Limits...
Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor.

Shiratori I, Ogasawara K, Saito T, Lanier LL, Arase H - J. Exp. Med. (2004)

Bottom Line: Transcripts of PILR ligand are present in many tissues, including some T cell lines.Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta.Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuoku, 260-8670, Japan.

ABSTRACT
Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of the immune system. Here, we report a mouse orthologue of the human activating paired immunoglobulin-like type 2 receptor (PILR) beta, which was cloned from a cDNA library of natural killer (NK) cells based on its ability to associate with the DAP12 signaling adaptor protein. The activating PILRbeta was expressed not only on NK cells but also on dendritic cells and macrophages. Furthermore, we have identified a novel CD99-like molecule as a ligand for the activating PILRbeta and inhibitory PILRalpha receptors. Transcripts of PILR ligand are present in many tissues, including some T cell lines. Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta. Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

Show MeSH
Molecular cloning of mouse PILR-L. (A) Nucleic and predicted amino acid sequences of mouse PILR-L are shown. The signal sequence and transmembrane domain are indicated by double and single underline, respectively. Sequence data of PILR-L are available from GenBank/EMBL/DDBJ under accession no. AB122023. (B) Ba/F3 and 293T cells were transfected with mouse PILR-L. Parental cells and the PILR-L transfectants were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. (C) SDS-PAGE analysis of PILR-L. PILR-L transfected (+) and parental (−) Ba/F3 cells were surface biotinylated, and cell lysates were precipitated with PILRα-Ig, PILRβ-Ig, or control Ig fusion protein as indicated. Precipitates were electrophoresed by SDS-PAGE and biotinylated proteins were detected by using horseradish peroxidase–conjugated streptavidin and the enhanced chemiluminescence detection reagent.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211832&req=5

fig5: Molecular cloning of mouse PILR-L. (A) Nucleic and predicted amino acid sequences of mouse PILR-L are shown. The signal sequence and transmembrane domain are indicated by double and single underline, respectively. Sequence data of PILR-L are available from GenBank/EMBL/DDBJ under accession no. AB122023. (B) Ba/F3 and 293T cells were transfected with mouse PILR-L. Parental cells and the PILR-L transfectants were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. (C) SDS-PAGE analysis of PILR-L. PILR-L transfected (+) and parental (−) Ba/F3 cells were surface biotinylated, and cell lysates were precipitated with PILRα-Ig, PILRβ-Ig, or control Ig fusion protein as indicated. Precipitates were electrophoresed by SDS-PAGE and biotinylated proteins were detected by using horseradish peroxidase–conjugated streptavidin and the enhanced chemiluminescence detection reagent.

Mentions: To elucidate the function of PILRβ, we sought to identify the ligand by expression cloning. Because EL-4 was stained well by PILRβ-Ig (Fig. 4 A), we constructed a retrovirus cDNA library from EL-4 and undertook expression cloning using PILRβ-Ig. The EL-4 retrovirus cDNA library was transfected into Ba/F3 cells, which did not stain with the PILRα-Ig or PILRβ-Ig, and Ba/F3 cells expressing the ligand for PILRβ were purified by using a fluorescence-activated cell sorter. Single cell clones that were stained with PILRβ-Ig were obtained and genes derived from the cDNA library were amplified by PCR using primers corresponding to the flanking retrovirus long terminal repeat and sequenced. Fig. 5 A shows the nucleic acid and predicted amino acid sequence of a PILR-L. PILR-L is a type I membrane protein with a signal sequence, a small extracellular domain, a transmembrane region, and a short 17–amino acid cytoplasmic domain with no evident signaling motifs. The sequence of PILR-L was remarkable in two aspects. Surprisingly, the GC content of PILR-L is 75%; GC-rich transcripts are rare in mammalian cells. In addition, the short extracellular domain of PILR-L contained no sites for N- or O-linked glycosylation, again unusual for proteins expressed on the cell surface. PILR-L showed 45% similarity to human CD99, 36% to human CD99L2, and 32% to mouse CD99L2 (26). This indicates that PILR-L belongs to CD99 family.


Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor.

Shiratori I, Ogasawara K, Saito T, Lanier LL, Arase H - J. Exp. Med. (2004)

Molecular cloning of mouse PILR-L. (A) Nucleic and predicted amino acid sequences of mouse PILR-L are shown. The signal sequence and transmembrane domain are indicated by double and single underline, respectively. Sequence data of PILR-L are available from GenBank/EMBL/DDBJ under accession no. AB122023. (B) Ba/F3 and 293T cells were transfected with mouse PILR-L. Parental cells and the PILR-L transfectants were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. (C) SDS-PAGE analysis of PILR-L. PILR-L transfected (+) and parental (−) Ba/F3 cells were surface biotinylated, and cell lysates were precipitated with PILRα-Ig, PILRβ-Ig, or control Ig fusion protein as indicated. Precipitates were electrophoresed by SDS-PAGE and biotinylated proteins were detected by using horseradish peroxidase–conjugated streptavidin and the enhanced chemiluminescence detection reagent.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211832&req=5

fig5: Molecular cloning of mouse PILR-L. (A) Nucleic and predicted amino acid sequences of mouse PILR-L are shown. The signal sequence and transmembrane domain are indicated by double and single underline, respectively. Sequence data of PILR-L are available from GenBank/EMBL/DDBJ under accession no. AB122023. (B) Ba/F3 and 293T cells were transfected with mouse PILR-L. Parental cells and the PILR-L transfectants were stained with PILRβ-Ig (solid line), PILRα-Ig (dashed line), or control Ig (dotted line), followed by PE-conjugated goat anti–human IgG. (C) SDS-PAGE analysis of PILR-L. PILR-L transfected (+) and parental (−) Ba/F3 cells were surface biotinylated, and cell lysates were precipitated with PILRα-Ig, PILRβ-Ig, or control Ig fusion protein as indicated. Precipitates were electrophoresed by SDS-PAGE and biotinylated proteins were detected by using horseradish peroxidase–conjugated streptavidin and the enhanced chemiluminescence detection reagent.
Mentions: To elucidate the function of PILRβ, we sought to identify the ligand by expression cloning. Because EL-4 was stained well by PILRβ-Ig (Fig. 4 A), we constructed a retrovirus cDNA library from EL-4 and undertook expression cloning using PILRβ-Ig. The EL-4 retrovirus cDNA library was transfected into Ba/F3 cells, which did not stain with the PILRα-Ig or PILRβ-Ig, and Ba/F3 cells expressing the ligand for PILRβ were purified by using a fluorescence-activated cell sorter. Single cell clones that were stained with PILRβ-Ig were obtained and genes derived from the cDNA library were amplified by PCR using primers corresponding to the flanking retrovirus long terminal repeat and sequenced. Fig. 5 A shows the nucleic acid and predicted amino acid sequence of a PILR-L. PILR-L is a type I membrane protein with a signal sequence, a small extracellular domain, a transmembrane region, and a short 17–amino acid cytoplasmic domain with no evident signaling motifs. The sequence of PILR-L was remarkable in two aspects. Surprisingly, the GC content of PILR-L is 75%; GC-rich transcripts are rare in mammalian cells. In addition, the short extracellular domain of PILR-L contained no sites for N- or O-linked glycosylation, again unusual for proteins expressed on the cell surface. PILR-L showed 45% similarity to human CD99, 36% to human CD99L2, and 32% to mouse CD99L2 (26). This indicates that PILR-L belongs to CD99 family.

Bottom Line: Transcripts of PILR ligand are present in many tissues, including some T cell lines.Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta.Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuoku, 260-8670, Japan.

ABSTRACT
Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of the immune system. Here, we report a mouse orthologue of the human activating paired immunoglobulin-like type 2 receptor (PILR) beta, which was cloned from a cDNA library of natural killer (NK) cells based on its ability to associate with the DAP12 signaling adaptor protein. The activating PILRbeta was expressed not only on NK cells but also on dendritic cells and macrophages. Furthermore, we have identified a novel CD99-like molecule as a ligand for the activating PILRbeta and inhibitory PILRalpha receptors. Transcripts of PILR ligand are present in many tissues, including some T cell lines. Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta. Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

Show MeSH