Several putative sites in the discoidin DS domain

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 Cy5.5 hydrazide (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. It is interesting to note that oligomerization of recombinant DDR1 ECD primarily occurred when the protein was incubated with collagen in solution and to a much lesser extent when collagen was pre-immobilized on a surface as reported in our earlier studies (Agarwal et al., 2007). We believe that pre-immobilization of collagen prevented accessibility of binding sites along all the faces of the collagen triple helix, and thus restricted receptor oligomerization. The use of neutral pH (conducive to this receptor–ligand interaction) did not enable strong attachment of collagen to mica required for fluid imaging. Therefore we had to employ AFM imaging of dried samples. Dry AFM imaging has previously been used to study binding of SPARC (Wang et al., 2005) and vWF (Novák et al., 2002) to collagen type I and of laminin to collagen type IV (Chen and Hansma, 2000). The basic morphology of DDR1-Fc dimers and collagen was found to be very similar in fluid vs. dry state based on our previous studies and our current results. Oligomerization of membrane receptors plays an important role in the receptor function and existing literature points toward specific downstream signaling that is unique to multivalent ligands (Cochran et al., 2000, Kiessling et al., 2006). Numerous receptors, including EGFR (Stabley et al., 2013), integrins (Boettiger, 2012) toll-like receptor (Triantafilou et al., 2006), ErbB family (Yarden and Sliwkowski, 2001) and Ephrin (Salaita et al., 2010) have been found to assemble into higher-order structures where downstream signaling events are often correlated to cluster formation. Consistent with our observations, a large heterogeneity in oligomer size has been reported for other RTKs, like the epidermal growth factor receptor (EGFR) (Abulrob et al., 2010). A recent study using a nano-patterned supported lipid bilayer technique to control EGFR clustering levels in living cells found that large-scale clustering of EGFR dampens its phosphorylation and that the cell endocytosis machinery contributes to this clustering behavior (Stabley et al., 2013).