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: We previously demonstrated that upon ligand stimulation, multiple RTKs phosphorylate GIV at two critical tyrosines, Y1764 and Y1798, and that both sites are capable of binding and activating class 1A PI3Ks (Lin et al., 2011). Although these two tyrosines are located within the boundaries of the SH2-like domain of GIV (Figure 5a and Supplemental Figure S6a), the homology model predicts that they are exposed and accessible to RTKs (Figure 5a). We asked whether receptor-mediated phosphorylation of GIV in cells requires an intact SH2-like domain of GIV, thereby bringing the substrate (GIV) in proximity to the kinase (EGFR). To answer this, we compared the extent of EGF-stimulated tyrosine phosphorylation of wild-type GIV to the SH2-deficient RL mutant and a previously described phosphorylation-deficient GIV-YF mutant (Lin et al., 2011); the latter is a negative control in which both tyrosines were replaced by Phe (F). Because nonreceptor TKs of the Src family can also phosphorylate GIV on those two tyrosines (Lin et al., 2011), we used the kinase inhibitor PP2 to abolish any contribution to GIV phosphorylation via the Src family of kinases, exactly as we used previously (Lin et al., 2011; Mittal et al., 2011). On EGF stimulation, wild-type GIV, but not the RL and the YF mutants, was tyrosine phosphorylated (Figure 5b), indicating that an intact SH2-like domain is essential for enhanced tyrosine phosphorylation in GIV. Because EGFR kinase phosphorylated the RL mutant just as efficiently as WT GIV-CT in vitro (Supplemental Figure S6b), its failure to be phosphorylated in cells (Figure 5b) indicates that the SH2-like function of GIV is required for enhanced phosphorylation of GIV by EGFR in cells.
Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.