Figure 5. The N-terminal SH3 domain of Grb2 mediates enhanced Ca2+ signalling downstream of the ITT.
(a) Schematic illustration of chimeric receptor variants used in this study. Chimeric constructs consisting of mouse YF-mutant γ2am fused to the N-terminal (b) or C-terminal (c) SH3 domains of Grb2 were retrovirally expressed in DG75 B cells. The SH3 domains were either wild type (wt, blue curves) or inactivated by tryptophan to lysine (W36K and W193K, respectively, red curves) or a phenylalanine to alanine (F165A, orange curve) mutations. Cells were either stimulated with anti-IgG or anti-IgM F(ab′)2 fragments (indicated by arrows) and Ca2+ mobilization was recorded in the presence of 1 mM extracellular CaCl2. (d) Chimeric γ2am-YF-chimeras containing the N-terminal SH3 domain of either Grb2 (wt: dark blue line, W36K: dark red line) or GRAP (wt: light blue line, W36K: light red line) were retrovirally expressed in murine splenic B cells and analysed as before. Data are representative of four independent experiments.