New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay

Background: B cell receptor (BCR) clusters modulate BCR signaling in B-lymphocytes. Results: We used a quantitative proteomic proximity assay to analyze the BCR cluster in DT40 cells. Conclusion: Our proximity labeling assay identified novel components of the BCR cluster linked to integrin signaling. Significance: We provide new insights into BCR assembly and identify new and unexpected targets for further functional analysis. In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30–40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.

• Publication year

  2014

• Venue

  Journal of Biological Chemistry

• Publication date

  2014-04-04

• Fields of study

  Biology, Medicine

• Identifiers
  DOI 10.1074/jbc.M113.529578PMID 24706754PMCID 4031500
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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