Members of the collapsin/semaphorin family play an important role in creating the complex pattern of neuronal connectivity. Inhibition of growth cone motility by chick collapsin is mediated by the intraneuronal protein CRMP-62. We have now isolated four rat sequences that are highly related to chick CRMP-62. All four genes are expressed exclusively in the nervous system and primarily during development. Rat CRMP-2/TOAD-64 is most closely related to chick CRMP-62 and is the most widely expressed CRMP within the nervous system. Rat CRMP-1 and CRMP-4/rUlip are expressed during discrete periods of neuronal development and are not found in the adult nervous system. Rat CRMP-3 has a distinct distribution, being expressed transiently in developing spinal cord and selectively in the postnatal cerebellum. The differential expression of these genes suggests that CRMPs may transduce signals from different semaphorins and that semaphorins may regulate the plasticity of the adult nervous system.

Discoidin domain receptor 2 (DDR2) is a tyrosine kinase receptor expressed in mesenchymal tissues, the ligand of which is fibrillar collagen. We have compared DDR2 signaling in skin fibroblasts derived from DDR2 ؊/؊ and DDR2 ؉/؊ mice. Proliferation of DDR2 ؊/؊ fibroblasts was significantly decreased compared with DDR2 ؉/؊ cells. DDR2 ؊/؊ fibroblasts exhibited markedly impaired capacity to migrate through a reconstituted basement membrane (Matrigel) in response to a chemotactic stimulus, which correlated with diminished matrix metalloproteinase-2 (MMP-2) activity by gelatin zymography and diminished MMP-2 transcription of a minimal MMP-2 promoter. In contrast, a lack of DDR2 had no effect on cell motility or ␣-smooth muscle actin or vinculin expression. Additionally, expression of type I collagen was greatly reduced in DDR2 ؊/؊ cells. Stable reconstitution of either wild-type DDR2 or constitutively active chimeric DDR2 in DDR2 ؊/؊ cells by retroviral infection restored cell proliferation, migration through a reconstituted basement membrane (Matrigel), and MMP-2 levels to those of DDR2 ؉/؊ fibroblasts. These data establish a role for DDR2 in critical events during wound repair.

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The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin‐1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two‐hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two‐hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two‐hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8–134 and 281–435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.

Discoidin domain receptor 2 (DDR2) is an unusual receptor tyrosine kinase in that its ligand is fibrillar collagen rather than a growth factor-like peptide. We examined signal transduction pathways of DDR2. Here we show that DDR2 is also unusual in that it requires Src activity to be maximally tyrosine-phosphorylated, and that Src activity also promotes association of DDR2 with Shc. The interaction with Shc involves a portion of Shc not previously implicated in interaction with receptor tyrosine kinases. These results identify Src kinase and the adaptor protein Shc as key signaling intermediates in DDR2 signal transduction. Furthermore, Src is required for DDR2-mediated transactivation of the matrix metalloproteinase-2 promoter. The data support a model in which Src and the DDR2 receptor cooperate in a regulated fashion to direct the phosphorylation of both the receptor and its targets. Receptor tyrosine kinases (RTKs)1 of the discoidin domain receptor (DDR) family are unlike most RTKs, in that they do not use typical peptide growth factors as ligands; instead, they signal in response to fibrillar collagens (1, 2), establishing the DDR family as receptors for extracellular matrix molecules. Thus far, two DDR receptors have been identified, DDR1 and DDR2. DDR1 is primarily expressed in epithelial cells in the brain, gastrointestinal tract, lung, and kidney, whereas DDR2 is expressed in interstitial cells in the heart, skeletal muscle, lung, brain, and kidney (3). DDR1 and DDR2 are differentially activated by collagens. DDR1 is activated primarily by collagen types I, II, III, V, and XI, whereas DDR2 is activated mainly by collagen types I and III (1, 2, 4).In addition to their unique ligand specification, several other features distinguish DDR receptors from other RTKs. The kinetics of DDR receptor activation by collagens differs significantly from other RTKs in response to their cognate ligands. For example, platelet-derived growth factor (PDGF) or epidermal growth factor stimulate receptor activation within seconds (4). In contrast, tyrosine phosphorylation of DDR receptors can be detected only after prolonged exposure to collagen (approximately 30 min), and then phosphorylation is sustained for an extended period (more than 16 h) (2, 5). This unique slow-on, slow-off phenomenon and receptor specificity raise important questions about the nature of downstream intracellular signals mediating the effects of DDR2.Receptor tyrosine kinases contain a catalytic domain that can autophosphorylate one or more tyrosine residues typically located in the noncatalytic region of the receptor. These phosphorylations lead to generation of docking sites for SH2 and PTB domains of signaling molecules that associate with the receptors (6). For example, PDGF receptor and fibroblast growth factor receptor associate with signaling molecules such as phospholipase C-␥, Src, Shc, and phosphatidylinositol 3-kinase. However, it is still unknown which molecules interact with DDR2.In this study, we have explored intracellular pathways medi...