An activating immunoreceptor complex formed by NKG2D and DAP10

Science

Volumn 285, Issue 5428, 1999, Pages 730-732

  Wu, Jun ^a   Song, Yaoli ^a   Bakker, Alexander B H ^a   Bauer, Stefan ^b   Spies, Thomas ^b   Lanier, Lewis L ^a   Phillips, Joseph H ^a  

^a MERCK RESEARCH LABORATORIES   (United States)

^b Fred Hutchinson Cancer Research Center   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADAPTOR PROTEIN; CELL RECEPTOR; MAJOR HISTOCOMPATIBILITY ANTIGEN CLASS 1; PHOSPHATIDYLINOSITOL 3 KINASE; RECEPTOR SUBUNIT;

ARTICLE; BINDING SITE; COMPLEX FORMATION; HUMAN; LYMPHOCYTE ACTIVATION; NATURAL KILLER CELL; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; RECEPTOR BINDING; SEQUENCE HOMOLOGY; SIGNAL TRANSDUCTION; T LYMPHOCYTE;

1-PHOSPHATIDYLINOSITOL 3-KINASE; AMINO ACID SEQUENCE; ANIMALS; BINDING SITES; CELL LINE; CYTOTOXICITY, IMMUNOLOGIC; HUMANS; KILLER CELLS, NATURAL; LIGANDS; LYMPHOCYTE ACTIVATION; MEMBRANE PROTEINS; MICE; MOLECULAR SEQUENCE DATA; NEOPLASMS; PHOSPHORYLATION; PHOSPHOTYROSINE; RECEPTORS, IMMUNOLOGIC; SIGNAL TRANSDUCTION; SRC HOMOLOGY DOMAINS; T-LYMPHOCYTES; TUMOR CELLS, CULTURED;

EID: 0033618624     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5428.730     Document Type: Article

References (28)

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21. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
22. Northern and Southern analysis using human DNA and RNA (Clontech) were performed as described (4).
23. Rabbits were immunized with peptides corresponding to the cytoplasmic domain of either human DAP10 (PAQEDGKVYINMPGRG) (Anaspec, San Jose, CA) or human NKG2D (KKSDFSTRWQKQR) (Research Genetics, Birmingham, AL) (13). Antisera were affinity-purified with the immunizing peptide. Specificity was determined by testing the affinity-purified antibodies by immunoprecipitation and protein immunoblot analysis on Ba/F3 cells transfected with human DAP10 and NKG2D (or both), compared with preimmune sera or control antibodies.
24. 3 (pH 7.8), and protease inhibitors] (4). Lysates were incubated (2 hours, 4°C) with Pansorbin (Calbiochem) coated with clg, affinity-purified anti-DAP10, and affinity-purified anti-NKG2D. For immunoprecipitation with mouse mAb, Pansorbin was precoated with rabbit antibody to mouse Ig (Sigma) and mouse NKG2D mAb 5C6, anti-Flag mAb M2 (Sigma), or clg. Immunoprecipitates were washed in buffer [5 mM CHAPS, 50 mM tris, 150 mM NaCl (pH 8.0), and protease inhibitors] (4).
25. A cDNA containing the human CD8 leader segment, followed by the Flag epitope (DYKDDDDK), and joined to the extracellular, transmembrane, and cytoplasmic segments of human DAP10, was subdoned into the pMX-neo retroviral vector (4, 13). Human NKG2D cDNA (8) was subdoned into pMX-puro. The D-A transmembrane Flag-DAP10 mutant cDNA with an A (GCC) substituted for D (GAT) and the R-L transmembrane NKG2D mutant cDNA with an L (CTT) substituted for R (CGT) were generated by PCR mutagenesis. Retroviruses were generated using the Phoenix packaging cell (4). Ba/F3 cells were infected and drug-selected, and transfectants were isolated by flow cytometry (4).
26. Phosphorylated or unphosphorylated biotinylated peptides of the cytoplasmic domain of DAP10 (PAQEDGKVYINMPGRG) were incubated with lysates from NKL, precipitated with avidin-agarose, and washed [1% NP-40, 50 mM tris, 150 mM NaCl (pH 7.8), and protease inhibitors) (4, 13). Immunoprecipitates were analyzed by protein immunoblot using either anti-p85 or anti-Shc (specificity control) (UBI, Lake Placid, NY).
27. + Ba/F3 cells were incubated with 100 mM pervanadate and lysed (0.875% Brij 97, 0.125% NP-40, 150 mM NaCl, 10 mM tris, and protease and phosphatase inhibitors) (4). Antigens precipitated with clg, Flag mAb M2, or NKG2D mAb 5C6 were analyzed by protein immunoblot using horseradish peroxidase (HRP)-conjugated phosphotyrosine mAb 4G10 (UBI) or antiserum to p85 (UBI).
28. We thank M. Robertson, J. P. Houchins, C. Turck, G. Nolan, and T. McClanahan for reagents; J. Katheiser, G. Burget, and M. Andonian for graphics; J. Cupp, E. Callas, and D. Polakoff for flow cytometry; and D. Gorman and D. Liggett for sequencing. DNAX is supported by Schering Plough. S.B. and T.S. are supported by NIH grant AI30581.