Murine CD9 is the receptor for pregnancy-specific glycoprotein 17

Abstract

Pregnancy-specific glycoproteins (PSGs) are a family of highly similar secreted proteins produced by the placenta. PSG homologs have been identified in primates and rodents. Members of the human and murine PSG family induce secretion of antiinflammatory cytokines in mononuclear phagocytes. For the purpose of cloning the receptor, we screened a RAW 264.7 cell cDNA expression library. The PSG17 receptor was identified as the tetraspanin, CD9. We confirmed binding of PSG17 to CD9 by ELISA, flow cytometry, alkaline phosphatase binding assays, and in situ rosetting. Anti-CD9 monoclonal antibody inhibited binding of PSG17 to CD9-transfected cells and RAW 264.7 cells. Moreover, PSG17 binding to macrophages from CD9-deficient mice was significantly reduced. We then tested whether PSG17 binds to other members of the murine tetraspanin family. PSG17 did not bind to cells transfected with CD53, CD63, CD81, CD82, or CD151, suggesting that PSG17-CD9 binding is a specific interaction. We have identified the first receptor for a murine PSG as well as the first natural ligand for a member of the tetraspanin superfamily.

Figure 1.

Binding of PSG17N to murine CD9-transfected 293T cells and inhibition of PSG17 binding to CD9 expressing cells by anti-CD9 mAb. (A) HEK 293T cells were transfected with CD9-pEF6/V5-His or empty plasmid after which they were incubated with varying concentrations of PSG17N-Myc-His. (B) HEK 293T-CD9-pEF/V5-His transfected cells were treated with increasing concentrations of anti-CD9 mAb or an isotype-matched control mAb before the addition of PSG17N-Myc-His (5 μg/ml). Bound PSG17N-Myc-His was detected after treatment with HRP-conjugated anti-myc mAb and TMB/peroxidase substrate. The data is expressed as mean absorbance ± SE. Each data point represents five identical wells and the experiment was repeated three independent times with similar results.

Figure 1.

Binding of PSG17N to murine CD9-transfected 293T cells and inhibition of PSG17 binding to CD9 expressing cells by anti-CD9 mAb. (A) HEK 293T cells were transfected with CD9-pEF6/V5-His or empty plasmid after which they were incubated with varying concentrations of PSG17N-Myc-His. (B) HEK 293T-CD9-pEF/V5-His transfected cells were treated with increasing concentrations of anti-CD9 mAb or an isotype-matched control mAb before the addition of PSG17N-Myc-His (5 μg/ml). Bound PSG17N-Myc-His was detected after treatment with HRP-conjugated anti-myc mAb and TMB/peroxidase substrate. The data is expressed as mean absorbance ± SE. Each data point represents five identical wells and the experiment was repeated three independent times with similar results.

Figure 2.

FACS^® analysis of PSG17N-Myc-His binding to CD9-transfected HEK 293T cells and BHK-21 cells. HEK 293T cells (A) and BHK-21 (B) cells transfected with empty plasmid (dotted line) or murine CD9-pEF6/V5-His; solid line) were treated with PSG17N-Myc-His, anti-myc mAb, biotin-conjugated goat anti–mouse IgG_2aκ, and FITC-conjugated streptavidin.

Figure 2.

FACS^® analysis of PSG17N-Myc-His binding to CD9-transfected HEK 293T cells and BHK-21 cells. HEK 293T cells (A) and BHK-21 (B) cells transfected with empty plasmid (dotted line) or murine CD9-pEF6/V5-His; solid line) were treated with PSG17N-Myc-His, anti-myc mAb, biotin-conjugated goat anti–mouse IgG_2aκ, and FITC-conjugated streptavidin.

Figure 3.

FACS^® analysis of PSG17 binding to RAW 264.7 cells pretreated with anti-CD9 mAb KMC8.8. RAW 264.7 cells were incubated with Fc block after which they were treated with anti-CD9 mAb KMC8.8 (dotted line), an isotype match control mAb (filled line), or no Ab (shaded area) before the addition of PSG17N-Myc-His (dotted and filled lines), or a control myc-tagged protein (shaded area). Binding was detected by the sequential addition of anti-myc mAb and PE-labeled rat anti–mouse IgG1.

Figure 4.

FACS^® analysis of PSG17 binding to peritoneal macrophages isolated from CD9-deficient and wild-type mice. Peritoneal macrophages isolated from wild-type mice (dotted line) or CD9-deficient mice (solid line) were sequentially incubated with Fc block, PSG17N-Myc-His, anti-myc mAb, and PE-labeled rat anti–mouse IgG1.