Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells.
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, 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-FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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.
Larson
Daniel R
DR
School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Gosse
Julie A
JA
Holowka
David A
DA
Baird
Barbara A
BA
Webb
Watt W
WW
D000818:Animals; D015550:Cell Degranulation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000502:physiology*; D008407:Mast Cells / Q000502:physiology; D021962:Membrane Microdomains / Q000502:physiology; D051379:Mice; D010766:Phosphorylation; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D017455:Receptors, IgE; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007073:Immunoglobulin E; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases