformed non-covalent homodimers [15]. 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 [7] and oligomeric state of DDR2 ECD enhanced its binding to collagen [22]. 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 [18]. Conversely, monomeric DDR2-V5-His exhibited reduced binding, consistent with earlier reports using monomeric His-DS2, which comprised only the DDR2 discoidin domain [7], [9]. 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 [7]. 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.