An unusual mechanism for ligad antagonism

Science

Volumn 281, Issue 5376, 1998, Pages 568-572

  Torigoe, Chikako ^a   Inman, John K ^b   Metzger, Henry ^a  

^a NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES   (United States)

^b NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

2,4 DINITROPHENOL; CELL SURFACE RECEPTOR; FC RECEPTOR; IMMUNOGLOBULIN E; IMMUNOGLOBULIN E ANTIBODY; DANSYL CHLORIDE; ENZYME PRECURSOR; FOCAL ADHESION KINASE 2; HAPTEN; IMMUNOGLOBULIN E RECEPTOR; LIGAND; MITOGEN ACTIVATED PROTEIN KINASE 1; NCK PROTEIN; ONCOPROTEIN; PHOSPHOTYROSINE; PROTEIN KINASE (CALCIUM,CALMODULIN); PROTEIN KINASE SYK; PROTEIN TYROSINE KINASE; PTK2B PROTEIN, RAT; SIGNAL PEPTIDE;

ANIMAL CELL; ANTIGEN RECOGNITION; ARTICLE; BINDING AFFINITY; BINDING KINETICS; LIGAND BINDING; MAST CELL; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; RAT; RECEPTOR BINDING; ANIMAL; ANTIBODY AFFINITY; ANTIGEN ANTIBODY REACTION; CAPPING PHENOMENON; CELL CULTURE; IMMUNOLOGY; METABOLISM; PHOSPHORYLATION; SIGNAL TRANSDUCTION; T LYMPHOCYTE;

ANIMALIA;

2,4-DINITROPHENOL; ANIMALS; ANTIBODY AFFINITY; ANTIGEN-ANTIBODY REACTIONS; CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; DANSYL COMPOUNDS; ENZYME PRECURSORS; FOCAL ADHESION KINASE 2; HAPTENS; IMMUNOGLOBULIN E; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; LIGANDS; MAST CELLS; MITOGEN-ACTIVATED PROTEIN KINASE 1; ONCOGENE PROTEINS; PHOSPHORYLATION; PHOSPHOTYROSINE; PROTEIN-TYROSINE KINASES; RATS; RECEPTOR AGGREGATION; RECEPTORS, IGE; SIGNAL TRANSDUCTION; T-LYMPHOCYTES; TUMOR CELLS, CULTURED;

EID: 0032562995     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5376.568     Document Type: Article

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52. RBL-2H3 cells were incubated with anti-DNP IgE, and, after washing, 6.2 × 106 cells/ml were incubated at 25°C in a buffer (9) containing Fab fragments (50 ng/ml) conjugated with DNP-or 2NP-caproate (10). Reactions were stopped by transferring cells directly into solubilization buffer at 0°C. All the methods used to analyze specific proteins for phosphotyrosine have been described (33). Antibodies not previously cited were anti-PLC γ1 from Upstate Biotechnology (Lake Placid, NY), a mixture of five monoclonal antibodies that cross reacts with Nck [D. Park and S. G. Rhee, Mol. Cell. Biol. 12, 5816 (1992)]; goat polyclonal anti-Erk2 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal IgG1 anti-Pyk2 (Transduction Laboratories, Lexington, KY); biotin-conjugated mouse monoclonal IgG2b anti-phosphotyrosine, 4G10 (Upstate Biotechnology); and horseradish peroxidase-conjugated avidin (Sigma). The cell equivalents analyzed were adjusted so that the samples would give approximately similar exposures on the autophotographs.
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54. Cells reacted with bispecific anti-DNP/anti-DNS IgE (0.3 μg/ml) (10) (a fivefold to sixfold excess of IgE per receptor) for ≈16 hours were washed and reacted with the alternative ligands at 28°C. After 30 min, release of hexosaminidase was measured. Cells in separate wells were stimulated identically and analyzed for phosphotyrosine after 3 min. The doses of ligand (adjusted to induce ample phosphorylation of the receptors) were 10, 100, and 2500 ng/ml for the DNP-, oDNCP-, and 2NP-protein conjugates, respectively. Separate experiments confirmed that higher doses of the oDNCP and 2NP conjugates gave no additional release. The experiments were conducted at a lower than optimal temperature (34) because of the unusual difference in the temperature response of the cells to the two ligands: Whereas the DNP conjugate stimulated increased phosphorylation with increasing temperature between 0° and 37°C, the 2NP conjugate yielded decreased phosphorylation above 15°C, although the ratio of modification in γ compared with β was always around 2:1 (35).
55. We thank D. Holowka and B. Baird for gifts of purified DNS-BSA and anti-DNS IgE, for quadroma cells, and for advice on preparing the bispecific antibody. 11 May 1998; accepted 24 June 1998