br Abbreviations br Introduction br Collagen induced DDR
Collagen-induced DDR activation A key feature of DDRs is their ability to bind both fibrillar and non-fibrillar collagens (Shrivastava et al., 1997, Vogel et al., 1997). DDR1 and DDR2 recognize the GVMGVO (O, hydroxyproline) motif within fibrillar collagens I–III and V [(Konitsiotis et al., 2008, Xu et al., 2011a) and reviewed in (Leitinger, 2011)]. Additional binding sites for DDR2 have been mapped on collagens II and III, although peptides encompassing these sites do not activate DDR2 (Xu et al., 2011a). DDR1 and DDR2 display distinct specificity for network forming collagens with DDR1 only binding to collagen IV (Vogel et al., 1997, Xu et al., 2011a) and DDR2 only binding to collagen X (Leitinger and Kwan, 2006). The amino acids in DDR1 and DDR2 that are critical for collagen binding are located within the DS domain and are well-conserved: Trp53 (Trp52 in DDR2), Thr57 (Thr56 in DDR2), Arg105 and Glu113 in both DDR1 and DDR2 (Leitinger, 2003, Abdulhussein et al., 2004, Carafoli et al., 2009). The collagen IV binding site in DDR1 has been mapped to 5 amino encorafenib residues within the receptor DS domain (Xu et al., 2011a); however the sequence(s) of the DDR1 biding sites on collagen IV are not well characterized. Although DDR2 can interact with collagen X, its DS domain is not sufficient for the binding (Leitinger and Kwan, 2006). Interestingly, DDR binding sites on fibrillar collagens map to residues which are distinct from the ones recognized by other matrix receptors (e.g., integrins) so that simultaneous binding and signaling from both DDRs and integrins can be achieved. Although a single DS domain of DDR1 and DDR2 contains the collagen binding site, high affinity binding to collagen requires DS domain dimerization (Leitinger, 2003). DDRs exist in dimers that form in a ligand independent manner through interactions mediated by their transmembrane domains (Noordeen et al., 2006). Thus, collagen interacts with pre-formed DDR dimers and upon binding induces dimer oligomerization and conformational changes that result in receptor activation. Unlike the interaction of soluble growth factors with receptor tyrosine kinases, collagen binding to DDRs induces a slow receptor tyrosine autophosphorylation that requires hours to reach full activation and can persist up to 18h. Upon collagen binding DDR1 undergoes internalization at a faster rate than receptor autophosphorylation (Mihai et al., 2009) and it has been proposed that DDR1 phosphorylation occurs in the endocytic vesicles (Fu et al., 2013b). In addition to tyrosine autophosphorylation, maximal receptor activation may require the involvement of additional tyrosine kinases or inhibition of tyrosine phosphatases. In this context, Src activates both DDR1 and DDR2 (Ikeda et al., 2002, Dejmek et al., 2003) and Src-mediated DDR2 phosphorylation is required for full receptor kinase activity (Yang et al., 2005). Furthermore, treatment of cells expressing DDR1 or DDR2 with tyrosine phosphatase inhibitors increases DDR tyrosine phosphorylation in a ligand-independent manner (Alves et al., 1995). Despite these studies, there are still outstanding questions regarding mechanisms of DDR activation that need to be answered. How DDRs are activated by collagens is still poorly understood and whether DDR activation requires a combination of internalization into endocytic vesicles and phosphorylation by additional tyrosine kinases or inactivation of tyrosine phosphatases is unknown. Furthermore the identity of the tyrosine phosphatases that negatively regulate DDR1 is not clear, although analysis with the PhosphoMotif finder program revealed that some of the tyrosine residues in DDR1 are putative substrates for tyrosine phosphatases such as TCPTP, PTP1b, and SHP1 (Borza and Pozzi, unpublished).
DDR tyrosine phosphorylation Upon collagen binding DDRs become phosphorylated on tyrosine residues and can activate various downstream signaling pathways. Since mutations of the critical residues within kinase domain, Lys618 in DDR1a or Thr664 in DDR2, effectively block collagen-induced DDR1 or DDR2 phosphorylation, it is likely that DDR phosphorylation is due to autophosphorylation (Vogel et al., 2000, Olaso et al., 2001). Residues Tyr792, Tyr796 and Tyr797 in DDR1 and Tyr736, Tyr740 and Tyr741 in DDR2 are located within the kinase activation loop, a region of 20–35 residues which starts with the conserved residues Asp-Phe-Gly (DFG) motif and contains tyrosine residues which upon phosphorylation regulate the kinase catalytic activity. Thus, these tyrosine residues are predicted to be critical for the receptor activation (Perez et al., 1994). Quantitative mass spectrometry-based phosphoproteomic analysis of collagen-stimulated DDR2 expressing cells showed that Tyr736 and Tyr740 become phosphorylated from 8 to 24h after collagen binding (Iwai et al., 2013b), which is in agreement with the activation kinetics of this receptor (Vogel et al., 1997). Moreover, in vitro kinase assays with DDR2 showed that Tyr740 and Tyr741 are only phosphorylated when DDR2 reaches the maximal kinase activity (Iwai et al., 2013b). In regard to DDR1, a phosphotyrosine profile of non-small cell lung cancer (NSCLC) tumors and cell lines revealed that DDR1 is phosphorylated in lung tumors and that phosphorylation occurs at the activation loop (Rikova et al., 2007). Similarly, analysis of pervanadate-treated DDR1-expressing cells with antibodies directed against phosphotyrosines within the activation loop indicated that these tyrosines are indeed phosphorylated (Lemeer et al., 2012). Moreover, proteomic analysis of pervanadate-treated DDR1-expressing cells revealed that Tyr484, Tyr513 and Tyr521 within the intracellular juxtamembrane region are phosphorylated (Lemeer et al., 2012). Moreover, Tyr792 in DDR1 is phosphorylated in response to collagen (Borza and Pozzi unpublished, Phospho-DDR1 (Tyr792) Antibody #11994 Cell Signaling). Interestingly, Tyr481 within the cytoplasmic juxtamembrane region of DDR2 is constitutively phosphorylated and does not follow the kinetic of receptor activation (Iwai et al., 2013b). Overall these studies demonstrate that phosphorylation of tyrosine residues within the DDR kinase domain activation loop is an indicator of receptor activation. Both receptors contain additional tyrosine residues in the cytoplasmic domain that can be phosphorylated and serve as docking sites for adaptor molecules. For instance, analysis for DDR1-expressing cells showed that DDR1 activation by collagen results in binding of ShcA to Tyr513, SHP-2 to Tyr703, Tyr796 and Tyr740, and the p85 subunit of PI3K to Tyr881 (Vogel et al., 1997, L\'Hote et al., 2002, Koo et al., 2006, Wang et al., 2006). These interactions were confirmed and additional binding proteins such as RasGAP, SHIP1, SHIP2, STATs and SRC family kinases were identified using proteomics approaches (Lemeer et al., 2012). Phosphoproteomic analysis of DDR2-expressing cells identified several candidate downstream signaling proteins including SHP-2, Nck1, Lyn, and SHIP-2 following collagen activation (Iwai et al., 2013b).