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CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes.

Huysamen C, Willment JA, Dennehy KM, Brown GD - J. Biol. Chem. (2008)

Bottom Line: We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex.CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis.These data indicate that CLEC9A functions as an activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, South Africa.

ABSTRACT
We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3(+) dendritic cells and on a small subset of CD14(+)CD16(-) monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.

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hCLEC9A is not a phagocytic receptor. A, schematic representation of the chimeric Dectin-1/CLEC9A receptor. B, fluorometric quantitation of zymosan (zym) binding by the transduced RAW264.7 macrophages or NIH3T3 fibroblasts, in the presence or absence of soluble β-glucan (β-glu), as indicated. RFU, relative fluorescence units. Shown are the mean ± S.D. of one representative experiment of three. C, flow cytometric quantitation of zymosan uptake (black histograms) by the chimera or Dectin-1 expressing RAW264.7 macrophages or NIH3T3 fibroblasts, as indicated. Cytochalasin D (gray-filled histograms) was used to inhibit actin polymerization and served as a control in this assay. The histograms shown are representative of at least three independent experiments, and the bars indicate the percentage of cells with internalized particles. *, p < 0.05 versus control (Student's t test). D, representative confocal images demonstrating FITC-zymosan (green) uptake in NIH3T3 fibroblasts transduced with Dectin-1, but not the chimeric receptor. Actin was stained with TRITC-phallodin, and is shown in red.
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fig5: hCLEC9A is not a phagocytic receptor. A, schematic representation of the chimeric Dectin-1/CLEC9A receptor. B, fluorometric quantitation of zymosan (zym) binding by the transduced RAW264.7 macrophages or NIH3T3 fibroblasts, in the presence or absence of soluble β-glucan (β-glu), as indicated. RFU, relative fluorescence units. Shown are the mean ± S.D. of one representative experiment of three. C, flow cytometric quantitation of zymosan uptake (black histograms) by the chimera or Dectin-1 expressing RAW264.7 macrophages or NIH3T3 fibroblasts, as indicated. Cytochalasin D (gray-filled histograms) was used to inhibit actin polymerization and served as a control in this assay. The histograms shown are representative of at least three independent experiments, and the bars indicate the percentage of cells with internalized particles. *, p < 0.05 versus control (Student's t test). D, representative confocal images demonstrating FITC-zymosan (green) uptake in NIH3T3 fibroblasts transduced with Dectin-1, but not the chimeric receptor. Actin was stained with TRITC-phallodin, and is shown in red.

Mentions: CLEC9A Does Not Mediate Phagocytosis—CLEC9A contains a tyrosine-based sequence, which is similar to that shown to mediate phagocytosis in Dectin-1 (19), so we investigated the possibility that CLEC9A could also mediate particle uptake. As the ligand for CLEC9A is unknown, we constructed a chimeric receptor expressing an HA-tagged extracellular domain of Dectin-1, coupled to the cytoplasmic tail and transmembrane regions of CLEC9A (Fig. 5A). This monomeric chimera would allow us to trigger CLEC9A signaling using zymosan, a defined particulate ligand of Dectin-1 (18).


CLEC9A is a novel activation C-type lectin-like receptor expressed on BDCA3+ dendritic cells and a subset of monocytes.

Huysamen C, Willment JA, Dennehy KM, Brown GD - J. Biol. Chem. (2008)

hCLEC9A is not a phagocytic receptor. A, schematic representation of the chimeric Dectin-1/CLEC9A receptor. B, fluorometric quantitation of zymosan (zym) binding by the transduced RAW264.7 macrophages or NIH3T3 fibroblasts, in the presence or absence of soluble β-glucan (β-glu), as indicated. RFU, relative fluorescence units. Shown are the mean ± S.D. of one representative experiment of three. C, flow cytometric quantitation of zymosan uptake (black histograms) by the chimera or Dectin-1 expressing RAW264.7 macrophages or NIH3T3 fibroblasts, as indicated. Cytochalasin D (gray-filled histograms) was used to inhibit actin polymerization and served as a control in this assay. The histograms shown are representative of at least three independent experiments, and the bars indicate the percentage of cells with internalized particles. *, p < 0.05 versus control (Student's t test). D, representative confocal images demonstrating FITC-zymosan (green) uptake in NIH3T3 fibroblasts transduced with Dectin-1, but not the chimeric receptor. Actin was stained with TRITC-phallodin, and is shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562446&req=5

fig5: hCLEC9A is not a phagocytic receptor. A, schematic representation of the chimeric Dectin-1/CLEC9A receptor. B, fluorometric quantitation of zymosan (zym) binding by the transduced RAW264.7 macrophages or NIH3T3 fibroblasts, in the presence or absence of soluble β-glucan (β-glu), as indicated. RFU, relative fluorescence units. Shown are the mean ± S.D. of one representative experiment of three. C, flow cytometric quantitation of zymosan uptake (black histograms) by the chimera or Dectin-1 expressing RAW264.7 macrophages or NIH3T3 fibroblasts, as indicated. Cytochalasin D (gray-filled histograms) was used to inhibit actin polymerization and served as a control in this assay. The histograms shown are representative of at least three independent experiments, and the bars indicate the percentage of cells with internalized particles. *, p < 0.05 versus control (Student's t test). D, representative confocal images demonstrating FITC-zymosan (green) uptake in NIH3T3 fibroblasts transduced with Dectin-1, but not the chimeric receptor. Actin was stained with TRITC-phallodin, and is shown in red.
Mentions: CLEC9A Does Not Mediate Phagocytosis—CLEC9A contains a tyrosine-based sequence, which is similar to that shown to mediate phagocytosis in Dectin-1 (19), so we investigated the possibility that CLEC9A could also mediate particle uptake. As the ligand for CLEC9A is unknown, we constructed a chimeric receptor expressing an HA-tagged extracellular domain of Dectin-1, coupled to the cytoplasmic tail and transmembrane regions of CLEC9A (Fig. 5A). This monomeric chimera would allow us to trigger CLEC9A signaling using zymosan, a defined particulate ligand of Dectin-1 (18).

Bottom Line: We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex.CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis.These data indicate that CLEC9A functions as an activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, South Africa.

ABSTRACT
We describe here the first characterization of CLEC9A, a group V C-type lectin-like receptor located in the "Dectin-1 cluster" of related receptors, which are encoded within the natural killer (NK)-gene complex. Expression of human CLEC9A is highly restricted in peripheral blood, being detected only on BDCA3(+) dendritic cells and on a small subset of CD14(+)CD16(-) monocytes. CLEC9A is expressed at the cell surface as a glycosylated dimer and can mediate endocytosis, but not phagocytosis. CLEC9A possesses a cytoplasmic immunoreceptor tyrosine-based activation-like motif that can recruit Syk kinase, and we demonstrate, using receptor chimeras, that this receptor can induce proinflammatory cytokine production. These data indicate that CLEC9A functions as an activation receptor.

Show MeSH
Related in: MedlinePlus