Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):E11914-E11923. doi: 10.1073/pnas.1817142115. Epub 2018 Dec 3.


The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

Keywords: T cell antigen receptor; phospholipase C; protein complex; signal transduction; tyrosine phosphorylation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Cell Membrane / metabolism
  • Humans
  • Membrane Lipids / metabolism
  • Membrane Proteins / metabolism
  • Multiprotein Complexes / metabolism*
  • Phospholipase C gamma / metabolism
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Binding*
  • Protein Interaction Domains and Motifs*
  • Receptors, Antigen, T-Cell / metabolism*
  • Recombinant Proteins
  • Signal Transduction / immunology*
  • Thermodynamics
  • Tissue Plasminogen Activator / metabolism
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / metabolism*


  • Adaptor Proteins, Signal Transducing
  • GRAP2 protein, human
  • Membrane Lipids
  • Membrane Proteins
  • Multiprotein Complexes
  • Phosphoproteins
  • Receptors, Antigen, T-Cell
  • Recombinant Proteins
  • SLP-76 signal Transducing adaptor proteins
  • Tumor Necrosis Factor Receptor Superfamily, Member 7
  • Phospholipase C gamma
  • PLAT protein, human
  • Tissue Plasminogen Activator