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The Ly-49D receptor activates murine natural killer cells.

Mason LH, Anderson SK, Yokoyama WM, Smith HR, Winkler-Pickett R, Ortaldo JR - J. Exp. Med. (1996)

Bottom Line: An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities.Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors.Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Immunology, SAIC Frederick, National Cancer Institute-FCRDC, Maryland 21702-1201, USA.

ABSTRACT
Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

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(A) Amino acid homology of Ly-49D vs. Ly-49A, C, and G2.  This figure is a schematic representation of the proposed amino acid sequences  of Ly-49D, A, C, and G2, as described by Smith et al. (3) The highly conserved  extracellular cysteine residues are represented (C) along with the proposed glycosylation sites (CHO). Potential serine/threonine phosphorylation sites are labeled in the cytoplasmic region (P). Possible tyrosine phosphorylation sites (Y)  are also designated in the cytoplasmic domain. Open spaces in these schematics  represent areas of homology between these four proteins, whereas the perpendicular lines shown in Ly-49A, C, and G2 represent individual amino acid differences compared to Ly-49D. (B) Homologous regions from the intracellular  domains of the four Ly-49 molecules compared to the human p58 inhibitory  receptor. Dashes represent amino acids identical to the p58 sequence. A consensus sequence (V/IxYxxL) for a possible immunoreceptor tyrosine inhibitory  motif found in p58 is also present in Ly-49A, C, and G2, but not in Ly-49D.
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Figure 7: (A) Amino acid homology of Ly-49D vs. Ly-49A, C, and G2. This figure is a schematic representation of the proposed amino acid sequences of Ly-49D, A, C, and G2, as described by Smith et al. (3) The highly conserved extracellular cysteine residues are represented (C) along with the proposed glycosylation sites (CHO). Potential serine/threonine phosphorylation sites are labeled in the cytoplasmic region (P). Possible tyrosine phosphorylation sites (Y) are also designated in the cytoplasmic domain. Open spaces in these schematics represent areas of homology between these four proteins, whereas the perpendicular lines shown in Ly-49A, C, and G2 represent individual amino acid differences compared to Ly-49D. (B) Homologous regions from the intracellular domains of the four Ly-49 molecules compared to the human p58 inhibitory receptor. Dashes represent amino acids identical to the p58 sequence. A consensus sequence (V/IxYxxL) for a possible immunoreceptor tyrosine inhibitory motif found in p58 is also present in Ly-49A, C, and G2, but not in Ly-49D.

Mentions: Because of the unique activating properties of Ly-49D+ NK cells compared to the inhibitory properties observed in Ly-49A, C, and G2+ NK cells, we compared the amino acid homology of these proteins. Fig. 7 A is a schematic comparison of Ly-49D vs. Ly-49 A, C, and G2, based on the proposed amino acid sequence of these molecules (3). There is obviously a high degree of homology between Ly49D and A that is highest in the extracellular domain (86%) and least homologous in the cytoplasmic domain (51%). The structural homology between Ly-49A and D in their extracellular domains probably accounts for the cross-reactivity that is observed with mAb 12A8, since it can bind to both Ly-49D and Ly-49A. Furthermore, we can also speculate that the lack of homology in the cytoplasmic region of these molecules accounts for the different signaling properties of these proteins. The cytoplasmic domains of Ly-49A, C, and G2 all share at least two potential serine/ threonine phosphorylation sites (P). These three molecules also share similar sites for tyrosine phosphorylation (Y) in their intracellular domains. A significant divergence in phosphorylation motifs is seen in the cytoplasmic domain of Ly-49D. Ly-49D contains only one potential serine/ threonine phosphorylation motif, and none of the tyrosine phosphorylation motifs found in either Ly-49A, C, or G2. Fig. 7 B presents a 13–amino acid consensus sequence comparing the Ly-49A, C, G2 and D molecules with a similar cytoplasmic region of the human p58 inhibitory receptors. Long et al. (23) have recently described a V/IxYxxL consensus sequence in p58 proteins that may represent an immunoreceptor tyrosine-based inhibitory motif (ITIM). This V/IxYxxL motif is present in the Ly-49 inhibitor receptors A, C, and G2. However, the Ly-49D receptor does not contain this ITIM motif, which further supports our functional data that Ly-49D is an activation receptor on murine NK cells.


The Ly-49D receptor activates murine natural killer cells.

Mason LH, Anderson SK, Yokoyama WM, Smith HR, Winkler-Pickett R, Ortaldo JR - J. Exp. Med. (1996)

(A) Amino acid homology of Ly-49D vs. Ly-49A, C, and G2.  This figure is a schematic representation of the proposed amino acid sequences  of Ly-49D, A, C, and G2, as described by Smith et al. (3) The highly conserved  extracellular cysteine residues are represented (C) along with the proposed glycosylation sites (CHO). Potential serine/threonine phosphorylation sites are labeled in the cytoplasmic region (P). Possible tyrosine phosphorylation sites (Y)  are also designated in the cytoplasmic domain. Open spaces in these schematics  represent areas of homology between these four proteins, whereas the perpendicular lines shown in Ly-49A, C, and G2 represent individual amino acid differences compared to Ly-49D. (B) Homologous regions from the intracellular  domains of the four Ly-49 molecules compared to the human p58 inhibitory  receptor. Dashes represent amino acids identical to the p58 sequence. A consensus sequence (V/IxYxxL) for a possible immunoreceptor tyrosine inhibitory  motif found in p58 is also present in Ly-49A, C, and G2, but not in Ly-49D.
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Related In: Results  -  Collection

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Figure 7: (A) Amino acid homology of Ly-49D vs. Ly-49A, C, and G2. This figure is a schematic representation of the proposed amino acid sequences of Ly-49D, A, C, and G2, as described by Smith et al. (3) The highly conserved extracellular cysteine residues are represented (C) along with the proposed glycosylation sites (CHO). Potential serine/threonine phosphorylation sites are labeled in the cytoplasmic region (P). Possible tyrosine phosphorylation sites (Y) are also designated in the cytoplasmic domain. Open spaces in these schematics represent areas of homology between these four proteins, whereas the perpendicular lines shown in Ly-49A, C, and G2 represent individual amino acid differences compared to Ly-49D. (B) Homologous regions from the intracellular domains of the four Ly-49 molecules compared to the human p58 inhibitory receptor. Dashes represent amino acids identical to the p58 sequence. A consensus sequence (V/IxYxxL) for a possible immunoreceptor tyrosine inhibitory motif found in p58 is also present in Ly-49A, C, and G2, but not in Ly-49D.
Mentions: Because of the unique activating properties of Ly-49D+ NK cells compared to the inhibitory properties observed in Ly-49A, C, and G2+ NK cells, we compared the amino acid homology of these proteins. Fig. 7 A is a schematic comparison of Ly-49D vs. Ly-49 A, C, and G2, based on the proposed amino acid sequence of these molecules (3). There is obviously a high degree of homology between Ly49D and A that is highest in the extracellular domain (86%) and least homologous in the cytoplasmic domain (51%). The structural homology between Ly-49A and D in their extracellular domains probably accounts for the cross-reactivity that is observed with mAb 12A8, since it can bind to both Ly-49D and Ly-49A. Furthermore, we can also speculate that the lack of homology in the cytoplasmic region of these molecules accounts for the different signaling properties of these proteins. The cytoplasmic domains of Ly-49A, C, and G2 all share at least two potential serine/ threonine phosphorylation sites (P). These three molecules also share similar sites for tyrosine phosphorylation (Y) in their intracellular domains. A significant divergence in phosphorylation motifs is seen in the cytoplasmic domain of Ly-49D. Ly-49D contains only one potential serine/ threonine phosphorylation motif, and none of the tyrosine phosphorylation motifs found in either Ly-49A, C, or G2. Fig. 7 B presents a 13–amino acid consensus sequence comparing the Ly-49A, C, G2 and D molecules with a similar cytoplasmic region of the human p58 inhibitory receptors. Long et al. (23) have recently described a V/IxYxxL consensus sequence in p58 proteins that may represent an immunoreceptor tyrosine-based inhibitory motif (ITIM). This V/IxYxxL motif is present in the Ly-49 inhibitor receptors A, C, and G2. However, the Ly-49D receptor does not contain this ITIM motif, which further supports our functional data that Ly-49D is an activation receptor on murine NK cells.

Bottom Line: An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities.Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors.Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Immunology, SAIC Frederick, National Cancer Institute-FCRDC, Maryland 21702-1201, USA.

ABSTRACT
Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Show MeSH
Related in: MedlinePlus