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The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer

Abstract

Binding of antigen to the B cell antigen receptor (BCR) triggers signaling that ultimately leads to B cell activation. Using quantitative fluorescence resonance energy transfer imaging, we provide evidence here that the BCR is a monomer on the surface of resting cells. Binding of multivalent antigen clustered the BCR, resulting in the simultaneous phosphorylation of and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation required immunoreceptor tyrosine-activation motif and continuous Src family kinase activity but not binding of the kinase Syk. Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by Src family kinase activity.

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Figure 1: The function and stoichiometry of the cell surface BCR composed of CFP- and YFP-containing chains.
Figure 2: FRET between BCR cytoplasmic domains measured by sensitized acceptor emission.
Figure 3: FRET between the cytoplasmic domains of the BCR chains in resting B cells.
Figure 4: Antigen-induced clustering of BCR extracellular domains.
Figure 5: Dynamic antigen-induced changes in FRET between the BCR intracellular domains.
Figure 6: The open conformation of the BCR cytoplasmic chains requires phosphorylation of the Ig-α and Ig-β ITAMs by Src kinases.
Figure 7: Syk activity and binding is not required for the drop in FRET, but recruitment of Syk correlates with the change in FRET.

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Acknowledgements

We thank T. Jin for the initial setup of the imaging system and J. Coligan for help with transfections. Supported by the Intramural Research program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.

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Correspondence to Susan K Pierce.

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Tolar, P., Sohn, H. & Pierce, S. The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer. Nat Immunol 6, 1168–1176 (2005). https://doi.org/10.1038/ni1262

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