טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentWaknin Cecilia
SubjectGads Dimerization and its Implications for TCR Signaling
DepartmentDepartment of Medicine
Supervisor Dr. Deborah Yablonski
Full Thesis textFull thesis text - English Version


Abstract

Generation of multiprotein complexes at adaptor proteins is essential for the activation of intracellular signaling pathways. In T lymphocytes, linker for activation of T cells (LAT), a membrane-bound adaptor protein, is the central adaptor protein used as a nucleating site. Upon T cell receptor activation, LAT is tyrosine-phosphorylated, creating docking sites for other signaling molecules such as PLC-γ1, Grb2 and Gads. In this study, we used biochemical and biophysical methods to examine the adaptor protein Gads and its binding to phosphorylated LAT.
Gads is a member of the Grb2 family of adaptor proteins. It contains a central SH2 domain and a linker region flanked by amino- and carboxy-terminal SH3 domains. Through its SH2 domain, Gads binds LAT at phosphotyrosines 171 and 191, bringing with it the adaptor protein SLP-76, which binds to the carboxy-terminal SH3 domain of Gads. Gads has a crucial role in T cell signaling, by promoting the formation of a protein complex containing LAT and SLP-76, which couples the upstream signaling to various downstream signals.

We expressed and purified recombinant WT Gads which purified as monomers and higher oligomers, probably dimers. Gads monomers and dimers were shown to exist in equilibrium. This equilibrium shifted completely to the dimer when Gads protein was incubated with a molar excess of LAT peptide doubly phosphorylated on tyrosines 171 and 191. This finding suggests that the dimeric form is preferred and stabilized upon binding to doubly phosphorylated LAT.

A series of domain deletions revealed that the minimum interface for Gads dimerization is the SH2 domain. Gads bearing a point mutation at Arginine 109 failed to form either spontaneous or inducible dimers.

Our results suggest that dimerization is mediated at least partly by electrostatic interactions, since disruption of a putative hydrogen bond between two Gads monomers by mutation of Arginine 109 disrupted Gads dimerization. 

SH2 domains are generally known to have one active site for binding one phosphotyrosine. However additional active sites were reported in few other cases, including an interface for binding to another protein, or for SH2 homodimerization, as in our case. Dimerization might be a way to regulate Gads recruitment to LAT once T cells are activated.  According to ITC experiments, the affinity of dimeric Gads to the doubly phosphorylated LAT is about 40nM, about 4-fold higher affinity than the reported affinity of Gads for single-phosphorylated peptides.  Therefore Gads dimerization may promote sustained recruitment of SLP-76 to LAT.