|M.Sc Student||Shalah Almour Rose|
|Subject||Regulatory Effects of Gads Domains in the T Cell Receptor|
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text|
T cells are a major component of the adaptive immune system, which require at least two signals for activation, T cell receptor (TCR) and co-stimulatory receptors. Both signals are integrated and are highly regulated by negative and positive feedback loops to precisely control T cell activity.
Gads is a Grb2 family adaptor proteins, which is composed of two SH3 domains connected by a central SH2 domain and a proline-rich linker and plays an important role in T cell signal transduction pathways. Recent work in our lab showed that the SH2 domain of Gads can dimerize and that the N terminal SH3 domain of Gads may negatively regulate T cell activation.
In order to investigate the role of Gads SH2 dimerization in the TCR signaling pathway, we stably reconstituted a Gads-deficient cell line with Gads alleles that were either wild-type, or bearing mutations that inactivate the SH2 dimerization interface. Mutation of the SH2 dimerization interface abolished TCR-induced association of Gads with pLAT, markedly impaired TCR-induced phosphorylation of PLC-ɣ1 and impaired TCR-induced CD69 expression. Together, these results suggest an important role of Gads SH2 domain dimerization in TCR signaling.
Given the marked effect of the SH2 dimerization interface on the interaction of Gads with LAT, we were interested in studying the regulation of this interaction in intact Jurkat cells. The idea was to create a cell line in which Gads and LAT are fused to different fluorescent proteins. Because of limited time, we created a cell line in which Gads protein is fused to CFP at its C terminal by CRISPR method. Using the CRISPR method, we found that the expression level of Gads in the different colonies is comparable to the expression level of endogenous Gads in Jurkat cells, which we have not been able to achieve by other genetic reconstitution methods. In addition, we optimized the CRISPR technique in Jurkat cells for future studies.
To probe the role of SH3 N-terminal domain of Gads, we created cell lines expressing WT, ∆N and Y45F Gads using three different constructs; one with an IRES sequence in which Gads and GFP are not fused together, a second one in which GFP is directly fused to the C terminus of Gads and a third in which YFP is directly fused to the N terminus of Gads. Removal of the N terminal SH3 domain increased TCR-induced CD69 expression and RE/AP transcription activity in IRES-GFP and fused-GFP constructs. Mutation at Y45 slightly decreased TCR-induced CD69 expression and RE/AP transcription activity in IRES-GFP construct and slightly increased TCR-induced CD69 expression and RE/AP transcription activity in fused-GFP construct. In N-term YFP construct, TCR-induced CD69 expression was similar in all the cell lines and RE/AP transcription activity increased upon removal of the N terminal SH3 domain but was not affected upon mutation of Y45. Based on the differences between the constructs, we deduced that tagging Gads with a fluorescent protein disrupts the regulatory functions of its N terminal SH3 domain which mediates a negative role in T cell signaling.