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Direct and Indirect Interactions of the Cytoplasmic Region of CD244 (2B4) in Mice and Humans with FYN Kinase

2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês

10.1074/jbc.m704483200

ISSN

1083-351X

Autores

Nicholas G. Clarkson, S. J. Simmonds, Michael J. Puklavec, Marion H. Brown,

Tópico(s)

Galectins and Cancer Biology

Resumo

Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged. Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged. Signaling by receptors that bind the intracellular adaptor SAP 2The abbreviations used are:EAT-2, Ewing sarcoma activated transcript-2.SAPSLAM-associated proteinITSMsimmunoreceptor tyrosine-based switch motifsSH2Src homologyNKnatural killermAbsmonoclonal antibodiesEGFPenhanced green fluorescence proteinRUresponse unitsLAKlymphokine-activated killerCHOChinese hamster ovaryIL-2interleukin-2 2The abbreviations used are:EAT-2, Ewing sarcoma activated transcript-2.SAPSLAM-associated proteinITSMsimmunoreceptor tyrosine-based switch motifsSH2Src homologyNKnatural killermAbsmonoclonal antibodiesEGFPenhanced green fluorescence proteinRUresponse unitsLAKlymphokine-activated killerCHOChinese hamster ovaryIL-2interleukin-2 is important for immune regulation in combating infection, and it is dysregulated in cancer and autoimmunity (1Veillette A. Nat. Rev. Immunol. 2006; 6: 56-66Crossref PubMed Scopus (176) Google Scholar, 4Thorley-Lawson D.A. J. Allergy. Clin. Immunol. 2005; 116: 251-261Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar); thus, an understanding of the mechanisms involved has wide implications. CD244 (also known as 2B4) is one of the group of leukocyte surface receptors, including CD244, NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9), that are characterized by related extracellular immunoglobulin superfamily domains and intracellular immunoreceptor tyrosine-based switch motifs (ITSMs) that can bind the SH2 adaptor protein SAP (see Fig. 1a) (3Engel P. Eck M.J. Terhorst C. Nat. Rev. Immunol. 2003; 3: 813-821Crossref PubMed Scopus (277) Google Scholar, 5van der Merwe P.A. Davis S.J. Annu. Rev. Immunol. 2003; 21: 659-684Crossref PubMed Scopus (425) Google Scholar). These receptors are differentially distributed on immune cells and are involved in cell/cell interactions through heterophilic or homophilic interactions (2Nichols K.E. Ma C.S. Cannons J.L. Schwartzberg P.L. Tangye S.G. Immunol. Rev. 2005; 203: 180-199Crossref PubMed Scopus (181) Google Scholar, 3Engel P. Eck M.J. Terhorst C. Nat. Rev. Immunol. 2003; 3: 813-821Crossref PubMed Scopus (277) Google Scholar, 6Romero X. Benitez D. March S. Vilella R. Miralpeix M. Engel P. Tissue Antigens. 2004; 64: 132-144Crossref PubMed Scopus (71) Google Scholar, 8Cao E. Ramagopal U.A. Fedorov A. Fedorov E. Yan Q. Lary J.W. Cole J.L. Nathenson S.G. Almo S.C. Immunity. 2006; 25: 559-570Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The CD48/CD244 interaction is the only well characterized heterophilic interaction among the receptors that contain SAP-binding motifs in their cytoplasmic regions (3Engel P. Eck M.J. Terhorst C. Nat. Rev. Immunol. 2003; 3: 813-821Crossref PubMed Scopus (277) Google Scholar). CD244, primarily characterized as a receptor on natural killer (NK) and activated CD8+ cells (6Romero X. Benitez D. March S. Vilella R. Miralpeix M. Engel P. Tissue Antigens. 2004; 64: 132-144Crossref PubMed Scopus (71) Google Scholar, 9Kambayashi T. Assarsson E. Chambers B.J. Ljunggren H.G. J. Immunol. 2001; 167: 6706-6710Crossref PubMed Scopus (72) Google Scholar, 10Assarsson E. Kambayashi T. Persson C.M. Chambers B.J. Ljunggren H.G. J. Immunol. 2005; 175: 2045-2049Crossref PubMed Scopus (34) Google Scholar), binds the glycophosphoinositol-anchored protein CD48 with similar affinity in mouse and human (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar). Rodent CD48 also retains a low but functionally significant affinity for CD2 (see Fig. 1a) (5van der Merwe P.A. Davis S.J. Annu. Rev. Immunol. 2003; 21: 659-684Crossref PubMed Scopus (425) Google Scholar, 11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar). The ligand for human CD2 (CD58) is missing in rodents.There has been confusion in the literature over the inhibitory and activating potential of CD244 (2B4) and over differences in behavior between mouse and human CD244. A study in a CD244-deficient mouse showed clearly that, when CD48 on a target cell is not able to bind to CD244 on an NK cell, killing is enhanced (12Lee K.M. McNerney M.E. Stepp S.E. Mathew P.A. Schatzle J.D. Bennett M. Kumar V. J. Exp. Med. 2004; 199: 1245-1254Crossref PubMed Scopus (166) Google Scholar). These results assisted with interpreting data with monoclonal antibodies (mAbs) and distinguishing between blocking and cross-linking effects (12Lee K.M. McNerney M.E. Stepp S.E. Mathew P.A. Schatzle J.D. Bennett M. Kumar V. J. Exp. Med. 2004; 199: 1245-1254Crossref PubMed Scopus (166) Google Scholar) and also suggested that the phenotype of the CD244-deficient mouse reflects loss of ligand engagement. Thus, binding of CD48 on the target cells to CD244 on NK cells has an inhibitory effect on NK cell killing. However, engagement of CD244 can also have positive effects in mice (10Assarsson E. Kambayashi T. Persson C.M. Chambers B.J. Ljunggren H.G. J. Immunol. 2005; 175: 2045-2049Crossref PubMed Scopus (34) Google Scholar, 13Lee K.M. Forman J.P. McNerney M.E. Stepp S. Kuppireddi S. Guzior D. Latchman Y.E. Sayegh M.H. Yagita H. Park C.K. Oh S.B. Wulfing C. Schatzle J. Mathew P.A. Sharpe A.H. Kumar V. Blood. 2006; 107: 3181-3188Crossref PubMed Scopus (68) Google Scholar). Likewise, in human, the functional outcome of CD48/CD244 interactions can be positive and negative (14Sivori S. Falco M. Marcenaro E. Parolini S. Biassoni R. Bottino C. Moretta L. Moretta A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4526-4531Crossref PubMed Scopus (167) Google Scholar, 15Morandi B. Costa R. Falco M. Parolini S. De Maria A. Ratto G. Mingari M.C. Melioli G. Moretta A. Ferlazzo G. J. Immunol. 2005; 175: 3690-3697Crossref PubMed Scopus (22) Google Scholar, 16Parolini S. Bottino C. Falco M. Augugliaro R. Giliani S. Franceschini R. Ochs H.D. Wolf H. Bonnefoy J.Y. Biassoni R. Moretta L. Notarangelo L.D. Moretta A. J. Exp. Med. 2000; 192: 337-346Crossref PubMed Scopus (413) Google Scholar, 17Nakajima H. Cella M. Langen H. Friedlein A. Colonna M. Eur. J. Immunol. 1999; 29: 1676-1683Crossref PubMed Scopus (202) Google Scholar). A model of CD244 signal transduction has to account for differences in functional outcome.The altered behavior of NK cells deficient in CD244 (12Lee K.M. McNerney M.E. Stepp S.E. Mathew P.A. Schatzle J.D. Bennett M. Kumar V. J. Exp. Med. 2004; 199: 1245-1254Crossref PubMed Scopus (166) Google Scholar), FYN (18Bloch-Queyrat C. Fondaneche M.C. Chen R. Yin L. Relouzat F. Veillette A. Fischer A. Latour S. J. Exp. Med. 2005; 202: 181-192Crossref PubMed Scopus (97) Google Scholar), SAP (16Parolini S. Bottino C. Falco M. Augugliaro R. Giliani S. Franceschini R. Ochs H.D. Wolf H. Bonnefoy J.Y. Biassoni R. Moretta L. Notarangelo L.D. Moretta A. J. Exp. Med. 2000; 192: 337-346Crossref PubMed Scopus (413) Google Scholar, 18Bloch-Queyrat C. Fondaneche M.C. Chen R. Yin L. Relouzat F. Veillette A. Fischer A. Latour S. J. Exp. Med. 2005; 202: 181-192Crossref PubMed Scopus (97) Google Scholar, 19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar), or EAT-2 and/or ERT (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar) has implicated CD244 in being a major player in regulation among the SAP-binding receptors. The CD244 cytoplasmic tail contains the highest number (four) of the particular tyrosine motif among the SAP-binding receptors that, when phosphorylated upon cell activation, can bind SAP and/or EAT-2 (see Fig. 1) (3Engel P. Eck M.J. Terhorst C. Nat. Rev. Immunol. 2003; 3: 813-821Crossref PubMed Scopus (277) Google Scholar, 19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar, 21Tassi I. Colonna M. J. Immunol. 2005; 175: 7996-8002Crossref PubMed Scopus (86) Google Scholar). These four motifs are present in mouse and human CD244 but are not identical in sequence. SAP can recruit FYN through an unusual interaction with the SH3 domain of FYN (22Chan B. Lanyi A. Song H.K. Griesbach J. Simarro-Grande M. Poy F. Howie D. Sumegi J. Terhorst C. Eck M.J. Nat. Cell Biol. 2003; 5: 155-160Crossref PubMed Scopus (237) Google Scholar, 23Latour S. Roncagalli R. Chen R. Bakinowski M. Shi X. Schwartzberg P.L. Davidson D. Veillette A. Nat. Cell Biol. 2003; 5: 149-154Crossref PubMed Scopus (225) Google Scholar). EAT-2 and ERT are structurally related to SAP, but their tail regions contain one or two tyrosine motifs in human and mouse, respectively (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar, 24Calpe S. Erdos E. Liao G. Wang N. Rietdijk S. Simarro M. Scholtz B. Mooney J. Lee C.H. Shin M.S. Rajnavolgyi E. Schatzle J. Morse H.C. II I Terhorst C. Lanyi A. Immunogenetics. 2006; 58: 15-25Crossref PubMed Scopus (26) Google Scholar). ERT is not expressed in human (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar, 24Calpe S. Erdos E. Liao G. Wang N. Rietdijk S. Simarro M. Scholtz B. Mooney J. Lee C.H. Shin M.S. Rajnavolgyi E. Schatzle J. Morse H.C. II I Terhorst C. Lanyi A. Immunogenetics. 2006; 58: 15-25Crossref PubMed Scopus (26) Google Scholar). Unlike the SH2 domain of SAP, that of EAT-2 does not bind the SH3 domain of FYN (22Chan B. Lanyi A. Song H.K. Griesbach J. Simarro-Grande M. Poy F. Howie D. Sumegi J. Terhorst C. Eck M.J. Nat. Cell Biol. 2003; 5: 155-160Crossref PubMed Scopus (237) Google Scholar, 23Latour S. Roncagalli R. Chen R. Bakinowski M. Shi X. Schwartzberg P.L. Davidson D. Veillette A. Nat. Cell Biol. 2003; 5: 149-154Crossref PubMed Scopus (225) Google Scholar). In mice, SAP and EAT-2 or ERT deficiencies have opposite phenotypes, with lack of SAP being associated with decreased responses and vice versa for EAT-2 or ERT (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar). In mice, the inhibitory effects of EAT-2 and ERT have been shown to be dependent on phosphorylation of the tail tyrosine motifs (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar). EAT-2 and ERT are expressed in NK cells (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar, 21Tassi I. Colonna M. J. Immunol. 2005; 175: 7996-8002Crossref PubMed Scopus (86) Google Scholar, 24Calpe S. Erdos E. Liao G. Wang N. Rietdijk S. Simarro M. Scholtz B. Mooney J. Lee C.H. Shin M.S. Rajnavolgyi E. Schatzle J. Morse H.C. II I Terhorst C. Lanyi A. Immunogenetics. 2006; 58: 15-25Crossref PubMed Scopus (26) Google Scholar), and CD244 is a major binding partner for EAT-2 and ERT (19Roncagalli R. Taylor J.E. Zhang S. Shi X. Chen R. Cruz-Munoz M.E. Yin L. Latour S. Veillette A. Nat. Immunol. 2005; 6: 1002-1010Crossref PubMed Scopus (115) Google Scholar), suggesting that the inhibitory function of CD244 is dependent on these interactions. Models for the mechanism of the inhibitory effects of SAP-binding receptors, particularly CD244, include direct binding of phosphatases competing with SAP and recruitment of negative regulators through EAT-2 (3Engel P. Eck M.J. Terhorst C. Nat. Rev. Immunol. 2003; 3: 813-821Crossref PubMed Scopus (277) Google Scholar, 25Tangye S.G. Lazetic S. Woollatt E. Sutherland G.R. Lanier L.L. Phillips J.H. J. Immunol. 1999; 162: 6981-6985PubMed Google Scholar, 26Colonna M. Nat. Immunol. 2005; 6: 961-962Crossref PubMed Scopus (8) Google Scholar).Conservation of extracellular and intracellular interactions with CD48 and the adaptors SAP and EAT-2 and expression in cytotoxic cells in both mouse and human (6Romero X. Benitez D. March S. Vilella R. Miralpeix M. Engel P. Tissue Antigens. 2004; 64: 132-144Crossref PubMed Scopus (71) Google Scholar, 9Kambayashi T. Assarsson E. Chambers B.J. Ljunggren H.G. J. Immunol. 2001; 167: 6706-6710Crossref PubMed Scopus (72) Google Scholar, 10Assarsson E. Kambayashi T. Persson C.M. Chambers B.J. Ljunggren H.G. J. Immunol. 2005; 175: 2045-2049Crossref PubMed Scopus (34) Google Scholar) suggest that the function of CD244 is conserved between the two species. We sought to identify the features of CD244 function that are conserved between mouse and human with the aim of establishing the fundamental mechanism by which CD244 regulates immune responses. An understanding of the differences between mouse and human CD244 will allow interpretation of the relevance of mouse model data to human. There is evidence that manipulating CD48/CD244 interactions may be useful therapeutically, as CD244-deficient mice are more effective in tumor clearance (12Lee K.M. McNerney M.E. Stepp S.E. Mathew P.A. Schatzle J.D. Bennett M. Kumar V. J. Exp. Med. 2004; 199: 1245-1254Crossref PubMed Scopus (166) Google Scholar, 27Vaidya S.V. Stepp S.E. McNerney M.E. Lee J.K. Bennett M. Lee K.M. Stewart C.L. Kumar V. Mathew P.A. J. Immunol. 2005; 174: 800-807Crossref PubMed Scopus (78) Google Scholar). We show that, in both mice and humans, engagement of mouse and human CD244 on normal NK cells by CD48 on target cells can be inhibitory. By determining the hierarchy of interactions likely to compete for direct binding to CD244 at physiological temperature, we show that intracellular interactions of CD244 are generally conserved between mouse and human CD244. We identify FYN kinase as a potential binding partner for the tail of EAT-2 and provide functional data to show that EAT-2 is not inhibitory per se. This suggests a model in which the functional outcome of CD48/CD244 engagement is a reflection of the potential to recruit FYN kinase and the balance between co-engaged inhibitory and activating receptors. The general mechanism is applicable to the other SAP-binding receptors.EXPERIMENTAL PROCEDURESRecombinant Proteins—The rat basophil leukemia cell line was stably transfected with the pBabe vector encoding a chimeric receptor containing the extracellular region of the C57/BL6 allele of mouse CD244 (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar) and the transmembrane and cytoplasmic regions of the mouse ζ-chain, CD244-ζ (28Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (250) Google Scholar), using FuGENE™ transfection reagent. Full-length mouse CD244 (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar) in pBabe was expressed in 2B4 and 171 mouse T cell hybridoma cells by transfection of ecoPhoenix cells and transduction. Cells expressing 2B4 were selected at 48 h by Dynal bead selection using anti-2B4 mAb (Pharmingen), and selection was maintained with puromycin (1 μg/ml) in RPMI 1640 medium containing 5–10% fetal calf serum. Constructs of full-length mouse SAP and the SH2 domain (amino acids 2–104; provided by Andre Veillette), full-length human SAP and the SH2 domain (amino acids 1–108; provided by Cox Terhorst), full-length mouse EAT-2 and the SH2 domain (amino acids 1–103; provided by Andre Veillette), full-length human EAT-2 and the SH2 domain (amino acids 1–108; provided by Marco Colonna), and the human FYN SH2 (amino acids 144–248) and SH3 (amino acids 82–149) domains (both provided by Rose Zamoyska, National Institute for Medical Research, London, UK) for production of soluble recombinant proteins were produced by PCR from templates. Fragments were cut with BamHI and SalI and cloned into BamHI and XhoI sites in the pTrcHISA vector (Invitrogen). The vector stop codon was used for the isolated domain constructs except for the mouse SAP SH2 domain, in which it was engineered directly after the final SAP amino acid. Constructs for His-tagged mouse FYN SH3-SH2 (amino acids 82–248) and human FYN SH3-SH2 (amino acids 82–248) proteins were provided by Louise Bird (Oxford Module Consortium). Proteins were expressed as N-terminally His-tagged fusion proteins and purified using nickel-agarose affinity chromatography. Proteins were subjected to gel filtration on Superdex 75 (GE Healthcare) prior to Biacore analysis. Concentration was determined using absorbance at 280 nm and the following theoretical extinction coefficients (Vector NTI): mouse SAP and SH2, 23,140 and 23,020 m–1 cm–1; human SAP and SH2, 24,660 and 24,540 M–1 cm–1; mouse EAT-2, 19,090 m–1 cm–1; human EAT-2 and SH2, 21,980 and 15,130 m–1 cm–1; human FYN SH2, 26,510 M–1 cm–1; human –1 FYN SH3, 29,280 m–1 cm–1; mouse FYN SH3-SH2, 35,680 m cm–1; and human FYN SH3-SH2, 37,561 m–1 cm–1 For mammalian expression as enhanced green fluorescence protein (EGFP) fusion proteins, BamHI-HindIII fragments from pTrcHISA constructs were cloned into the retroviral vector pLEGFP-C1 (Invitrogen) and expressed in 2B4 hybridoma cells as described for the pBabe vector, and selection was maintained with G418 (0.4 mg/ml).Biacore™ Analyses—Biacore analyses using a Biacore™ 2000 were carried out essentially as described previously (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar). Analyses of CD244 mAb blocking were carried out at 25 °C (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar), and all other experiments were carried out at 37 °C. Streptavidin (∼3000 response units (RU)) and mAbs (∼15,000 RU) were directly coupled to CM5 chips by amine coupling for immobilization of biotinylated phosphorylated peptides (Peptide Protein Research Ltd. (Fareham, UK) and Sigma) (see Tables 1 and 2) and native receptors, respectively. The FYN SH3 domain (∼1250–2000 RU) was immobilized by amine coupling. In measurements of binding to the four ITSM peptides, one flow cell was used as a negative control and then coated with the fourth peptide, and equilibrium binding was repeated. At the level of immobilization of peptide (∼25 RU), there was no difference between negative controls of streptavidin or an irrelevant phosphorylated peptide. Native receptor was captured from cell lysate (108 cell eq/ml) by passing the lysate at 1 μl/min over a mAb-coated flow cell. Cell lysates were prepared from lymphokine-activated killer (LAK) cells that had been treated with pervanadate or not at 108 cells/ml for 7–10 min at 37 °C; washed with phosphate-buffered saline; and lysed at 108 cells/ml in 10 mm Tris-HCl (pH 7.4) supplemented with 140 mm NaCl, 1 mm EDTA, 10% (v/v) glycerol, 0.02% (w/v) NaN3, 1% Brij 96, 1 mm sodium pervanadate, mammalian protease inhibitor mixture (Sigma), and 1 mm NaF for ∼10 min at 4 °C. The lysate was centrifuged at 20,800 × g in a microcentrifuge for 10 min at 4 °C. Lysates were used immediately in Biacore experiments.TABLE 1SAP, EAT-2, and FYN bind phosphorylated CD244 peptides with affinities consistent with a direct functional interactionImmobilized peptideMouse SAP SH2Mouse SAPMouse EAT-2Human SAP SH2Human SAPHuman EAT-2 SH2Human EAT-2Mouse FYN SH3-SH2Human FYN SH2CD244 ITSM10.20.230.70.160.750.10.430.850.55 m: EPLTIYEYVKD (93%) h: EFLTIYEDVKD (81%)CD244 ITSM214.65.50.080.30.250.61.623.6 m: DRGTMYSMIQC (70%) h: GGSTIYSMIQS (77%)CD244 ITSM30.51.40.90.08170.171.41.52 m: EKCTVYSVVQP (41%) h: PAYTLYSLIQP (100%)CD244 ITSM411.521.751.20.540.651.111.7 m: LSCTVYEEVGN (>70%) h: FNSTIYEVIGK (100%) Open table in a new tab TABLE 2The FYN SH2 domain binds phosphorylated EAT-2 tail peptides with higher affinity than SAP binds the FYN SH3 domainImmobilized peptide/proteinmFYN SH3-SH2hFYN SH3-SH2hFYN SH2hSAP SH2hSAPEAT-2 Tyr/Phe1190.5>100 m: LELNVYENTDE (84%) h: LELETFVNSNS (87%)EAT-2 Tyr128113 m: NTDEEYVDVLP (97%) h: NSNSDYVDVLP (94%)FYN SH311, 9aIn the same experiment, the human SAP SH2 domain bound the phosphorylated peptide of the membrane-proximal motif of human CD244 (KD = 0.14 μm at 37 °C)., 6bThe human SAP SH2 domain and full-length SAP were compared in the same experiment.30bThe human SAP SH2 domain and full-length SAP were compared in the same experiment.a In the same experiment, the human SAP SH2 domain bound the phosphorylated peptide of the membrane-proximal motif of human CD244 (KD = 0.14 μm at 37 °C).b The human SAP SH2 domain and full-length SAP were compared in the same experiment. Open table in a new tab Antibodies—AO rats (haplotype RT1u; Harlan UK) were initially immunized intravenously with 4 × 106 irradiated rat basophil leukemia cells (haplotype RT1u) expressing the CD244-ζ chimera, as it was more highly expressed than native CD244. The rats were given three additional immunizations, once with complete and twice with incomplete Freund's adjuvant of 20 μg of CD244-CD4d3+4 soluble fusion protein purified from Chinese hamster ovary (CHO) cells stably transfected with a CD244-CD4d3+4-PEE14 construct (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar) subcutaneously. Rats were boosted three times intravenously with 4 × 106 rat basophil leukemia cells expressing CD244-ζ and 20 μgof CD244-CD4d3+4 soluble fusion protein. 3 days after the final boost, the spleen was removed, and a single cell suspension was prepared and fused with the Lewis rat myeloma cell line Y3 AG8.153. Three hybridomas producing mAb that stained CD244/171 cell lines but not the 171 untransfected cell line were isolated and subcloned twice by limiting dilution. The mAbs were of the IgG1-κ (OX120) and IgG2a-κ (OX121 and OX122) isotypes as determined by enzyme-linked immunosorbent assay (Pharmingen). mAbs OX121 and OX122 were purified on protein G-Sepharose (GE Healthcare). Fab fragments were prepared by papain digestion and gel filtration on a Sephadex HR 200 column (GE Healthcare) in phosphate-buffered saline. C57/BL6 and BALB/c mice obtained from Sir William Dunn School of Pathology were used for flow cytometric analysis of normal CD244.Other mAbs used were as follows: anti-mouse 2B4 (Pharmingen), anti-mouse CD3 (KT3) rat IgG2a, anti-mouse CD2 (RM2.1) rat IgG2a, anti-mouse CD48 (OX78) rat IgG2a, antirat κ-chain (OX11) rat IgG2a, anti-mouse κ-chain (OX20) rat IgG1, anti-mouse B220, anti-human CD244 (C1.7) mouse IgG1 (Beckman Coulter), anti-human CD48 (6.28) mouse IgG3, antihuman CD2 (X53) mouse IgG1, negative controls mouse IgG1 (OX21) and mouse IgG3 (OX61), anti-human CD56 (TA181 H12), anti-human CD3 (OKT3), anti-human CD4 (OKT4), anti-CD8 (OKT8), anti-phosphotyrosine (clone PT66; Sigma), phycoerythrin-coupled secondary antibodies (Sigma), and fluorescein isothiocyanate-coupled secondary antibodies (Serotec). Antibodies used in Western blotting and immunoprecipitation experiments included anti-human EAT-2 polyclonal antibody raised against an unphosphorylated (Santa Cruz Biotechnology, Inc.) or tyrosine-phosphorylated (Everest Biotech) peptide from the tail of human EAT-2, anti-FYN polyclonal antibody (Upstate), biotinylated anti-phosphotyrosine mAb (clone PT66; Sigma), and horseradish peroxidase-coupled secondary reagents (Sigma).Immunoprecipitation and Western Blotting—Cell lysates were prepared at 108 cells/ml using Triton X-100 detergent as described above (29Hassan N.J. Simmonds S.J. Clarkson N.G. Puklavec M.J. Hanrahan S. Bomb M. Barclay A.N. Brown M.H. Mol. Cell. Biol. 2006; 26: 6727-6738Crossref PubMed Scopus (72) Google Scholar). Immunoprecipitates were prepared using Dynal beads (Dynal, Oslo, Norway). Western blot analysis was carried out using 4–12% SDS-polyacrylamide gels under reducing conditions and ECL detection methods (Amersham Biosciences) (29Hassan N.J. Simmonds S.J. Clarkson N.G. Puklavec M.J. Hanrahan S. Bomb M. Barclay A.N. Brown M.H. Mol. Cell. Biol. 2006; 26: 6727-6738Crossref PubMed Scopus (72) Google Scholar).Cellular Assays—Murine NK cells were enriched by passing a splenic cell suspension from C57/BL6 mice through a nylon wool column for negative depletion of adherent cells. LAK cells were generated by culturing the passaged cells in RPMI 1640 medium with l-glutamine supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 50 μm 2-mercaptoethanol (complete RPMI medium) and containing 0.5% spent tissue culture supernatant from a rat interleukin-2 (IL-2)-producing CHO line at 37 °C and 5% CO2. On day 3, half of the medium was replaced with fresh medium. Murine LAK cells were used on day 6. Human NK cells were purified from non-adherent peripheral blood cells (National Blood Service, Bristol, UK) by depletion with anti-human CD3, CD4, and CD8 mAbs and cultured for ∼12 days with an irradiated Epstein-Barr virus-positive B cell line (221.721) and irradiated allogeneic peripheral blood cells. 2 × 104 RMA-S or 221.721 cells, target and effector NK cells (LAK cells), or human NK cells at the indicated effector/target ratios were incubated in complete RPMI medium in 96-well plates. mAb or Fab fragments were added to the wells at a final concentration of 10 μg/ml. After 4–5 h of incubation at 37 °C, the supernatants were removed, and the amount of lysis was measured using a nonradioactive cytotoxicity assay (CytoTox 96, Promega Corp.). Antigen-specific IL-2 production of hybridoma cells (105 cells/well) using mouse CD48+ and CD48–, major histocompatibility complex Class II+ CHO cells (30Wild M.K. Cambiaggi A. Brown M.H. Davies E.A. Ohno H. Saito T. van der Merwe P.A. J. Exp. Med. 1999; 190: 31-41Crossref PubMed Scopus (99) Google Scholar) (105 cells/well) with a final concentration of 0.1 μm moth cytochrome c peptide was performed as described (29Hassan N.J. Simmonds S.J. Clarkson N.G. Puklavec M.J. Hanrahan S. Bomb M. Barclay A.N. Brown M.H. Mol. Cell. Biol. 2006; 26: 6727-6738Crossref PubMed Scopus (72) Google Scholar). In all cellular experiments, triplicate samples were assayed, and the mean is displayed with S.E. Each experiment was performed at least four times.RESULTSCD244 Can Be Inhibitory in Killing by Murine NK Cells—To distinguish between the effects of CD244 and CD2 engagement by CD48 in rodents, we produced an anti-CD244 mAb that was more effective at blocking CD48/CD244 interactions than that characterized previously (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 2083-2090Crossref PubMed Scopus (350) Google Scholar). Of three mAbs that were specific for mouse CD244 and bound to a mouse T cell hybridoma transduced with mouse CD244 (Fig. 1b) (data not shown), one (mAb OX122) completely blocked binding of CD48-CD4d3+4-coated fluorescent beads to cells (30Wild M.K. Cambiaggi A. Brown M.H. Davies E.A. Ohno H. Saito T. van der Merwe P.A. J. Exp. Med. 1999; 190: 31-41Crossref PubMed Scopus (99) Google Scholar) expressing CD244 (Fig. 1c). mAb OX121 partially blocked binding of CD48-CD4d3+4-coated beads, similar to a mAb used in our previous study (11Brown M.H. Boles K. van der Merwe P.A. Kumar V. Mathew P.A. Barclay A.N. J. Exp. Med. 1998; 188: 208

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