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Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells.

Vance RE, Jamieson AM, Raulet DH - J. Exp. Med. (1999)

Bottom Line: Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells.Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events.These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of California, Berkeley, California 94720, USA.

ABSTRACT
The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1(b), a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1(b). Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93-95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1(b) receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

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CD94/NKG2C and CD94/NKG2E are receptors for Qa-1b. CHO cells were stably transfected with expression vectors encoding CD94 and NKG2 cDNAs as described in Materials and Methods. Untransfected CHO cells or stable CHO transfectants were stained with PE-complexed tetramers of soluble Qa-1b/Qdm class I molecules or with streptavidin–PE as a negative control. The same cells were also stained with FITC-conjugated 20d5 mAb (anti-NKG2) or with FITC-conjugated 18d3 mAb (anti-CD94). The mean fluorescence intensity (MFI) is indicated for each histogram.
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Figure 3: CD94/NKG2C and CD94/NKG2E are receptors for Qa-1b. CHO cells were stably transfected with expression vectors encoding CD94 and NKG2 cDNAs as described in Materials and Methods. Untransfected CHO cells or stable CHO transfectants were stained with PE-complexed tetramers of soluble Qa-1b/Qdm class I molecules or with streptavidin–PE as a negative control. The same cells were also stained with FITC-conjugated 20d5 mAb (anti-NKG2) or with FITC-conjugated 18d3 mAb (anti-CD94). The mean fluorescence intensity (MFI) is indicated for each histogram.

Mentions: Based on the high degree of similarity between the extracellular ligand binding domains of NKG2A, -C, and -E, it is reasonable to hypothesize that CD94/NKGC and CD94/NKG2E heterodimers, like CD94/NKG2A heterodimers, would recognize Qa-1b. We generated stable transfectants of CHO cells expressing high levels of CD94/NKG2A, CD94/NKG2C, and CD94/NKG2E (Fig. 3). All three stable cell lines, but not untransfected CHO cells, stained brightly with soluble, tetramerized Qa-1/β2 microglobulin/Qdm complexes. We previously showed that CD94 alone is incapable of binding Qa-1b 9. The results therefore provide direct evidence that both CD94/NKG2C and CD94/NKG2E recognize Qa-1b–Qdm complexes. The degree of staining appeared to correlate roughly with the levels of NKG2 and CD94, as independently assessed by staining with anti-NKG2 and anti-CD94 mAbs. In particular, the CD94/NKG2A transfectant stained most brightly with tetramer and with anti-CD94 and anti-NKG2 antibodies, whereas the CD94/NKG2C transfectant stained least brightly with all three staining reagents. Thus, our data do not reveal a gross difference in the avidity of the various CD94/NKG2 subunits for Qa-1b. Evidence in humans using quantitative surface plasmon resonance techniques suggests that CD94/NKG2A binds HLA-E with higher affinity than does CD94/NKG2C 21. Such differences may also exist for the mouse CD94/NKG2 receptors, as our data is not quantitative. Because the Qa-1 protein was produced in Escherichia coli and is therefore unglycosylated, our results also demonstrate that CD94/NKG2C and CD94/NKG2E, like CD94/NKG2A 9, can recognize carbohydrate-independent epitopes on their ligands.


Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells.

Vance RE, Jamieson AM, Raulet DH - J. Exp. Med. (1999)

CD94/NKG2C and CD94/NKG2E are receptors for Qa-1b. CHO cells were stably transfected with expression vectors encoding CD94 and NKG2 cDNAs as described in Materials and Methods. Untransfected CHO cells or stable CHO transfectants were stained with PE-complexed tetramers of soluble Qa-1b/Qdm class I molecules or with streptavidin–PE as a negative control. The same cells were also stained with FITC-conjugated 20d5 mAb (anti-NKG2) or with FITC-conjugated 18d3 mAb (anti-CD94). The mean fluorescence intensity (MFI) is indicated for each histogram.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195720&req=5

Figure 3: CD94/NKG2C and CD94/NKG2E are receptors for Qa-1b. CHO cells were stably transfected with expression vectors encoding CD94 and NKG2 cDNAs as described in Materials and Methods. Untransfected CHO cells or stable CHO transfectants were stained with PE-complexed tetramers of soluble Qa-1b/Qdm class I molecules or with streptavidin–PE as a negative control. The same cells were also stained with FITC-conjugated 20d5 mAb (anti-NKG2) or with FITC-conjugated 18d3 mAb (anti-CD94). The mean fluorescence intensity (MFI) is indicated for each histogram.
Mentions: Based on the high degree of similarity between the extracellular ligand binding domains of NKG2A, -C, and -E, it is reasonable to hypothesize that CD94/NKGC and CD94/NKG2E heterodimers, like CD94/NKG2A heterodimers, would recognize Qa-1b. We generated stable transfectants of CHO cells expressing high levels of CD94/NKG2A, CD94/NKG2C, and CD94/NKG2E (Fig. 3). All three stable cell lines, but not untransfected CHO cells, stained brightly with soluble, tetramerized Qa-1/β2 microglobulin/Qdm complexes. We previously showed that CD94 alone is incapable of binding Qa-1b 9. The results therefore provide direct evidence that both CD94/NKG2C and CD94/NKG2E recognize Qa-1b–Qdm complexes. The degree of staining appeared to correlate roughly with the levels of NKG2 and CD94, as independently assessed by staining with anti-NKG2 and anti-CD94 mAbs. In particular, the CD94/NKG2A transfectant stained most brightly with tetramer and with anti-CD94 and anti-NKG2 antibodies, whereas the CD94/NKG2C transfectant stained least brightly with all three staining reagents. Thus, our data do not reveal a gross difference in the avidity of the various CD94/NKG2 subunits for Qa-1b. Evidence in humans using quantitative surface plasmon resonance techniques suggests that CD94/NKG2A binds HLA-E with higher affinity than does CD94/NKG2C 21. Such differences may also exist for the mouse CD94/NKG2 receptors, as our data is not quantitative. Because the Qa-1 protein was produced in Escherichia coli and is therefore unglycosylated, our results also demonstrate that CD94/NKG2C and CD94/NKG2E, like CD94/NKG2A 9, can recognize carbohydrate-independent epitopes on their ligands.

Bottom Line: Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells.Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events.These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of California, Berkeley, California 94720, USA.

ABSTRACT
The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1(b), a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1(b). Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93-95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1(b) receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

Show MeSH
Related in: MedlinePlus