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Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

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Related in: MedlinePlus

The C-terminus of GIV directly binds autophosphorylated cytoplasmic tails of multiple RTKs. (a) Recombinant cytoplasmic tails of EGFR, InsR, and VEGFR were autophosphorylated and subsequently used in binding assays with His-GIV-CT (aa 1660–1870) or His-Sec22 (a negative control) prebound to cobalt beads. Bound proteins were analyzed for His-tagged proteins by Ponceau staining (bottom) and autophosphorylated receptors by IB with anti-pTyr mAb (top). (b) Schematic representation of GST-tagged, phospho–EGFR-tail peptides used in this work. (c, d) Equal aliquots (25 μg) of GST and GST-EGFR peptides containing the indicated tyrosines were autophosphorylated in vitro using recombinant EGFR kinase and subsequently used in pull-down assays with His-GIV-CT as in a. (c) An aliquot of the GST proteins were analyzed for GST and pTyr by IB. Single-channel images for GST and pTyr are displayed in grayscale. Yellow pixels in the overlay images (merge panels) confirm that Y1068, 86, Y1148, and Y1173 are autophosphorylated on tyrosine(s) by EGFR (lanes 2, 4, 5), whereas GST (lane 1) and GST-Y1114 (lane 3) are not. (d) Bound proteins were visualized by IB for His. His-GIV-CT bound strongly to GST-Y1148 (lane 7) and with GST-Y1173 (lane 8) but not to GST alone or other GST-EGFR peptides (lanes 4–6).
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Figure 1: The C-terminus of GIV directly binds autophosphorylated cytoplasmic tails of multiple RTKs. (a) Recombinant cytoplasmic tails of EGFR, InsR, and VEGFR were autophosphorylated and subsequently used in binding assays with His-GIV-CT (aa 1660–1870) or His-Sec22 (a negative control) prebound to cobalt beads. Bound proteins were analyzed for His-tagged proteins by Ponceau staining (bottom) and autophosphorylated receptors by IB with anti-pTyr mAb (top). (b) Schematic representation of GST-tagged, phospho–EGFR-tail peptides used in this work. (c, d) Equal aliquots (25 μg) of GST and GST-EGFR peptides containing the indicated tyrosines were autophosphorylated in vitro using recombinant EGFR kinase and subsequently used in pull-down assays with His-GIV-CT as in a. (c) An aliquot of the GST proteins were analyzed for GST and pTyr by IB. Single-channel images for GST and pTyr are displayed in grayscale. Yellow pixels in the overlay images (merge panels) confirm that Y1068, 86, Y1148, and Y1173 are autophosphorylated on tyrosine(s) by EGFR (lanes 2, 4, 5), whereas GST (lane 1) and GST-Y1114 (lane 3) are not. (d) Bound proteins were visualized by IB for His. His-GIV-CT bound strongly to GST-Y1148 (lane 7) and with GST-Y1173 (lane 8) but not to GST alone or other GST-EGFR peptides (lanes 4–6).

Mentions: We previously demonstrated that GIV is recruited to ligand-activated EGFR (Ghosh et al., 2010). To discern whether GIV can interact directly with multiple ligand-activated RTKs, we tested the ability of the C-terminus of GIV to bind recombinant, autophosphorylated cytoplasmic tails of EGFR, VEGFR, and insulin receptor β (InsRβ) containing their respective kinase domains in pull-down assays. These three RTKs were chosen because we and others have demonstrated that GIV modulates Akt signaling and triggers cell migration when any of these three receptors is activated by its respective ligand (Enomoto et al., 2005; Jiang et al., 2008; Kitamura et al., 2008; Garcia-Marcos et al., 2009, 2010, 2011a, 2012; Ghosh et al., 2010; Lin et al., 2011). We found that a purified ∼200–amino acid (aa)–long C-terminal fragment of GIV is sufficient for GIV to bind all three recombinant active RTKs (Figure 1a), demonstrating that GIV can directly bind multiple autophosphorylated RTKs via its C-terminus. Next we asked which autophosphorylated tyrosine on the RTK tail GIV binds. Among the various RTKs, we focused on EGFR for two reasons: 1) it is a prototype RTK, and 2) EGFR autophosphorylation is best characterized, with in-depth understanding of the function of each autophosphorylation site and the signaling intermediates/adaptors that they recruit (Helin et al., 1991; Decker, 1993; Batzer et al., 1994; Okabayashi et al., 1994; Okutani et al., 1994).


Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin.

Lin C, Ear J, Midde K, Lopez-Sanchez I, Aznar N, Garcia-Marcos M, Kufareva I, Abagyan R, Ghosh P - Mol. Biol. Cell (2014)

The C-terminus of GIV directly binds autophosphorylated cytoplasmic tails of multiple RTKs. (a) Recombinant cytoplasmic tails of EGFR, InsR, and VEGFR were autophosphorylated and subsequently used in binding assays with His-GIV-CT (aa 1660–1870) or His-Sec22 (a negative control) prebound to cobalt beads. Bound proteins were analyzed for His-tagged proteins by Ponceau staining (bottom) and autophosphorylated receptors by IB with anti-pTyr mAb (top). (b) Schematic representation of GST-tagged, phospho–EGFR-tail peptides used in this work. (c, d) Equal aliquots (25 μg) of GST and GST-EGFR peptides containing the indicated tyrosines were autophosphorylated in vitro using recombinant EGFR kinase and subsequently used in pull-down assays with His-GIV-CT as in a. (c) An aliquot of the GST proteins were analyzed for GST and pTyr by IB. Single-channel images for GST and pTyr are displayed in grayscale. Yellow pixels in the overlay images (merge panels) confirm that Y1068, 86, Y1148, and Y1173 are autophosphorylated on tyrosine(s) by EGFR (lanes 2, 4, 5), whereas GST (lane 1) and GST-Y1114 (lane 3) are not. (d) Bound proteins were visualized by IB for His. His-GIV-CT bound strongly to GST-Y1148 (lane 7) and with GST-Y1173 (lane 8) but not to GST alone or other GST-EGFR peptides (lanes 4–6).
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Figure 1: The C-terminus of GIV directly binds autophosphorylated cytoplasmic tails of multiple RTKs. (a) Recombinant cytoplasmic tails of EGFR, InsR, and VEGFR were autophosphorylated and subsequently used in binding assays with His-GIV-CT (aa 1660–1870) or His-Sec22 (a negative control) prebound to cobalt beads. Bound proteins were analyzed for His-tagged proteins by Ponceau staining (bottom) and autophosphorylated receptors by IB with anti-pTyr mAb (top). (b) Schematic representation of GST-tagged, phospho–EGFR-tail peptides used in this work. (c, d) Equal aliquots (25 μg) of GST and GST-EGFR peptides containing the indicated tyrosines were autophosphorylated in vitro using recombinant EGFR kinase and subsequently used in pull-down assays with His-GIV-CT as in a. (c) An aliquot of the GST proteins were analyzed for GST and pTyr by IB. Single-channel images for GST and pTyr are displayed in grayscale. Yellow pixels in the overlay images (merge panels) confirm that Y1068, 86, Y1148, and Y1173 are autophosphorylated on tyrosine(s) by EGFR (lanes 2, 4, 5), whereas GST (lane 1) and GST-Y1114 (lane 3) are not. (d) Bound proteins were visualized by IB for His. His-GIV-CT bound strongly to GST-Y1148 (lane 7) and with GST-Y1173 (lane 8) but not to GST alone or other GST-EGFR peptides (lanes 4–6).
Mentions: We previously demonstrated that GIV is recruited to ligand-activated EGFR (Ghosh et al., 2010). To discern whether GIV can interact directly with multiple ligand-activated RTKs, we tested the ability of the C-terminus of GIV to bind recombinant, autophosphorylated cytoplasmic tails of EGFR, VEGFR, and insulin receptor β (InsRβ) containing their respective kinase domains in pull-down assays. These three RTKs were chosen because we and others have demonstrated that GIV modulates Akt signaling and triggers cell migration when any of these three receptors is activated by its respective ligand (Enomoto et al., 2005; Jiang et al., 2008; Kitamura et al., 2008; Garcia-Marcos et al., 2009, 2010, 2011a, 2012; Ghosh et al., 2010; Lin et al., 2011). We found that a purified ∼200–amino acid (aa)–long C-terminal fragment of GIV is sufficient for GIV to bind all three recombinant active RTKs (Figure 1a), demonstrating that GIV can directly bind multiple autophosphorylated RTKs via its C-terminus. Next we asked which autophosphorylated tyrosine on the RTK tail GIV binds. Among the various RTKs, we focused on EGFR for two reasons: 1) it is a prototype RTK, and 2) EGFR autophosphorylation is best characterized, with in-depth understanding of the function of each autophosphorylation site and the signaling intermediates/adaptors that they recruit (Helin et al., 1991; Decker, 1993; Batzer et al., 1994; Okabayashi et al., 1994; Okutani et al., 1994).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, School of Medicine, CA 92093.

Show MeSH
Related in: MedlinePlus