Subject(s)
Heterotrimeric GTP-Binding Proteins/physiology , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/physiology , Animals , Cell Membrane/physiology , Cell Separation/methods , Cells, Cultured , Guanosine Triphosphate/metabolism , Humans , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
We have investigated the role of the hematopoietically expressed G16 GTP-binding protein on T cell activation. We constructed transfectants of Jurkat T cells that express a function-deficient mutant of G alpha 16 predicted to prevent activation of this G protein. Upon stimulation with anti-CD3 epsilon antibodies, mutant G alpha 16 transfectants display a profound defect in the production of IL-2 and IL-10, as well as in the expression of CD69. In contrast, the phorbol 12-myristate 13-acetate (PMA)-induced IL-10 production and CD69 expression, and the ionomycin plus PMA-induced IL-2 production are not affected. Consistent with the reduction in cytokine production is the inhibition of early signaling events in the mutant G alpha 16-expressing cells. There are significant reductions in anti-epsilon-induced tyrosine phosphorylation of zeta, epsilon, ZAP-70, and phospholipase C gamma 1, as well as in intracellular Ca2+ mobilization. In accordance with the effects on tyrosine phosphorylation is the reduction of TCR/CD3-mediated Fyn and Lck activities in G alpha 16 mutant cells. Even though the mechanism through which the G alpha 16 mutant mediates inhibition of T cell activation is not known, the data suggest a model where G proteins become activated upon TCR/CD3 engagement and regulate the activation of tyrosine kinases and subsequent downstream signaling events that lead to the activation of cytokine genes.
Subject(s)
GTP-Binding Proteins/biosynthesis , GTP-Binding Proteins/genetics , Hematopoietic Stem Cells/metabolism , Mutagenesis, Site-Directed , Receptor-CD3 Complex, Antigen, T-Cell/physiology , Signal Transduction/immunology , Alanine/genetics , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , CD3 Complex/metabolism , Calcium/metabolism , Enzyme Activation/genetics , Gene Expression/immunology , Glycine/genetics , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/immunology , Humans , Interleukin-10/antagonists & inhibitors , Interleukin-10/biosynthesis , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Intracellular Fluid/enzymology , Intracellular Fluid/metabolism , Isoenzymes/metabolism , Jurkat Cells , Lectins, C-Type , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Phospholipase C gamma , Phosphorylation , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-fyn , Signal Transduction/genetics , Transfection/immunology , Type C Phospholipases/metabolism , Tyrosine/antagonists & inhibitors , Tyrosine/metabolismABSTRACT
Engagement of the T cell receptor (TCR).CD3 complex results in the induction of multiple intracellular events, with protein tyrosine kinases playing a pivotal role in their initiation. Biochemical studies also exist suggesting the involvement of heterotrimeric GTP-binding proteins (G proteins); however, the functional consequence of this participation in TCR.CD3-mediated signaling is unresolved. Here, we report TCR.CD3-mediated guanine nucleotide exchange among the 42-kDa G protein alpha subunits of the G alpha q/11 family, their physical association with CD3 epsilon, and the G alpha 11-dependent activation of phospholipase C beta. Protein tyrosine kinase inhibitors, however, abrogate TCR.CD3-mediated G protein activation. Quite interesting is the observation that cells transfected with a function-deficient mutant of G alpha 11 display diminished tyrosine phosphorylation of TCR.CD3 zeta and epsilon chains, as well as ZAP-70, upon anti-CD3 antibody triggering. These data indicate the involvement of the G alpha q/11 family in TCR.CD3 signaling at a step proximal to the receptor and suggest a reciprocal regulation between tyrosine kinases and G proteins in T cells.
Subject(s)
GTP-Binding Proteins/metabolism , Protein-Tyrosine Kinases/metabolism , Receptor-CD3 Complex, Antigen, T-Cell/physiology , Signal Transduction , T-Lymphocytes/physiology , Antibodies, Monoclonal , Base Sequence , Blotting, Western , Cell Line , Cell Membrane/metabolism , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/biosynthesis , Genistein , Guanosine Triphosphate/metabolism , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Isoflavones/pharmacology , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Phosphorylation , Phosphotyrosine/analysis , Protein-Tyrosine Kinases/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , T-Lymphocytes/immunology , TransfectionABSTRACT
In this report, we investigate G protein alpha subunit diversity in human thymocytes, utilizing common properties shared by these genes and reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis of PCR amplified gene portions, indicate the presence of members from all four G-protein families that have been described thus far. The alpha subunit genes identified are: G alpha i1-3 and G alpha z but not G alpha o from the Gi family, G alpha s from the Gs family, G alpha 11, G alpha q, and G alpha 16 from the Gq family, and G alpha 12 and G alpha 13 from the G12 family. Also in this report we present the nucleotide and predicted amino acid sequences of the human G alpha 13 cloned from a thymocyte cDNA library. The sequence of the human G alpha 13 has not been previously reported. Comparison of this sequence with the reported murine G alpha 13 shows > 90% identity at the deduced amino acid sequence level. We conclude that thymocytes represent a useful experimental system for the study of G protein involvement in immune responses and lymphocyte development.