It is interesting to note
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 hydrazide 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). Much less is known about the mechanism of DDR clustering and its role in the receptor function. It is understood that DDR1 exists as an inactive dimer on the cell surface and undergoes further oligomerization upon ligand binding (Abdulhussein et al., 2008, Noordeen et al., 2006, Mihai et al., 2009). Thus oligomerization seems to be a prerequisite for receptor activation. At present we are unable to define the reasons behind the heterogeneity observed in DDR1 oligomer sizes. We speculate that like the EGFR receptor, the large-scale (>10nm) clusters of DDR1 may dampen receptor activation and only the tetramer–octamer population (4–5nm in height) may be responsible for signaling. It is interesting to note that the time taken (4h) for formation of maximum number of DDR1 ECD tetramer–octamer in-vitro, coincides with that for maximal receptor phosphorylation (Vogel et al., 1997). An increase in oligomer size with collagen stimulation time was also observed for full length DDR1b–YFP in our earlier studies (Mihai et al., 2009). Further studies using antibodies against phophorylated DDR1 may help elucidate how oligomer size correlates with receptor phosphorylation. Endocytosis of the oligomerized receptor may also contribute to receptor function. In our previous study (Mihai et al., 2009), DDR1b oligomers formed upon ligand binding underwent rapid internalization and incorporation into the early endosomal compartments, an important site of receptor-initiated signal transduction (von Zastrow and Sorkin, 2007). In addition to their role as active signaling kinases it has been suggested (Fu et al., 2013) that DDRs, may act as molecular scaffolds similar to other kinases such as integrin-linked kinase (ILK) and kinase suppressor of ras1 (KSR1) (Hannigan et al., 2011, Zhang et al., 2012). Integrin clustering was shown to modulate downstream signaling at focal adhesion sites where these molecular scaffolds develop and it is possible that DDR clustering could play a similar role (Shi and Boettiger, 2003, Ye et al., 2010). Further studies would be needed to fully characterize the role played by DDR oligomerization in receptor function and signaling.