Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.
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.
- GTP-Binding Protein alpha Subunits, Gi-Go/chemistry*/genetics/metabolism
- Microfilament Proteins/chemistry*/genetics/metabolism
- Receptor, Epidermal Growth Factor/chemistry*/genetics/metabolism
- Vesicular Transport Proteins/chemistry*/genetics/metabolism
- Amino Acid Sequence
- Cell Movement
- Gene Expression Regulation
- HeLa Cells
- Models, Molecular
- Molecular Sequence Data
- Protein Binding
- Protein Folding
- Protein Interaction Domains and Motifs
- Signal Transduction
- Structural Homology, Protein
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Figure 7: EGFR degradation is delayed and actin remodeling and cell migration are impaired in the absence of an intact SH2 domain in GIV. (a) HeLa control, HeLa GIV-WT, HeLa GIV-RL, and HeLa GIV-FA cells stably expressing siRNA-resistant GIV constructs were either treated with control (Scr) siRNA or GIV siRNA, starved, and stimulated with EGF as in Figure 4e before fixation. Cells were costained for EEA1 (green), tEGFR (red; anti-EGFR cytoplasmic tail), and the nucleus/DAPI (blue). At 60 min after ligand stimulation, EGFR is virtually undetectable in GIV-WT cells (e, j), but significant staining is seen in GIV-RL cells (t) compared with controls (e). Images are representative of four independent repeats. Bar, 10 μm. (b) Serum-starved control, GIV-WT, GIV-RL, and GIV-FA HeLa cells were stimulated with EGF as in a before lysis. Equal aliquots of lysates were analyzed for total EGFR (tEGFR, anti-EGFR cytoplasmic tail) and tubulin by immunoblotting. (c) The amount of tEGFR present at 30 min in b was quantified by Odyssey infrared imaging, normalized to tubulin, and expressed as percentage remaining compared with 0 min. Results are shown as mean ± SEM (n = 4). (d) Control HeLa cells or HeLa GIV-WT, GIV-FA, and GIV-RL cells expressing various siRNA-resistant GIV constructs were treated with scrambled or GIV siRNA as indicated. Fixed cells were costained with phalloidin–Texas red (F-actin, red) and DAPI (DNA, blue) and visualized by confocal microscopy. Stress fibers were reduced when endogenous GIV was depleted in control, HeLa GIV-FA (f), and HeLa GIV-RL (h) cells but not in HeLa GIV-WT cells. Both HeLa GIV-FA and GIV-RL cells show a paucity of stress fibers even without depletion of endogenous GIV (e, g), indicating that these GIV mutants have a dominant negative effect on actin remodeling. Bar, 10 μM. (e) HeLa control or HeLa GIV-WT and GIV-RL cells expressing various siRNA-resistant GIV constructs were treated with scrambled or GIV siRNA as in d. Cell migration was determined as described in Materials and Methods. Depletion of GIV inhibited migration in control and HeLa GIV-RL cells but not in HeLa GIV-WT cells.
We previously demonstrated (Ghosh et al., 2010) that GIV induces rapid degradation of EGFR after receptor endocytosis, thereby reducing the duration of signaling from endosomes. Because the SH2-like domain of GIV alters key early events in EGF signaling, we reasoned that it might also be responsible for altering the rate of receptor degradation. When we investigated this in HeLa-GIV-WT, -FA, and -RL cells by immunofluorescence (Figure 7a) and immunoblotting (Figure 7b), both approaches showed that rates of receptor degradation were faster in GIV-WT cells but slower in GIV-RL cells, with significant amounts of receptor lingering at 30 and 60 min (Figure 7, a and b) within EEA1-positive endocytic compartments (Figure 7a). Consistent with our prior work, the rate of receptor degradation was delayed also in GIV-FA cells. Because both GIV-RL and GIV-FA are deficient in assembling ternary RTK-GIV-Gαi complexes, these results indicate that both SH2 and GEF domains, which cooperatively assemble such ternary complexes (Figure 4), are required for efficient down-regulation of activated receptor in endocytic compartments.