Subcellular localization of T-cell receptor complexes containing tyrosine-phosphorylated zeta proteins in immature CD4+CD8+ thymocytes.
The T-cell antigen receptor (TCR) is a complex of at least six different proteins (alpha, beta, gamma, delta, epsilon, and zeta) that is assembled in the endoplasmic reticulum (ER) and transported to the cell surface. Unlike mature T cells, most immature CD4+CD8+ thymocytes retain within the ER and degrade greater than 90% of some of the TCR components they synthesize, resulting in low surface expression of TCR complexes. The few surface TCR complexes that most immature CD4+CD8+ thymocytes do express are only marginally capable of transducing signals mobilizing intracellular calcium. The inverse relationship with TCR expression and function suggested that phosphorylated zeta (P-zeta) molecules might function in CD4+CD8+ thymocytes either as an ER retention signal for newly synthesized TCR complexes or as a negative regulatory modification of TCR complexes present on the cell surface. The present study sought to evaluate these two possibilities by determining the subcellular location of TCR complexes containing P-zeta chains. We found that, unlike unmodified zeta chains, all P-zeta chains in CD4+CD8+ thymocytes existed in assembled TCR complexes and that all TCR complexes containing P-zeta molecules had undergone carbohydrate processing events indicative of transit through the Golgi apparatus. These results demonstrate that P-zeta chains are exclusively associated with mature TCR complexes, excluding the possibility that P-zeta serves as an ER retention signal in immature thymocytes. Although we could not directly determine the representation of P-zeta chains among surface TCR complexes, we found that 60-70% of surface TCR complexes on immature CD4+CD8+ thymocytes were associated with tyrosine-phosphorylated protein(s) and that this percentage was inversely correlated with their signaling competence. These results support the concept that tyrosine phosphorylation serves as a negative regulatory modification of certain TCR-associated proteins.
Kearse
K P
KP
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Wiest
D L
DL
Singer
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens / Q000276:immunology*; D016827:CD8 Antigens / Q000276:immunology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000302:isolation & purification; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D013950:Thymus Gland / Q000276:immunology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D006023:Glycoproteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009844:Oligosaccharides; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D019000:Phosphotyrosine; D014443:Tyrosine