Comparison of phosphorylation and internalization of the antigen receptor/CD3 complex, CD8, and class I MHC-encoded proteins on T cells. Role of intracytoplasmic domains analyzed with hybrid CD8/class I molecules.
We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.
Boyer
C
C
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, France.
Auphan
N
N
Gabert
J
J
Blanc
D
D
Malissen
B
B
Schmitt-Verhulst
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism* / Q000502:physiology; D016827:CD8 Antigens; D002999:Clone Cells / Q000378:metabolism / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism / Q000502:physiology; D006183:H-2 Antigens / Q000378:metabolism* / Q000502:physiology; D006825:Hybridomas / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D000945:Antigens, Differentiation, T-Lymphocyte; D016827:CD8 Antigens; D006183:H-2 Antigens; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins