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  • In vitro work by us and others revealed

    2020-09-16

    In vitro work by us and others revealed that high affinity interaction with collagen requires dimerization and/or pre-oligomerization of DDR1 (Agarwal et al., 2007, Abdulhussein et al., 2008, Leitinger, 2003). It has also been reported that a significant percentage of the DDR1 population forms ligand independent dimers on the cell-surface (Abdulhussein et al., 2008, Noordeen et al., 2006, Mihai et al., 2009). Using fluorescence microscopy and live cell imaging, we had also shown that ligand binding results in receptor oligomerization and Palifosfamide (Mihai et al., 2009). The specific sites in DDR1 responsible for receptor dimerization have been described to be the leucine zipper motif in the transmembrane domain (Noordeen et al., 2006) and the cysteine residues in the JM region of the DDR1 extracellular domain (ECD) (Abdulhussein et al., 2008). While it is speculated that regions in DDR1 ECD may contribute to receptor dimerization (Carafoli et al., 2012), the role of the ECD in receptor oligomerization is not completely understood.
    Materials and methods
    Results DDR1-Fc dimers and antibody induced DDR1-Fc oligomers have previously been characterized by us using atomic force microscopy (AFM) and SDS PAGE (Agarwal et al., 2007). DDR1-Fc appears as 91 and 197kD in reducing and non-reducing conditions respectively on SDS PAGE. The anti-Fc mediated oligomers are approximately 600kD as ascertained using size exclusion chromatography (Mihai et al., 2006). AFM analysis on samples in a fluid environment revealed that DDR1-Fc dimers and antibody mediated oligomers have an average topographic height of 3.24(±0.7) and 4.02(±0.8)nm, respectively.
    Discussion Our results demonstrate that oligomerization of DDR1 ECD is crucial for high-affinity receptor–ligand binding. Previous studies by us (Agarwal et al., 2007) and others (Leitinger, 2003) have shown conflicting results where DDR1-Fc dimers failed to bind to collagen in one study and did bind in another. These discrepancies were likely due to differences in the techniques used (surface plasmon resonance (SPR) vs. solid-phase binding assays) for analyzing receptor binding. In this study, by utilizing solid-phase binding assays we confirm that dimeric DDR1-Fc does indeed bind to collagen. The IC50 determined for binding of DDR1-Fc dimers (∼7.5nM) to bovine dermal collagen type 1 in this study was comparable to that for rat tail collagen type 1 (∼10nM) as reported earlier (Leitinger, 2003). We also demonstrate that pre-oligomerization of DDR1-Fc enhances its binding to collagen (IC50 for oligomers was three times lower than that for DDR1 dimers) consistent with our earlier SPR results (Agarwal et al., 2007). Using AFM, we show that the recombinant DDR1 ECD (which lacks the transmembrane and intracellular domains) undergoes oligomerization upon binding to collagen in-vitro. Although we could not determine the stoichiometry of DDR1 oligomers formed, our AFM results show that the mode value for DDR1-Fc size distribution before and after collagen binding was 2–3 and 4–5nm respectively. Based on the previously determined topographic heights of antibody-induced oligomers of DDR1 (Agarwal et al., 2007), we estimate that ligand binding results in formation of tetramers, hexamers or octamers consisting of two to four DDR1-Fc dimers. However, besides tetramers–octamers, higher order oligomers of DDR1-Fc (10–15nm in height) are also formed upon collagen binding. Our cell based studies confirmed that DDR1 oligomerization occurs at the cell surface upon ligand binding and the oligomers thus formed were heterogeneous in size. Several putative sites in the discoidin (DS) domain, DS-like domain and the JM region of DDR1 ECD may mediate oligomerization of DDR1-Fc dimers. In a recent study, Carafoli et al. (2012) have shown that monoclonal antibodies (mAbs) that bind to the DS-like domain of DDR1, inhibit collagen-induced receptor activation. They propose that mAbs prevent the proximity of the two DS-like domains and the JM regions in the collagen-bound, signaling, state of the DDR1 dimer. A conserved patch between the DS and DS-like domain is understood to mediate protomer contacts in the signaling DDR1 dimer, either by forming a direct DS-DS interface or by providing a secondary collagen-binding site. In addition, Arg32 and Leu152 in the DS domain were also shown to mediate dimer formation in the crystal state and were required for DDR1 signaling, even though they are not part of the primary collagen-binding site. Thus far, soluble versions of monomeric DDR1 ECD have shown little (Leitinger, 2003) or reduced (Abdulhussein et al., 2004) binding to collagen in solid-phase binding assays. It remains to be investigated if monomeric DDR1 ECD can undergo ligand-induced oligomerization as elucidated for DDR1-Fc dimers in this study.