The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

• Published in: The Journal of biological chemistry Vol. 286; no. 37; pp. 31993 - 32001
• Main Authors: James, John R., McColl, James, Oliveira, Marta I., Dunne, Paul D., Huang, Elizabeth, Jansson, Andreas, Nilsson, Patric, Sleep, David L., Gonçalves, Carine M., Morgan, Sara H., Felce, James H., Mahen, Robert, Fernandes, Ricardo A., Carmo, Alexandre M., Klenerman, David, Davis, Simon J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.09.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Bioluminescence Resonance Energy Transfer; CD4 Antigens - immunology; CD4 Antigens - metabolism; Cell Membrane - immunology; Cell Membrane - metabolism; Cell Surface; Cytosol - immunology; Cytosol - metabolism; HEK293 Cells; HLA Antigens - immunology; HLA Antigens - metabolism; Humans; Immunology; Jurkat Cells; Leukocyte Common Antigens - immunology; Leukocyte Common Antigens - metabolism; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - immunology; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism; Membrane Proteins; Models, Immunological; Natural sciences; Naturvetenskap; Protein Stoichiometry; Protein Structure; Receptors; Receptors, Antigen - immunology; Receptors, Antigen - metabolism; Signal Transduction; Signal Transduction - immunology; Single Molecule Biophysics; T Cell Receptor; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; Membrane Proteins; Bioluminescence Resonance Energy Transfer; Receptors; Cell Surface; Signal Transduction; Single Molecule Biophysics; T Cell Receptor; Protein Structure; Protein Stoichiometry
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M111.219212

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.