Abstract
Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction1. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli2,3. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-ε (IgE) receptor FcεRI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of FcεRI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to FcεRI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.
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Acknowledgements
We thank J. Lawitts for blastocyst injections; V. Petkova for help with Real Time PCR analysis; D. P. Liu for technical assistance; and. L. C. Cantley and C. Carpenter for comments. This work was supported by grants from the N.I.H. (B.G.N, J.-P.K. and B.L.). H.G. was supported by fellowships from the N.I.H. and the American Association for Cancer Research. J.C.P. was supported by grants from the Cancer League of Colorado and the Association for International Cancer Research.
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Targeted disruption of the murine Gab2 gene. a, Targeting strategy. A P1 clone containing exon 1 of murine Gab2 was purchased from Genome Systems. The 10.5 kb EcoRI DNA fragment harboring this exon was excised, subcloned, and restriction-mapped (Supp. Fig.1). A targeting vector was designed to replace the first exon with an SAbgal pgkneo cassette, using the Loxp-SAbgal pgkneo-loxp-DTA plasmid as the backbone (provided by Sheila Thomas, BIDMC, Boston). Further details are available from H.G. upon request. b, Southern blot of EcoRI-digested genomic DNA with the probe indicated as "-P". Gab2 targeting construct was electroporated into J1 ES cells (provided by Dr. Arlene Sharpe, Brigham and Women’s Hospital, Boston). Proper targeting was verified in 6 clones (out of 100 G418R clones) by Southern blot. ES clone 14 with an unrearranged Gab2 locus shows only the 9.4 kb endogenous band. ES clones (23, 31, 68) shown exhibit homologous recombination indicated by the additional 6.5 kb band. c, Gab2-/- mice are viable. Two independent targeted clones were injected into C57/Bl6 blastocysts. High percentage chimeras were bred to wild type C57/Bl6 mice or 129Sv mice to obtain Gab2+/-mice (Sv129 X C57/Bl6 or 129Sv, respectively). Gab2 -/- mice were generated interbreeding Gab2 +/- mice. PCR genotyping reveals Gab2+/+, Gab2+/- and Gab2-/- mice. The protocol for genotyping Gab2 mice is available from HG upon request. d, Gab2 protein is undetectable in tissues of Gab2-/- mice. Gab2 immunoprecipitates from lysates (1 mg) of brain (B), spleen (S), and testis (T) were immunoblotted with anti-Gab2 antibodies. Similar results were obtained with direct Gab2 immunoblots from liver, lung, heart and muscle tissues. Lysates from each of the same samples were immunoblotted with anti-Shp-2 antibodies to control for loading.
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Gu, H., Saito, K., Klaman, L. et al. Essential role for Gab2 in the allergic response. Nature 412, 186–190 (2001). https://doi.org/10.1038/35084076
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DOI: https://doi.org/10.1038/35084076
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