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Intracellular Single-Chain Variable Fragments Directed to the Src Homology 2 Domains of Syk Partially Inhibit FcRI Signaling in the RBL-2H3 Cell Line¹

Stéphanie Dauvillier^*, Peggy Mérida^*, Michela Visintin, Antonino Cattaneo, Christian Bonnerot and Piona Dariavach^2,*

^* Institut de Génétique Moléculaire de Montpellier, Unité Mixte de Recherche 5535 Centre National de la Recherche Scientifique, Montpellier, France; International School for Advanced Studies, Neuroscience Program, Trieste, Italy; and Institut Curie, Unité 520, Institut National de la Santé et de la Recherche Médicale, Paris, France

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcRI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton’s tyrosine kinase and phospholipase C-2 tyrosine phosphorylation and activation. Interestingly, FcRI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-2 activation.

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