No abstract

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structurefunction analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.Collagens are the most abundant proteins found in the animal kingdom. Whereas some collagens are key structural components in load-bearing tissues, others are essential elements of basement membranes. Collagens have a pivotal role in regulating cellular differentiation and pattern formation during embryogenesis and postnatal development. Increased synthesis of fibrillar collagens or perturbed turnover correlates with a variety of human diseases, including liver fibrosis, glomerulonephritis, vascular diseases, or tumor angiogenesis (1). Three different types of collagen-receptors are currently known: the tyrosine kinases discoidin domain receptor 1 and 2 (DDR1 and DDR2), 1 four integrin heterodimers containing the ␤1 subunit, and glycoprotein VI (2). Although glycoprotein VI is only found on platelets, both integrins and DDR are widely expressed and trigger an array of signaling pathways upon collagen binding.DDR1 and DDR2 are characterized by a ϳ155-amino acid discoidin homology domain (DiscD) in the extracellular region of the protein. The discoidin domain is followed by a 200-amino acid stretch termed the stalk region, a single transmembrane peptide, a juxtamembrane region, and the catalytic tyrosine kinase domain (Fig. 1A). DDR1 was isolated from a number of diff...

Discoidin domain receptor 1 (DDR1) is a transmembrane receptor tyrosine kinase activated by triple-helical collagen. So far six different isoforms of DDR1 have been described. Aberrant expression and signaling of DDR1 have been implicated in several human diseases linked to accelerated matrix degradation and remodeling, including tumor invasion, atherosclerosis, and lung fibrosis. Here we show that DDR1 exists as a disulfidelinked dimer in transfected as well as endogenously expressing cells. This dimer formation occurred irrespective of its kinase domain, as dimers were also found for the truncated DDR1d isoform. A deletion analysis of the extracellular domain showed that DDR1 mutants lacking the stalk region failed to form dimers, whereas deletion of the discoidin domain did not prevent dimerization. Point mutagenesis within the stalk region suggested that cysteines 303 and 348 are necessary for dimerization, collagen binding, and activation of kinase function. The identification of DDR1 dimers provides new insights into the molecular structure of receptor tyrosine kinases and suggests distinct signaling mechanisms of each receptor subfamily. Discoidin domain receptors (DDRs)3 are a subfamily of transmembrane collagen-binding receptor tyrosine kinases (RTK). DDRs are distinguished from other RTKs by a discoidin domain in their extracellular region, which functions as a lectin in the slime mold Dictyostelium discoideum (1). There are two genes that code for DDRs in humans, DDR1 and DDR2 (2). Each DDR contains an extracellular region containing the ligandbinding discoidin domain, a stalk region, a single transmembrane region, as well as a cytoplasmic domain consisting of a juxtamembrane region and a tyrosine kinase domain.Through alternative splicing in the juxtamembrane or kinase domain of the human gene, at least five isoforms DDR1a-DDR1e are generated (3). The longest DDR1 transcript codes for the c-isoform with 919 amino acids. Compared with the c-isoform, the a-isoform lacks 37 amino acids in the juxtamembrane region, and the b-isoform lacks six amino acids in the kinase domain because of alternative splicing (4, 5). The DDR1d and e-isoforms are truncated variants that either lack the entire kinase region (d-isoform) or parts of the juxtamembrane region and the ATP-binding site (e-isoform (3)). A sixth isoform lacking parts of the extracellular domain has been described from rat testis (6). DDR1 is expressed in a variety of cell types and tissues, specifically in the mammary gland, brain, kidney, lung, and colon mucosa and has been isolated from several carcinoma cell lines, including MCF7 mammary carcinoma cells, ovarian, lung, and esophageal cancer cells, and primary pediatric brain tumor samples (for review see Ref.2). Immune cells, including macrophages, vascular smooth muscle cells, as well as oligodendrocytes also express DDR1 (7-10). From experiments with cultured cells, it was found that DDR1 regulates cell migration and branching morphogenesis within collagen-rich matrices (11,12).To address the ro...

Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators.Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domaincontaining proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.