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Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.

ABSTRACT: Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by single-particle electron microscopy to obtain a three-dimensional structure of the N-terminal part of the protein at a resolution of 17 A and reveal, for the first time, the interactions between non-adjacent domains in the mannose receptor family. We show that for Endo180, the cysteine-rich domain contacts the second C-type lectin-like domain, thus providing structural insight into how modulation of its several ligand interactions may regulate Endo180 receptor function.

SUBMITTER: Rivera-Calzada A

PROVIDER: S-EPMC1326338 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.

Rivera-Calzada Angel A Robertson David D MacFadyen John R JR Boskovic Jasminka J Isacke Clare M CM Llorca Oscar O

EMBO reports 20030704 8

Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by ...[more]

PMID: 12856000

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Project description:Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Project description:Data from clinical studies, cell culture, and animal models implicate the urokinase (uPA)/Plasminogen (Plg) system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/Plg stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression to clarify mechanisms of uPA/Plg-accelerated atherosclerosis and aneurysm formation. Microarray studies were performed to identify potential mediators of uPA-accelerated atherosclerosis. These studies identified S100A8 and S100A9 mRNA as the most highly upregulated transcripts in uPA-overexpressing macrophages; upregulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also upregulated in aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is upregulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Bioinformatics analysis of the microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm—in a second animal model—that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. Six independent biological replicate RNA samples were prepared from thioglycollate-elicited peritoneal macrophages from mice of two different genotypes: SR-uPA+/0 transgenic (overexpress uPA in macrophages) and nontransgenic, respectively), for a total of 12 independent RNA samples. Both transgenic and nontransgenic mice were Apoe-/- and were fed Western diet from 5-15 weeks before peritoneal macrophages were elicited. In addition, from the six samples of each genotype, a pooled sample was prepared by combining the six genotype-specific samples. Each of the two pooled samples was assayed on the BeadChip in two technical replicates, for a total of 16 hybridizations performed using two Illumina Mouse Ref-8 v1.1 chips.

Dataset Information

Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.

Publications

Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180.

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