Analysis of the individual role of the TCRzeta chain in transgenic mice after conditional activation with chemical inducers of dimerization.
Signaling through the TCR/CD3 complex plays a critical role in T-cell development and activation. Gene-targeted mice lacking particular components of this complex show arrested T-cell development in the thymus. As all TCR/CD3 components are required for efficient surface expression of the complex, it is difficult to assess the specific signaling role of each receptor component. To overcome this problem, we designed a strategy to examine the specific role(s) of individual receptor chains. A chimeric protein, containing binding domains for chemical inducers of dimerization fused to the cytoplasmic tail of TCRzeta, was generated. Activation of the chimeric receptor after stimulation with chemical dimerizers in Jurkat cells showed tyrosine phosphorylation of the TCRzeta chain chimera, recruitment of phosphorylated Zap70, and generation of NFAT in a reporter assay. Analysis of thymocytes from transgenic mice expressing this chimeric receptor showed that intracytoplasmic crosslinking of the chimera induced tyrosine phosphorylation of the protein, as well as a slow and very weak calcium mobilization response. However, this signaling did not lead to increased expression of activation markers, T-cell proliferation, or apoptosis. In addition, stimulation of thymocytes in suspension or in fetal thymic organ cultures with chemical inducers of dimerization did not lead to alterations in positive or negative selection. We conclude that signaling through the TCRzeta chain alone is not sufficient to generate downstream events leading to full T-cell activation or thymocyte selection; instead, additional CD3 components must be required to induce a functional response in primary thymocytes and peripheral T cells.
Soldevila
G
G
Departamento de Inmunología, Universidad Nacional Autónoma de México, México DF, México. soldevi@servidor.unam.mx
Castellanos
C
C
Malissen
M
M
Berg
L J
LJ
D020816:Amino Acid Motifs; D000818:Animals; D017637:Clonal Deletion; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009924:Organ Culture Techniques; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics; D013950:Thymus Gland / Q000166:cytology / Q000196:embryology / Q000276:immunology; D014157:Transcription Factors / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
C119717:AP 1510; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C054979:antigen T cell receptor, zeta chain; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus