|M.Sc Student||Ifrach Talia|
|Subject||Function and Regulation of Gads in the T cell Receptor and|
FC Epsilon Receptor Signaling Pathways
|Department||Department of Medicine||Supervisor||Dr. Deborah Yablonski|
|Full Thesis text|
Many cells derived from the hematopoietic lineage collaborate to generate the defense role of the immune system. T and mast cells are major players in adaptive and innate immune systems, mediating inflammatory reactions and immune-tolerance maintenance. Activation of these cells occurs mostly through antigen-recognition receptors, the best-known of which are the T-cell receptor (TCR) of T cells, and the high-affinity immunoglobulin-E receptor (FcεR1) of mast cells. Since the responses of T and mast cells are very potent and sometimes irreversible, they are highly regulated by positive and negative feedback loops.
Antigen-receptor activation is initiated with extracellular stimulation which is transduced into intracellular signals and transmitted to the nucleus. The transduction of the signal is accomplished by the cooperation of signaling molecules, which are governed by interactions and post translational modifications (PTM) to assemble a wide and complex signaling network.
Grb2-related adaptor downstream of Shc (Gads) is an adaptor protein, which is used as a molecular scaffold and has no enzymatic activity; instead, it mediates protein-protein interactions that are highly important in TCR and FcεR1 signaling pathways. Gads is a member of the Grb2 family of adaptor proteins, and is composed of two SH3 domain flanked by a central SH2 domain and a proline-rich linker. Gads interacts with signaling molecules in the cell via its domains to promote signal transduction. The interaction of Gads with linker for activation of T-cells (LAT) via its SH2 domain, and with the SH2 domain-containing leukocyte protein of 76 kDa (SLP76) via its C-terminal SH3 domain, assemble the important LAT-signaling complex. Although the function of Gads through its structural domains is well studied, its precise regulatory role in antigen-receptor signaling is still not fully clear.
Here we investigated the functional significance of unstudied regions and motifs in Gads structure-domains, and their influence on TCR and FcεR1 signaling. We examined the influence of Gads linker, and further investigated the role of the N-SH3 domain and F92-dimerization site on Gads activity in cell function. We compared the function of T and mast cells expressing WT Gads to those expressing different mutants of Gads.
Deletion of a conserved region in Gads linker increased activation of signals downstream of TCR. We suggest that this sequence contain a single motif, based on a previously-studied threonine residue, which acts to down regulate Gads activity.
An F92D mutation, which abolishes Gads dimerization and disrupts TCR-induced signaling events, was found to interrupt FcεR1 signaling as well, confirming the importance of dimerization in Gads activation.