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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

Validation of the homology model of GIV's SH2 domain. (a, b) Based on the generated three-dimensional model shown in Figure 2, d and e, a series of GIV C-terminal mutations were predicted to decrease, increase, or have no effect on the recognition and binding to the phosphotyrosine 1148 on the cytoplasmic tail of EGFR. These residues are highlighted in red, green, and gold, and listed along with the preferred amino acid substitution in b. R1745 of GIV corresponds to the invariant Arg residue at the βB5 position within the conserved GXFXXR motif that is characteristic of the entire SH2 family of adaptors (Songyang et al., 1993, 1994; Schlessinger, 1994). The βC-βD loop, which is predicted to not affect phosphotyrosine binding, was either deleted or replaced with a neutral flexible linker, SGS. F1765 was mutated to Thr (T) to resemble the mouse sequence; this substitution is predicted to increase the depth of the binding pocket and improve binding. α, helix; β, β-sheets. (c, d) Equal aliquots (25 μg) of GST and GST-pY1148 were phosphorylated in vitro using recombinant EGFR kinase and used in pull-down assays with purified WT or various mutants of His-GIV-CT listed in b. (c) An aliquot of the GST proteins was analyzed for GST and pTyr by IB. Yellow pixels in the overlay images (merge panels) confirm that GST-pY1148 is autophosphorylated on tyrosine(s) by EGFR kinase in vitro. (d) Bound GIV CT was visualized by IB for His. Equal loading of GST and GST-pY1148 was confirmed by Ponceau S staining. A representative experiment is shown; n = 4. (e) Schematic representation of EGFR-VC and VN-GIV-SH2 constructs used for BiFC assay. (f) Cos7 cells were cotransfected with indicated complementary pairs of probes, grown in 10% FBS, fixed, and analyzed for fluorescence by confocal microscopy. Images representative of each condition are shown. Fluorescence is observed at the PM (arrowheads) and on vesicles (arrows; likely endolysosomal compartments) exclusively when complementary VN-GIV-SH2 WT, but not the SH2-deficient RL mutant probe, was cotransfected with EGFR-VC. Paired transfection of other complementary VN- and VC-control probes did not show discernible fluorescence (∼400 cells/experiment; n = 4).
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Figure 3: Validation of the homology model of GIV's SH2 domain. (a, b) Based on the generated three-dimensional model shown in Figure 2, d and e, a series of GIV C-terminal mutations were predicted to decrease, increase, or have no effect on the recognition and binding to the phosphotyrosine 1148 on the cytoplasmic tail of EGFR. These residues are highlighted in red, green, and gold, and listed along with the preferred amino acid substitution in b. R1745 of GIV corresponds to the invariant Arg residue at the βB5 position within the conserved GXFXXR motif that is characteristic of the entire SH2 family of adaptors (Songyang et al., 1993, 1994; Schlessinger, 1994). The βC-βD loop, which is predicted to not affect phosphotyrosine binding, was either deleted or replaced with a neutral flexible linker, SGS. F1765 was mutated to Thr (T) to resemble the mouse sequence; this substitution is predicted to increase the depth of the binding pocket and improve binding. α, helix; β, β-sheets. (c, d) Equal aliquots (25 μg) of GST and GST-pY1148 were phosphorylated in vitro using recombinant EGFR kinase and used in pull-down assays with purified WT or various mutants of His-GIV-CT listed in b. (c) An aliquot of the GST proteins was analyzed for GST and pTyr by IB. Yellow pixels in the overlay images (merge panels) confirm that GST-pY1148 is autophosphorylated on tyrosine(s) by EGFR kinase in vitro. (d) Bound GIV CT was visualized by IB for His. Equal loading of GST and GST-pY1148 was confirmed by Ponceau S staining. A representative experiment is shown; n = 4. (e) Schematic representation of EGFR-VC and VN-GIV-SH2 constructs used for BiFC assay. (f) Cos7 cells were cotransfected with indicated complementary pairs of probes, grown in 10% FBS, fixed, and analyzed for fluorescence by confocal microscopy. Images representative of each condition are shown. Fluorescence is observed at the PM (arrowheads) and on vesicles (arrows; likely endolysosomal compartments) exclusively when complementary VN-GIV-SH2 WT, but not the SH2-deficient RL mutant probe, was cotransfected with EGFR-VC. Paired transfection of other complementary VN- and VC-control probes did not show discernible fluorescence (∼400 cells/experiment; n = 4).

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).


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)

Validation of the homology model of GIV's SH2 domain. (a, b) Based on the generated three-dimensional model shown in Figure 2, d and e, a series of GIV C-terminal mutations were predicted to decrease, increase, or have no effect on the recognition and binding to the phosphotyrosine 1148 on the cytoplasmic tail of EGFR. These residues are highlighted in red, green, and gold, and listed along with the preferred amino acid substitution in b. R1745 of GIV corresponds to the invariant Arg residue at the βB5 position within the conserved GXFXXR motif that is characteristic of the entire SH2 family of adaptors (Songyang et al., 1993, 1994; Schlessinger, 1994). The βC-βD loop, which is predicted to not affect phosphotyrosine binding, was either deleted or replaced with a neutral flexible linker, SGS. F1765 was mutated to Thr (T) to resemble the mouse sequence; this substitution is predicted to increase the depth of the binding pocket and improve binding. α, helix; β, β-sheets. (c, d) Equal aliquots (25 μg) of GST and GST-pY1148 were phosphorylated in vitro using recombinant EGFR kinase and used in pull-down assays with purified WT or various mutants of His-GIV-CT listed in b. (c) An aliquot of the GST proteins was analyzed for GST and pTyr by IB. Yellow pixels in the overlay images (merge panels) confirm that GST-pY1148 is autophosphorylated on tyrosine(s) by EGFR kinase in vitro. (d) Bound GIV CT was visualized by IB for His. Equal loading of GST and GST-pY1148 was confirmed by Ponceau S staining. A representative experiment is shown; n = 4. (e) Schematic representation of EGFR-VC and VN-GIV-SH2 constructs used for BiFC assay. (f) Cos7 cells were cotransfected with indicated complementary pairs of probes, grown in 10% FBS, fixed, and analyzed for fluorescence by confocal microscopy. Images representative of each condition are shown. Fluorescence is observed at the PM (arrowheads) and on vesicles (arrows; likely endolysosomal compartments) exclusively when complementary VN-GIV-SH2 WT, but not the SH2-deficient RL mutant probe, was cotransfected with EGFR-VC. Paired transfection of other complementary VN- and VC-control probes did not show discernible fluorescence (∼400 cells/experiment; n = 4).
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Figure 3: Validation of the homology model of GIV's SH2 domain. (a, b) Based on the generated three-dimensional model shown in Figure 2, d and e, a series of GIV C-terminal mutations were predicted to decrease, increase, or have no effect on the recognition and binding to the phosphotyrosine 1148 on the cytoplasmic tail of EGFR. These residues are highlighted in red, green, and gold, and listed along with the preferred amino acid substitution in b. R1745 of GIV corresponds to the invariant Arg residue at the βB5 position within the conserved GXFXXR motif that is characteristic of the entire SH2 family of adaptors (Songyang et al., 1993, 1994; Schlessinger, 1994). The βC-βD loop, which is predicted to not affect phosphotyrosine binding, was either deleted or replaced with a neutral flexible linker, SGS. F1765 was mutated to Thr (T) to resemble the mouse sequence; this substitution is predicted to increase the depth of the binding pocket and improve binding. α, helix; β, β-sheets. (c, d) Equal aliquots (25 μg) of GST and GST-pY1148 were phosphorylated in vitro using recombinant EGFR kinase and used in pull-down assays with purified WT or various mutants of His-GIV-CT listed in b. (c) An aliquot of the GST proteins was analyzed for GST and pTyr by IB. Yellow pixels in the overlay images (merge panels) confirm that GST-pY1148 is autophosphorylated on tyrosine(s) by EGFR kinase in vitro. (d) Bound GIV CT was visualized by IB for His. Equal loading of GST and GST-pY1148 was confirmed by Ponceau S staining. A representative experiment is shown; n = 4. (e) Schematic representation of EGFR-VC and VN-GIV-SH2 constructs used for BiFC assay. (f) Cos7 cells were cotransfected with indicated complementary pairs of probes, grown in 10% FBS, fixed, and analyzed for fluorescence by confocal microscopy. Images representative of each condition are shown. Fluorescence is observed at the PM (arrowheads) and on vesicles (arrows; likely endolysosomal compartments) exclusively when complementary VN-GIV-SH2 WT, but not the SH2-deficient RL mutant probe, was cotransfected with EGFR-VC. Paired transfection of other complementary VN- and VC-control probes did not show discernible fluorescence (∼400 cells/experiment; n = 4).
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).

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