Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.
Bottom Line: We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins.Using a combination of homology modeling, protein-protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands.Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV-Akt enhancement, actin remodeling, and cell migration.
Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.Show MeSH
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Mentions: To determine whether GIV-CT folds into a SH2-like module and recognizes/binds to phosphotyrosine ligands, as predicted by the homology model in Figure 2, we made a number of predictions by computational modeling regarding the effect of strategically placed mutations within GIV's SH2-like domain (Figure 3, a and b): 1) mutations in the core GXFXXR motif and phosphotyrosine binding pocket that were predicted to abolish the GIV:EGFR interaction, 2) mutations within adjacent sequences that do not participate in phosphotyrosine recognition and were predicted to be inconsequential, and 3) a mutation within the pocket that was expected to favor binding. We found all these predictions to be accurate when we carried out binding assays using various His-tagged GIV mutants and in vitro–phosphorylated, GST-tagged pY1148 EGFR tail peptide (Figure 3, c and d); that is, compared with GIV–wild type (WT), some mutants abolished binding (lanes 2, 4–6, and 8), whereas others bound equally well (lanes 3, 9, and 10) or consistently better (F1765T; ∼1.45-fold, p < 0.01). These findings validate our homology model of GIV-SH2 (Figure 2, d and f) and demonstrate that the conserved ‘GXFXXR' core motif and the flanking sequence that the C-terminus of GIV shares with other SH2 adaptors can function as a SH2-like domain, in that they recognize and directly bind phosphotyrosine ligands. These results also indicate that the ∼100- to 110-aa stretch within GIV's C-terminus is likely to assume a SH2-like domain structure as predicted by homology modeling (Figure 2).
Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.