Activation of T lymphocytes and the subsequent signal transduction events mediated through the TCR/CD3 molecule complex are important events in the immune response against tumors and in autoimmune diseases. Protein tyrosine kinases are intimately involved in T cell signaling through TCR/CD3. However, the role of heterotrimeric G proteins in this process is not clearly established. Therefore, in the present application we are proposing to study the involvement of G proteins in T cell signal transduction. We have observed that TCR/CD3 ligation induces the interaction between G proteins and the CD3 chain. We propose to study this interaction by the use of chimeras and mutants of the intracytoplasmic tail of the CD3 epsilon chain in an attempt to define the critical sites involved in this physical association. We will also use synthetic peptides representing specific sites on G alpha subunits, as well as point mutants of G alpha proteins to determine whether they can interfere with the G alpha-CD3 epsilon association. Co-immunoprecipitation and western blotting analyses with specific antibodies, and CD3 epsilon-GST fusion proteins will be additional tools utilized in order to address this issue. We have also observed that upon TCR/CD3 perturbation, G proteins associate with tyrosine kinases and they regulate their function. We proposed to study this process by focusing on the specific tyrosine kinase EMT, a Tec family member. We will examine the TCR/CD3-mediated physical association and the functional consequences of EMT - G beta and EMT-CD3 epsilon interactions. We will utilize truncation mutants of EMT in order to determine sites that are important for these interactions. The association of src kinases fyn and lck with the EMT-G beta-CD3 epsilon complex will be assessed, and the consequences of this association on src kinase activity will be examined. The effects of G protein function-deficient mutants on the above processes will be also determined. The above analyses will be extended to human thymocytes and peripheral T cells in order to better establish the biological relevance of these events.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM056374-01
Application #
2385691
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1997-09-30
Project End
2000-09-29
Budget Start
1997-09-30
Budget End
1998-09-29
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
San Diego State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
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Ching, K A; Grasis, J A; Tailor, P et al. (2000) TCR/CD3-Induced activation and binding of Emt/Itk to linker of activated T cell complexes: requirement for the Src homology 2 domain. J Immunol 165:256-62
Tsoukas, C D; Stanners, J; Ching, K A (2000) Activation of heterotrimeric GTP-binding proteins upon TCR/CD3 engagement. Methods Mol Biol 134:319-24
Ching, K A; Kawakami, Y; Kawakami, T et al. (1999) Emt/Itk associates with activated TCR complexes: role of the pleckstrin homology domain. J Immunol 163:6006-13
Zhou, J; Stanners, J; Kabouridis, P et al. (1998) Inhibition of TCR/CD3-mediated signaling by a mutant of the hematopoietically expressed G16 GTP-binding protein. Eur J Immunol 28:1645-55