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Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

¹ School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
² Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853

Correspondence to Barbara A. Baird: bab13{at}cornell.edu; or Watt W. Webb: www2{at}cornell.edu

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein–protein interactions in intact, living cells.

D.R. Larson's present address is Dept. of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.

Abbreviations used in this paper: FCS, fluorescence correlation spectroscopy; huIgE, human IgE; ITAM, immunoreceptor tyrosine-based activation motif; moIgE, mouse monoclonal DNP-specific IgE; RBL, rat basophilic leukemia.

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