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Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing

J J Perez-Villar1, G S Whitney, M A Bowen

  • 1Immunology and Inflammation Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA. perezvj@bms.com

Molecular and Cellular Biology
|March 19, 1999
PubMed

Insights

CD5 negatively regulates T-cell receptor signaling by binding the SHP-1 phosphatase. This interaction, mediated by CD5

Area of Science:

  • Immunology
  • Cell Signaling
  • Molecular Biology

Background:

  • Previous studies suggested CD5 negatively regulates T-cell and B-cell receptor signaling.
  • The precise mechanism of CD5-mediated regulation remained unclear.

Purpose of the Study:

  • To elucidate the molecular mechanism by which CD5 negatively regulates T-cell receptor (TCR) signaling.
  • To identify the specific interactions and signaling pathways involved in CD5 function.

Main Methods:

  • Co-immunoprecipitation assays to identify interacting proteins with CD5.
  • T-cell stimulation assays with and without CD5 cross-linking.
  • Calcium mobilization assays.
  • Western blotting to assess protein tyrosine phosphorylation.
  • Site-directed mutagenesis of CD5 intracellular tyrosine residues.

Main Results:

  • CD5 constitutively associates with the phosphotyrosine phosphatase SHP-1 in T cells.
  • CD5-SHP-1 interaction is enhanced upon TCR-CD3 stimulation.
  • CD5 co-cross-linking with TCR-CD3 down-regulates calcium mobilization and tyrosine phosphorylation of key signaling molecules, including CD3zeta, ZAP-70, Syk, and phospholipase Cgamma1.
  • A specific tyrosine residue at position 378 (Y378) in CD5's ITIM-like motif is critical for SHP-1 binding and CD5's inhibitory function.

Conclusions:

  • CD5 functions as a negative regulator of TCR signaling through its association with SHP-1 phosphatase.
  • The CD5 ITIM-like motif at Y378 is essential for recruiting SHP-1 and mediating signal down-regulation.
  • These findings reveal a key mechanism for controlling T-cell activation thresholds.

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