Proteomic Characterization of Plasma Membrane-proximal T Cell Activation Responses

Ben De Wet,Tobias Zech,Mogjiborahman Salek,Oreste Acuto,Thomas Harder

doi:10.1074/jbc.m110.165415

Abstract

Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.

Highlights

T cell activation is triggered by T cell antigen receptor (TCR)2 signals that are induced by its interaction with a cognate peptide-MHC presented on the surface of an antigen-presenting cell or target cell
Bead-cell conjugates were mechanically homogenized by nitrogen cavitation, and plasma membrane fragments harboring TCR-proximal signaling complexes were recovered with a magnet
We present the first proteomic characterization of an isolated physical network of cell surface receptor signaling proteins anchored in its native plasma membrane lipid bilayer platform

Summary

Introduction

T cell activation is triggered by T cell antigen receptor (TCR)2 signals that are induced by its interaction with a cognate peptide-MHC (pMHC) presented on the surface of an antigen-presenting cell or target cell. Downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR.

Results

Conclusion