Grb2 directly binds to HGAL via the pYEN motif. (A) Reciprocal co-IP of HGAL and Grb2 from unstimulated Raji, Bjab, VAL, and OCILY19 lymphoma cells. (B) BCR stimulation of Raji cells with goat anti-human IgM F(ab′)₂ increases HGAL and Grb2 co-IP at the indicated times. (C) γ-Phosphatase decreases co-IP of Grb2 with HGAL in Raji cells. (D) Grb2 is not interacting with HGAL(FEN) mutant. HeLa cells were transfected with plasmids encoding wild-type HGAL or its mutant HGAL (FEN) and 48 hours later subjected to Grb2 IP followed by HGAL western blotting. (E) GST-Grb2 pull-down assays with recombinant Trx-HGAL and Trx-HGAL(FEN) mutant proteins in the presence or absence of active Syk or Lyn kinases demonstrate that the HGAL-Grb2 interaction is direct and occurs only with phosphorylated HGAL protein. (F) The SH2 domain of Grb2 binds to the phosphorylated (pYEN) 12-mer peptide derived from HGAL with an affinity of 5 μM using isothermal titration calorimetry (red curve with solid dots), but not the unphosphorylated (YEN) peptide (curve with empty dots). In the inset, Grb2 SH2 domain binding to HGAL is governed by favorable enthalpic contributions accompanied by entropic penalty to the overall free energy, implying that the Grb2-HGAL interaction is largely governed by electrostatic interactions with a minor hydrophobic force contribution. Data in panels A-F are representative of 3 independent experiments. IgG, immunoglobulin G.