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The Journal of Immunology, 1998, 160: 4148-4152.
Copyright © 1998 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Characterization of an Associated 16-kDa Tyrosine Phosphoprotein Required for Ly-49D Signal Transduction3

Llewellyn H. Mason4,*, Jami Willette-Brown*, Stephen K. Anderson{dagger}, Pierre Gosselin*, Elizabeth W. Shores{ddagger}, Paul E. Love§, John R. Ortaldo* and Daniel W. McVicar*

* Laboratory of Experimental Immunology, Division of Basic Sciences, and {dagger} Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Reseach and Development Center, Frederick, MD 21702; {ddagger} Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Washington, DC; and § Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-{zeta}/Fc{epsilon}RI{gamma} double knockout mice confirm that pp16 is not TCR-{zeta}, TCR-{eta}, or Fc{epsilon}RI{gamma}. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49DR54L) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49DR54L transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly-49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Murine NK cells express Ly-49 receptors that can initiate either inhibitory or activating signals to regulate lytic function. Ly-49A, Ly-49C, and Ly-49G2 have been shown to inhibit NK cell function on recognition of class I ligands on target cells (1, 2, 3). These inhibitory receptors contain ITIMs1 in their cytoplasmic domains that are phosphorylated on stimulation, leading to the recruitment of SHP-1 phosphatase and attenuation of intracellular signals (4, 5). A similar system exists in human NK cells, where signaling by the p58 receptor family also uses SHP-1 phosphatase to inhibit NK cell activation (6). However, receptors exist in both systems that can activate NK cells. In the human, p50 receptors have truncated cytoplasmic domains that lack ITIMs (7) and have been shown to mediate reverse Ab-dependent cellular cytotoxicity and mobilize intracellular Ca2+ in the presence of specific mAb (8). Olcese et al. have recently demonstrated that p50 receptors are associated with a multimeric complex of proteins that when appropriately stimulated become phosphorylated (killer cell-activatory receptor-associated polypeptides) (9). These molecules exist as a 12–16 kDa group of polypeptides that can be detected upon internal radiolabeling of NK cells and immunoprecipitation. Since the human killer cell-activatory receptors contain a lysine in their transmembrane domain, they speculated that this amino acid is important for the association of killer cell-activatory receptor with killer cell-activatory receptor-associated polypeptides (9).

Our laboratory has shown that murine Ly-49D, in the presence of mAb 12A8, activates NK cells by inducing reverse Ab-dependent cellular cytotoxicity of FcR+ targets (10). Ly-49D contains a cytoplasmic domain that is comparable in length with other Ly-49 receptors. We have recently demonstrated that Ly-49D is not phosphorylated following pervanadate stimulation, consistent with its lack of an ITIM. However, stimulation of NK cells with pervanadate and immunoprecipitation of Ly-49D did reveal associated tyrosine-phosphorylated proteins (pp16). These associated phosphoproteins exist as disulfide-linked dimers that are highly phosphorylated following either pervanadate stimulation of NK cells or specific receptor cross-linking. In this report, we characterize these phosphoproteins and provide data to prove that the arginine in the transmembrane domain of Ly-49D is critical for pp16 association and subsequent signaling by this receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
NK cell isolation

Splenic NK cells were isolated from C57BL/6 (B6) mice2 and grown for 7 to 10 days in 1000 U/ml Cetus recombinant IL-2 as previously described (11).

Antibodies

The following mAb were used: 4D11 (Ly-49G2 (3)); 12A8 (Ly-49A/D (10)); 4E5 (Ly-49D; manuscript in preparation); and RM2-1 (CD2 (12)). Antisera to Fc{epsilon}RI{gamma} and TCR-{zeta} were kindly provided by Dr. J. P. Kinet and Dr. A. Weissman, respectively.

Stimulation, immunoprecipitation, electrophoresis, and blotting

Cell stimulation, immunoprecipitation, and immunoblotting were performed as previously described (5). Pervanadate stimulation of cells utilized 1 mM pervanadate for 15 min at 37°C.

Site-directed mutagenesis and transfection

A substitution mutant was generated within the Ly-49D transmembrane domain in which the arginine at position 54 was mutated to a leucine (Ly-49DR54L). Mutation of the Ly-49D cDNA was performed with the Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. The mutant construct was confirmed by sequencing. P815 cells were electroporated as previously described (5).

Calcium flux

Analysis of the changes in intracellular Ca2+ concentration ([Ca2+]i) was conducted using a DeltaScan fluorometer (Photon Technologies, Princeton, NJ) and the calcium-sensitive fluorochrome Indo-1 as previously described (13). The cells were stimulated with primary mAb followed 25 s later by rabbit anti-rat Ab. Data are expressed as the ratio of bound dye to free dye, a direct indication of [Ca2+]i.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Previous work from our laboratory has demonstrated that phosphorylation of the tyrosine within the ITIMs of Ly-49A, Ly-49C, and Ly-49G2 occurs following pervanadate stimulation of NK cells (5). Ly-49D, however, does not contain any tyrosines and is therefore not phosphorylated. Although anti-phosphotyrosine blotting of mAb 12A8 immunoprecipitates of NK cells did not reveal phosphorylation of Ly-49D, associated phosphoproteins with molecular masses of 28 to 32 kDa were readily observed. Since mAb 12A8 reacts with both Ly-49A and Ly-49D, we repeated these experiments with a new mAb (4E5) that reacts with Ly-49D, but not Ly-49A, Ly-49B, Ly-49C/I, Ly-49H, or Ly-49G2 (data not shown). Figure 1GoA shows a representative experiment in which pervanadate-treated B6 NK cells were surface biotinylated, immunoprecipitated, separated on SDS-PAGE without reduction, and blotted with SA-HRP. Immunoprecipitation with mAb 4D11 (Ly-49G2) and mAb 12A8 (Ly-49A&D) resulted in 90- to 100-kDa proteins, consistent with the reported molecular masses for these receptors. Immunoprecipitation with mAb 4E5 yields a band of 95 to 100 kDa, consistent with that reported for Ly-49D. Furthermore, transfection of Cos-7 cells with Ly-49D cDNA, and immunoprecipitation with both mAb12A8 and 4E5 detected proteins of similar size (data not shown). These data confirmed the ability of both mAb 12A8 and 4E5 to immunoprecipitate Ly-49D. In Figure 1GoB, a parallel blot with anti-phosphotyrosine blotting of immunoprecipitates from pervanadate-treated NK cells demonstrates that both mAb 12A8 and 4E5 coimmunoprecipitated Ly-49D-associated phosphoproteins of ~28 to 32 kDa under nonreducing conditions. Control immunoprecipitations included mAb 4D11 that detects phosphorylated Ly-49G2 and mAb 12A8 that detects phosphorylated Ly-49A with which it cross-reacts. Ly-49D, which is not phosphorylated, is not seen after immunoprecipitation with mAb 4E5 and anti-phosphotyrosine blotting. Figure 1GoC depicts the Ly-49D-associated phosphoproteins under reducing conditions at an apparent molecular mass of ~16 kDa. Therefore, immunoprecipitation of Ly-49D, with either of two mAb reacting with different receptor epitopes, detects a strongly associated tyrosine phosphoprotein that exists as a ~32-kDa dimer in its fully phosphorylated form.



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  		FIGURE 1. Immunoprecipitation and anti-phosphotyrosine blotting of Ly-49D receptor-associated phosphoproteins. A, IL-2-cultured C57BL/6 (B6) NK cells were surface biotinylated, lysed, and immunoprecipitated, and proteins were separated on 8 to 16% SDS-PAGE under nonreducing conditions. Proteins were transferred to Immobilon and blotted with SA-HRP. B, NK cells were treated with pervanadate (1 mM), lysed in Triton X-100, immunoprecipitated, separated on SDS-PAGE as above, and immunoblotted with biotinylated 4G10 followed by SA-HRP. C, NK cells treated as in B were separated on 16% SDS-PAGE under reducing conditions.

 
To compare the kinetics between pervanadate- and receptor-mediated induction of pp16 phosphorylation, a time course experiment was performed. After stimulation for various lengths of time, aliquots of cells were lysed, immunoprecipitated, separated on SDS-PAGE under nonreducing conditions, and blotted with anti-phosphotyrosine. Figure 2GoA shows a time course experiment using pervanadate to stimulate B6 NK cells. These experiments suggest that at least four phosphorylated species of pp16 are observed within 2 min after pervanadate stimulation. From 4 to 8 min after stimulation, the maximally phosphorylated form is predominant. More importantly, phosphorylation of pp16 can be induced through cross-linking of Ly-49D as seen in Figure 2GoB. In these experiments, phosphorylation of the dimeric form of pp16 occurs, within 30 s, and is maximal by 2 min after cross-linking. Phosphorylation of pp16 is not mediated by the Ly-49D-specific mAb 4E5 alone or by rabbit anti-rat antisera. Only cross-linking by a secondary anti-rat Ab mediates efficient pp16 phosphorylation.



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  		FIGURE 2. Kinetics of pp16 phosphorylation by pervanadate stimulation and Ly-49D receptor cross-linking. A, IL-2-cultured B6 NK cells (3 x 106/lane) were stimulated with pervanadate for the indicated time periods followed by lysis, immunoprecipitation with mAb 4E5/protein G-agarose, and treated as in Figure 1GoB for blotting with anti-phosphotyrosine. B, 5 x 106 NK cells in 100 µl of HBSS were placed on ice for 5 min, after which 5 µg of mAb 4E5 were added and allowed to react for 5 min. A secondary rabbit anti-rat antiserum (RART) (10 µg) was added to each tube, and the tubes were placed in a 37°C water bath for the indicated times. The NK cells were immediately lysed by the addition of 1 ml of ice-cold lysis buffer for 30 min. Lysates were cleared, immunoprecipitated by the addition of mAb 4E5/protein G-agarose, and processed as above.

 
Phosphoproteins in the range of 14 to 28 kDa are reminiscent of proteins involved in TCR and FcR signaling. Therefore, immunoprecipitation experiments were performed on B6 NK cells to determine whether pp16 might be TCR-{zeta} or Fc{epsilon}RI{gamma}, both of which are found in NK cells (13). Figure 3GoA depicts such an experiment in which pervanadate-treated NK cells were lysed, immunoprecipitated with antisera to TCR-{zeta} or Fc{epsilon}RI{gamma}, and blotted with anti-phosphotyrosine. This experiment suggests that pp16 is not the same as the phosphorylated forms of TCR-{zeta} or Fc{epsilon}RI{gamma}. While immunoprecipitation with antisera to Fc{epsilon}RI{gamma} yielded 2 phosphorylated bands of ~14 and ~18 kDa, antisera to TCR-{zeta} yielded a single phosphorylated protein of ~21 kDa, consistent with the reported sizes of these phosphorylated receptors. However, both mAb 12A8 and 4E5 immunoprecipitated a 16-kDa phosphoprotein that migrated to a different position than phosphoproteins recognized by antisera to TCR-{zeta} or Fc{epsilon}RI{gamma}. These results suggest that pp16 is a novel phosphoprotein that may function as a signal transduction molecule in murine NK cells.



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  		FIGURE 3. pp16 is not TCR-{zeta} or Fc{epsilon}RI{gamma}. A, 9 x 106 B6 NK cells were stimulated with pervanadate, lysed, immunoprecipitated as indicated, and blotted with anti-phosphotyrosine after separation on 16% SDS-PAGE under reducing conditions. Control immunoprecipitations are RM2–1 (CD-2/lane 1), 4D11 (Ly-49G2/lane 2), anti- Fc{epsilon}RI{gamma} Ab alone (no lysate/lane 3), and anti-TCR-{zeta} Ab alone (no lysate/lane 8). B, 4 x 106 B6 NK cells from TCR-{zeta} and Fc{epsilon}RI{gamma} double knockout (K.O.) mice were treated with pervanadate lysed and blotted for tyrosine- phosphorylated proteins after separation on 8 to 16% SDS-PAGE under nonreducing conditions. Controls consisted of RM2–1 (CD-2, lane 1) and 4D11 (Ly-49G2, lane 2).

 
To confirm the immunologic data indicating that pp16 was neither TCR-{zeta} nor Fc{epsilon}RI{gamma}, spleens were obtained from mice that possessed null mutations of both the TCR-{zeta} and Fc{epsilon}RI{gamma} loci (double knockouts). Splenic adherent lymphokine-activated killer cells were prepared and after 7 days of culture in IL-2, were analyzed for expression of Ly-49s by flow cytometry analysis. IL-2-cultured NK cells from the double knockout mice appeared to express a normal Ly-49 receptor phenotype (data not shown). We next performed immunoprecipitation experiments and anti-phosphotyrosine immunoblotting to determine whether pp16 was associated with Ly-49D in the mutant mice. Figure 3GoB demonstrates that pp16 is definitely present in these double knockout mice, because both mAb 12A8 and 4E5 immunoprecipitate pp16. These results clearly illustrate that pp16 is not a product of the TCR-{zeta} or Fc{epsilon}RI{gamma} loci and may therefore represent a novel signaling molecule.

Comparison of the structure of Ly-49D with other Ly-49 family members revealed two significant differences: the lack of an appropriate ITIM; and an arginine residue within the transmembrane domain. We have demonstrated previously that tyrosine phosphorylation of Ly-49D does not occur following pervanadate stimulation, most likely due to its lack of an ITIM. However, the presence of a charged residue in the Ly-49D transmembrane region suggested that this residue might mediate the association of this receptor with pp16 in a manner analogous to the association of TCR-{zeta} and Fc{epsilon}RI{gamma} with their receptors. Therefore, we constructed a point mutation changing arginine-54 to leucine (Ly-49DR54L) and generated stable transfectants of the mutant and wild-type Ly-49D in the P815 mastocytoma cell line, which was previously found to express pp16 (data not shown). Figure 4GoA presents the data obtained after pervanadate stimulation of transfected P815 cells followed by lysis, immunoprecipitation with mAb 12A8 or 4E5, and anti-phosphotyrosine blotting. The data demonstrate that although both forms of the receptor are expressed (data not shown), immunoprecipitation of Ly-49D coimmunoprecipitates pp16 only in cells transfected with wild-type Ly-49D and not with Ly-49DR54L. Moreover, Western blotting with a rabbit antiserum raised against pp16 detects pp16 in wild-type Ly-49D immunoprecipitates but not immunoprecipitates of Ly-49DR54L (data not shown). These data demonstrate that physical association of pp16 with Ly-49D is mediated by the arginine within the transmembrane domain of Ly-49D.



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  		FIGURE 4. A, Mutation of the Ly-49D transmembrane arginine abolishes pp16 association. The Ly-49D transmembrane region was mutated by a single substitution at amino acid 54 from an arginine to a leucine (Ly-49DR54L) using site-directed mutagenesis. Both wild-type (WT) Ly-49D and Ly-49DR54L were stably transfected into P815 cells by electroporation and selection in G418. Cells were stimulated with pervanadate for 15 min, lysates were immunoprecipitated with the designated mAb, and proteins were separated by electrophoresis under reducing conditions and immunoblotted with anti-phosphotyrosine as described previously. B, Mutation of the arginine residue of the Ly-49D transmembrane domain ablates calcium mobilization in P815. Parental P815 or P815 stably expressing wild-type Ly-49D or Ly-49DR54L were stimulated with either anti-Ly-49D (4E5) or anti-Ly-49G mAb (4D11) as indicated (arrow 1). Primary Ab was cross-linked with rabbit anti-rat Ab after 25 s (arrow 2). [Ca2+]i was monitored over time and is presented as the ratio of dye bound to Ca2+ (emission at 402 nm) over dye free of Ca2+ (emission at 486 nm) in arbitrary units.

 
Having established that Ly-49D is capable of physically interacting with pp16 in P815, we asked whether this ectopically expressed receptor was able to couple to the P815 signal transduction mechanism. Figure 4GoB demonstrates that stimulation of parental P815 with cross-linked mAb 4E5 had no effect on the intracellular calcium concentration ([Ca2+]i). In contrast, cross-linking mAb 4E5 on P815 cells stably expressing wild-type Ly-49D showed a substantial increase in [Ca2+]i consistent with coupling of Ly-49D to the P815 signaling apparatus. This response was specific in that treatment of Ly-49D transfectants with mAb recognizing Ly-49G (4D11) showed no increase in calcium levels. Next we used this model to test the importance of pp16 in Ly-49D signaling by cross-linking Ly-49DR54L, a receptor incapable of physically interacting with pp16. Cross-linking of Ly-49DR54L in two independent clones failed to induce calcium mobilization in P815, even though these cells express higher levels of surface Ly-49D than their wild-type counterparts (data not shown).

Together our data suggest the absolute requirement for pp16 association in Ly-49D signal transduction. These findings suggest that analogous to other multichain immune receptors such as the TCR, B cell receptor, and FcR, Ly-49D lacks intrinsic signaling properties and instead forms multimeric complexes with low m.w., tyrosine-phosphorylated polypeptides. Within other receptor systems, these low m.w. proteins serve as tyrosine kinase substrates that recruit additional src homology 2 (SH2)-containing kinases such as members of the Syk/Zap70 family (14, 15). Preliminary data using anti-pp16 antisera directly demonstrates expression of pp16 not only in NK cells but also in cells of the myeloid lineage. This leads to the speculation that pp16 may serve as the Syk/Zap70 docking site within receptor complexes other than Ly-49D. Candidate receptors include the recently described positive signaling receptors of the ILT/MIR (16, 17) and PIRA families (18, 19). Recently, Lanier et al. (20) cloned an ITAM-containing polypeptide, DAP12, that associates with noninhibitory isoforms of human killer cell-inhibitory receptor molecules and appears to be involved in NK cell activation. We are currently examining the possibility that the Ly-49D-associated pp16 represents the murine DAP12.


   Acknowledgments
 
We thank Dr. Deborah N. Burshtyn for critical reading of the manuscript, Dr. Kazuo Maruyama for the PMKit vector, and Ms. Joyce Vincent for manuscript preparation. In addition, we thank Dr. Jean-Pierre Kinet for rabbit antisera to Fc{epsilon}RI{gamma} and Dr. Allan Weissman for rabbit antisera to the {zeta}-chain of the TCR.


   Footnotes
 
1 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory motif; pp16, 16-kDa tyrosine-phosphorylated protein; SA-HRP, streptavidin-horseradish peroxidase. Back

2 Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985). Back

3 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Back

4 Address correspondence and reprint requests to Dr. L. H. Mason, NCI-FCRDC Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: Back

Received for publication January 15, 1998. Accepted for publication February 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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A. P. Makrigiannis, J. Etzler, R. Winkler-Pickett, A. Mason, J. R. Ortaldo, and S. K. Anderson
Identification of the Ly49L protein: evidence for activating counterparts to inhibitory Ly49 proteins
J. Leukoc. Biol., November 1, 2000; 68(5): 765 - 771.
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J. Wu, H. Cherwinski, T. Spies, J. H. Phillips, and L. L. Lanier
Dap10 and Dap12 Form Distinct, but Functionally Cooperative, Receptor Complexes in Natural Killer Cells
J. Exp. Med., October 2, 2000; 192(7): 1059 - 1068.
[Abstract] [Full Text] [PDF]

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L. H. Mason
Recognition of CHO cells by inhibitory and activating Ly-49 receptors
J. Leukoc. Biol., October 1, 2000; 68(4): 583 - 586.
[Abstract] [Full Text]

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N. Aoki, S. Kimura, Y. Takiyama, Y. Atsuta, A. Abe, K. Sato, and M. Katagiri
The Role of the DAP12 Signal in Mouse Myeloid Differentiation
J. Immunol., October 1, 2000; 165(7): 3790 - 3796.
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L. H. Mason, J. Willette-Brown, A. T. Mason, D. McVicar, and J. R. Ortaldo
Interaction of Ly-49D+ NK Cells with H-2Dd Target Cells Leads to Dap-12 Phosphorylation and IFN-{gamma} Secretion
J. Immunol., January 15, 2000; 164(2): 603 - 611.
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G. Sconocchia, J. A. Titus, A. Mazzoni, A. Visintin, F. Pericle, S. W. Hicks, F. Malavasi, and D. M. Segal
CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer Cells
Blood, December 1, 1999; 94(11): 3864 - 3871.
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J. R. Ortaldo, R. Winkler-Pickett, J. Willette-Brown, R. L. Wange, S. K. Anderson, G. J. Palumbo, L. H. Mason, and D. W. McVicar
Structure/Function Relationship of Activating Ly-49D and Inhibitory Ly-49G2 NK Receptors ,2
J. Immunol., November 15, 1999; 163(10): 5269 - 5277.
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C. Chang, J. Dietrich, A. G. Harpur, J. A. Lindquist, A. Haude, Y. W. Loke, A. King, M. Colonna, J. Trowsdale, and M. J. Wilson
Cutting Edge: KAP10, a Novel Transmembrane Adapter Protein Genetically Linked to DAP12 but with Unique Signaling Properties
J. Immunol., November 1, 1999; 163(9): 4651 - 4654.
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M. C. Nakamura, C. Naper, E. C. Niemi, S. C. Spusta, B. Rolstad, G. W. Butcher, W. E. Seaman, and J. C. Ryan
Natural Killing of Xenogeneic Cells Mediated by the Mouse Ly-49D Receptor
J. Immunol., November 1, 1999; 163(9): 4694 - 4700.
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A. P. Makrigiannis, P. Gosselin, L. H. Mason, L. S. Taylor, D. W. McVicar, J. R. Ortaldo, and S. K. Anderson
Cloning and Characterization of a Novel Activating Ly49 Closely Related to Ly49A ,2 ,3
J. Immunol., November 1, 1999; 163(9): 4931 - 4938.
[Abstract] [Full Text] [PDF]

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N. S. Williams, J. Klem, I. J. Puzanov, P. V. Sivakumar, M. Bennett, and V. Kumar
Differentiation of NK1.1+, Ly49+ NK Cells from flt3+ Multipotent Marrow Progenitor Cells
J. Immunol., September 1, 1999; 163(5): 2648 - 2656.
[Abstract] [Full Text] [PDF]

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L. S. Taylor and D. W. McVicar
Functional Association of Fcvarepsilon RIgamma With Arginine632 of Paired Immunoglobulin-Like Receptor (PIR)-A3 in Murine Macrophages
Blood, September 1, 1999; 94(5): 1790 - 1796.
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 Proc. Natl. Acad. Sci. USAHome page
A. B. H. Bakker, E. Baker, G. R. Sutherland, J. H. Phillips, and L. L. Lanier
Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells
PNAS, August 17, 1999; 96(17): 9792 - 9796.
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T. C. George, J. R. Ortaldo, S. Lemieux, V. Kumar, and M. Bennett
Tolerance and Alloreactivity of the Ly49D Subset of Murine NK Cells
J. Immunol., August 15, 1999; 163(4): 1859 - 1867.
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 Proc. Natl. Acad. Sci. USAHome page
A. H. Idris, H. R. C. Smith, L. H. Mason, J. R. Ortaldo, A. A. Scalzo, and W. M. Yokoyama
The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing
PNAS, May 25, 1999; 96(11): 6330 - 6335.
[Abstract] [Full Text] [PDF]

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S. K. P. Kung, R.-C. Su, J. Shannon, and R. G. Miller
The NKR-P1B Gene Product Is an Inhibitory Receptor on SJL/J NK Cells
J. Immunol., May 15, 1999; 162(10): 5876 - 5887.
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K. Bernard, A. Cambiaggi, S. Guia, F. Bertucci, S. Granjeaud, R. Tagett, C. N'Guyen, B. R. Jordan, and E. Vivier
Engagement of Natural Cytotoxicity Programs Regulates AP-1 Expression in the NKL Human NK Cell Line
J. Immunol., April 1, 1999; 162(7): 4062 - 4068.
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T. C. George, L. H. Mason, J. R. Ortaldo, V. Kumar, and M. Bennett
Positive Recognition of MHC Class I Molecules by the Ly49D Receptor of Murine NK Cells
J. Immunol., February 15, 1999; 162(4): 2035 - 2043.
[Abstract] [Full Text] [PDF]

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H. Kubagawa, C.-C. Chen, Le Hong Ho, T. Shimada, L. Gartland, C. Mashburn, T. Uehara, J. V. Ravetch, and M. D. Cooper
Biochemical Nature and Cellular Distribution of the Paired Immunoglobulin-like Receptors, PIR-A and PIR-B
J. Exp. Med., January 18, 1999; 189(2): 309 - 318.
[Abstract] [Full Text] [PDF]

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A. H. Idris, K. Iizuka, H. R.C. Smith, A. A. Scalzo, and W. M. Yokoyama
Genetic Control Of Natural Killing and In Vivo Tumor Elimination by the Chok Locus
J. Exp. Med., December 21, 1998; 188(12): 2243 - 2256.
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E. Tomasello, L. Olcese, F. Vely, C. Geourgeon, M. Blery, A. Moqrich, D. Gautheret, M. Djabali, M.-G. Mattei, and E. Vivier
Gene Structure, Expression Pattern, and Biological Activity of Mouse Killer Cell Activating Receptor-associated Protein (KARAP)/DAP-12
J. Biol. Chem., December 18, 1998; 273(51): 34115 - 34119.
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D. W. McVicar, L. S. Taylor, P. Gosselin, J. Willette-Brown, A. I. Mikhael, R. L. Geahlen, M. C. Nakamura, P. Linnemeyer, W. E. Seaman, S. K. Anderson, et al.
DAP12-mediated Signal Transduction in Natural Killer Cells. A DOMINANT ROLE FOR THE Syk PROTEIN-TYROSINE KINASE
J. Biol. Chem., December 4, 1998; 273(49): 32934 - 32942.
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K. M. Smith, J. Wu, A. B. H. Bakker, J. H. Phillips, and L. L. Lanier
Cutting Edge: Ly-49D and Ly-49H Associate with Mouse DAP12 and Form Activating Receptors
J. Immunol., July 1, 1998; 161(1): 7 - 10.
[Abstract] [Full Text] [PDF]

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