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C-type lectins that contain collagen-like domains are known as collectins. These proteins are present both in the circulation and in extravascular compartments and are central players of the innate immune system, contributing to first-line defenses against viral, bacterial, and fungal pathogens. The collectins mannose-binding lectin (MBL) and surfactant protein D (SP-D) are regulated by tissue fibroblasts at extravascular sites via an endocytic mechanism governed by urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180), which is also a collagen receptor. Here, we investigated the molecular mechanisms that drive the uPARAP-mediated cellular uptake of MBL and SP-D. We found that the uptake depends on residues within a protruding loop in the fibronectin type-II (FNII) domain of uPARAP that are also critical for collagen uptake. Importantly, however, we also identified FNII domain residues having an exclusive role in collectin uptake. We noted that these residues are absent in the related collagen receptor, the mannose receptor (MR or CD206), which consistently does not interact with collectins. We also show that the second C-type lectin-like domain (CTLD2) is critical for the uptake of SP-D, but not MBL, indicating an additional level of complexity in the interactions between collectins and uPARAP. Finally, we demonstrate that the same molecular mechanisms enable uPARAP to engage MBL immobilized on the surface of pathogens, thereby expanding the potential biological implications of this interaction. Our study reveals molecular details of the receptor-mediated cellular regulation of collectins and offers critical clues for future investigations into collectin biology and pathology., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nørregaard et al.)

A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.

Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1β identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.

DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes.

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 structure-function 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.

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.

Endo180/urokinase plasminogen activator receptor-associated protein together with the mannose receptor, the phospholipase A(2) receptor, and DEC-205/MR6-gp200 comprise the four members of the mannose receptor family. These receptors have a unique structural composition due to the presence of multiple C-type lectin-like domains within a single polypeptide backbone. In addition, they are all constitutively internalized from the plasma membrane via clathrin-mediated endocytosis and recycled back to the cell surface. Endo180 is a multifunctional receptor displaying Ca(2+)-dependent lectin activity, collagen binding, and association with the urokinase plasminogen activator receptor, and it has a proposed role in extracellular matrix degradation and remodeling. Within their short cytoplasmic domains, all four receptors contain both a conserved tyrosine-based and dihydrophobic-based putative endocytosis motif. Unexpectedly, Endo180 was found to be distinct within the family in that the tyrosine-based motif is not required for efficient delivery to and recycling from early endosomes. By contrast, receptor internalization is completely dependent on the dihydrophobic motif and modulated by a conserved upstream acidic residue. Furthermore, unlike the mannose receptor, Endo180 does not function as a phagocytic receptor in vitro. These findings demonstrate that despite an overall structural similarity, members of this receptor family employ distinct trafficking mechanisms that may reflect important differences in their physiological functions.

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.

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 alpha-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.

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.

Vascular endothelial growth factor (VEGF) is an important mediator of ocular neovascularization by exerting its endothelial specific mitogenic effects through high affinity tyrosine kinase receptors. By screening a rat retina cDNA library, we have isolated a clone encoding the full-length prototypic form of the rat VEGF receptor-2/Flk-1, as well as a short form of the mRNA that encodes the complete seven N-terminal immunoglobulin-like extracellular ligand-binding domains, transmembrane region, NH2-terminal half of the intracellular kinase domain, and kinase insert domain but does not encode the COOH-terminal half of the intracellular kinase domain and carboxyl-terminal region. Both forms of mRNA are detected in rat retina, although the short form is expressed at a lower level. VEGF induced a biphasic increase of cytoplasmic calcium with both forms in HK 293 transfected cells, indicating that both forms of the VEGF receptor-2/Flk-1 are functional and that the COOH-terminal half of the intracellular kinase domain and carboxyl region of VEGF receptor-2/Flk-1 are not strictly necessary for either ligand binding or this biological activity.

The endothelial cell type-specific tyrosine kinase KDR/flk-1 is a receptor for vascular endothelial growth factor and a critical regulator of endothelial cell growth and development. To study mechanisms of endothelial cell differentiation and gene regulation, we have analyzed the topology of the proximal promoter of human KDR/flk-1. A protected sequence between base pairs -110 and -25 was defined by in vitro DNase I footprinting analysis in human umbilical vein endothelial cells (HUVECs). Purified Sp1 alone produced similar protection, and electrophoretic mobility shift assays demonstrated that Sp1 was indeed the major nuclear protein binding to this region. Despite the cell type specificity of KDR/flk-1 expression, no cell type differences were observed in DNA-protein interactions in vitro. In contrast, in vivo footprinting assays demonstrated marked differences in core promoter interactions between cell types. Protection of Sp1 binding sites was observed in HUVECs by in vivo DNase I footprinting, whereas in human fibroblasts and HeLa cells a pattern consistent with nucleosomal positioning was observed. In vivo dimethylsulfate footprinting confirmed that DNA-protein interactions occurred within Sp1 elements in HUVECs but not in nonendothelial cells. It is possible that distant elements coordinate Sp1 binding and chromatin structure to regulate cell type-specific expression of KDR/flk-1.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.

Interleukin 4 (IL-4) is a potent, pleiotropic lymphokine that affects a variety of cells, especially those of hematopoietic origin. Although murine and human IL-4 are homologous proteins, they display a species specificity in which murine IL-4 acts only upon mouse cells, and human IL-4 only upon human cells. We have used a mutagenesis strategy to define both the structural determinants of this specificity and a receptor binding domain of murine IL-4. To do this, we developed convenient solid-phase binding assays for mouse and for human IL-4, each utilizing receptor-immunoglobulin fusion proteins and alkaline phosphatase-tagged ligands. These were employed to assess the receptor binding activities of wild type and mutant forms of IL-4. In a separate biological assay, we measured the ability of each version of IL-4 to induce proliferation of a cultured mouse T-cell line. By replacing regions of mouse IL-4 with homologous segments of human IL-4, we found that the amino-terminal 16 residues and the carboxyl-terminal 20 residues of murine IL-4 are required for species-specific receptor binding as well as for T-cell proliferation. A major portion of the amino acid sequence between these regions can be substituted between mouse and human without loss of receptor binding or biological activity. Further, alanine-scanning mutagenesis revealed specific residues in the amino- and carboxyl-terminal regions (Glu-12, Ile-14, Leu-104, Asp-106, Phe-107, and Leu-111) that bear side chains critical for function. An analysis of the carboxyl-terminal region of murine IL-4 and its comparison with carboxyl-terminal regions of other related cytokines suggest an evolutionary conservation of structural and functional features.

We have previously reported the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in the internalization of ricin (Ray, B., and Wu, H.C. (1982) Mol. Cell. Biol. 2, 535-544). These mutants also do not exhibit the enhancement of ricin internalization by nigericin pretreatment at a low concentration, which is observed in the wild-type CHO cells. An analysis of somatic cell hybrids between the mutant and the toxin-sensitive wild-type CHO cell line shows that all of the phenotypes associated with the toxin resistance mutation are dominant in the hybrid cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]palmitic acid-labeled cell extracts from the mutant and toxin-resistant hybrid cell lines has revealed an increased incorporation of [3H] palmitic acid into two proteins with apparent molecular weights near 30,000 in the mutant and hybrid cells as compared to that in the wild-type cell line. Our studies indicate that these two fatty acyl proteins might be related to a dominant mutation(s) which results in a decreased uptake of ricin.

Using the lambda gt11 expression library of murine teratocarcinoma cells, we isolated cDNA clones encoding a core protein carrying a developmentally regulated carbohydrate marker, namely the binding site for Dolichos biflorus agglutinin. The deduced amino acid sequence of the polypeptide (molecular weight, 37,102) revealed a leader sequence, nine potential asparagine glycosylation sites, and a transmembrane region. Sequence homology to variable domain of immunoglobulin kappa chain has been detected in a domain; homologous amino acids near cysteine residues are those conserved in many members of the immunoglobulin gene superfamily. In contrast to other members of the superfamily, the core protein gene was significantly expressed in embryonal carcinoma cells, which are similar to undifferentiated cells of early embryos.

The T lymphocytes that expand with age in the peripheral lymphoid organs of autoimmune disease-prone mice homozygous for the lpr mutation display deficient activation and proliferation in response to mitogenic lectins or antigen. In the present study, an attempt was made to correlate the deficient agonist-induced proliferation of these lpr T cells with early transmembrane signaling events mediated by receptor-coupled phosphoinositide hydrolysis. lpr T cells were capable of binding the agonistic lectin, phytohemagglutinin, in a normal manner. In addition, they expressed on their surface the antigen-specific T cell receptor-CD3 complex, which is required for T cell activation, albeit at a lower density than that found on congenic +/+ T cells. Furthermore, lpr T cells contained normal levels of the Ca2+- and phospholipid-dependent enzyme, protein kinase C, and the enzyme was translocated from the cytosol to the particulate fraction upon phorbol ester treatment. On the other hand, the lpr T cells displayed a markedly deficient agonist-induced phosphoinositide hydrolysis in comparison with their congenic +/+ counterparts, as indicated by the minimal accumulation of the phosphoinositide-derived second messengers, inositol phosphates and diacylglycerol. The defective step(s) in transmembrane signaling was bypassed by a combination of phorbol ester plus Ca2+ ionophore, which reconstituted proliferative responses of lpr T cells to normal levels, suggesting that: (a) the phosphoinositide signaling pathway plays an obligatory role in T cell activation; and (b) signaling events subsequent to phosphoinositide hydrolysis are, for the most part, intact in lpr T cells. The deficient step(s) in lpr T cell activation precedes, therefore, the generation of phosphoinositide-derived second messengers and could be due to defective function of the T cell receptor-CD3 complex, GTP-binding proteins, and/or phosphoinositide-specific phosphodiesterase. It remains to be determined whether the deficient signaling event(s) in lpr T cells is a direct pathologic consequence of the lpr gene, or rather, reflects the immature status of a normally minor thymic subset that is aberrantly exported and expanded in lpr mice.

Human interleukin 4 (IL-4) specifically induces the low affinity receptor for IgE (Fc epsilon R2/CD23) on the surface of the Burkitt lymphoma cell line Jijoye. At 4 degrees C 125I-IL-4 specifically binds to high affinity receptors (Kd = 4-10 x 10(-11) M; Bmax, 600-1,200 sites/cell). Following a rapid temperature shift from 4 to 37 degrees C, 80% of the receptor-bound 125I-IL-4 disappeared from the cell surface within 20 min (t1/2 = 8.9 min). For every two internalized molecules of IL-4 (t1/2 = 13 min), one molecule of IL-4 dissociated from the cell surface (t1/2 = 25 min). More than 90% of the internalized IL-4 was released in a degraded form into the medium following first order kinetics (t1/2 = 68 min). Internalization was inhibited by cytoskeletal disrupting and lysosomotropic agents. Incubating cells with 1 nM IL-4 resulted in a rapid down-regulation of IL-4 receptors (75% loss after 2 h); a reexpression of receptor to control level occurred after 20 h in spite of the presence of a large excess of IL-4. Reexpression was delayed by chloroquine and blocked by cycloheximide and actinomycin D. By using the cross-linking agent bis(sulfosuccinimidyl)suberate, three polypeptides of Mr 130,000, 80,000, and 70,000 were specifically labeled with 125I-IL-4. These three polypeptides coordinately disappeared and reappeared with the 125I-IL-4 binding sites. The induction of Fc epsilon R2/CD23 required prolonged incubation (greater than 8 h) with IL-4 and thus may be dependent on the reexpression of IL-4 receptor.