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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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GIV's SH2 and GEF domains are required for the recruitment of Gαi3 to ligand-activated EGFR and activation of G protein after EGF stimulation. (a) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro using 5 ng of recombinant EGFR kinase (GST-pY EGFR-T; lanes 5–7) or mock treated (GST-EGFR-T; lane 4) and subsequently used in pull-down assays with equal amounts of purified WT, FA (GEF-deficient), or RL (SH2-deficient) mutants of His-GIV-CT (aa 1660–1870; lanes 1–3, inputs). Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. As anticipated, GST-pY EGFR-T (lane 5) but not GST-EGFR-T (lane 4) directly binds His-GIV-CT WT. The FA (lane 6) but not RL (lane 7) mutant of GIV-CT binds GST-pY EGFR-T. (b) WT, FA, and RL mutants of His-GIV-CT were incubated with 5 μg of GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by IB for GIV-CT using anti-His mAb. His-GIV-CT WT (lane 3) and RL mutant (lane 9) bound GST-Gαi3 equally, and, as anticipated, the FA mutant did not (lane 6). (c) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of WT (∆), FA (Δ), and RL mutant (•) His-GIV-CT. Both WT and RL mutants increased the steady-state GTPase activity of His-Gαi3 efficiently and equally in a dose-dependent manner, whereas the FA mutant did not. The basal steady-state GTPase activity of His-Gαi3 is 0.021 ± 0.04 mol Pi mol Gαi3−1 min−1 and the results are displayed as percentage of basal activity. (d) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro with recombinant EGFR kinase (GST-pY EGFR-T; lanes 2 and 4–7) or mock treated (GST-EGFR-T; lanes 1 and 3) and subsequently used in pull-down assays with purified His-Gαi3 alone (lanes 1 and 2), His-GIV-CT alone (lanes 3 and 4), or a mix of His-Gαi3 and WT, FA, or RL mutants of His-GIV-CT as indicated. Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. Gαi3 binds GST-pY EGFR in the presence of WT (lane 5) but not FA (lane 6) or RL (lane 7). (e) HeLa GIV-WT, HeLa GIV-FA, and HeLa GIV-RL cells stably expressing the indicated siRNA-resistant GIV constructs were depleted of endogenous GIV by siRNA, serum starved for 16 h, and then stimulated with EGF for the indicated durations before lysis. Equal aliquots of lysates (bottom) were incubated with anti-EGFR mAb (#225 IgG). Immune complexes (top) were analyzed for total (t-EGFR) and Gαi3 by IB. (f) Equal aliquots of lysates (bottom) from starved or EGF-treated HeLa GIV-WT and GIV-RL cells were incubated with anti-Gαi:GTP mAb. Immune complexes (top) were analyzed for Gαi3 by IB. (g) HeLa-GIV-WT and GIV-RL cells were depleted of endogenous GIV as in e, serum starved (0.2% FBS), and subsequently stimulated with EGF and analyzed for cAMP by RIA (see Materials and Methods). Results are displayed as fold change in cAMP (y-axis) in each cell line normalized to their respective starved state. (h) HeLa-GIV-WT and GIV-RL cells were stimulated with EGF before lysis as in e. Top, equal aliquots of whole-cell lysates were analyzed for phosphorylated CREB (pCREB) and tubulin by IB. Bottom, bar graphs display the quantification of pCREB/tubulin by band densitometry. All values are normalized to the starved HeLa-GIV-WT cells. Results are shown as mean ± SEM; n = 3.
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Figure 4: GIV's SH2 and GEF domains are required for the recruitment of Gαi3 to ligand-activated EGFR and activation of G protein after EGF stimulation. (a) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro using 5 ng of recombinant EGFR kinase (GST-pY EGFR-T; lanes 5–7) or mock treated (GST-EGFR-T; lane 4) and subsequently used in pull-down assays with equal amounts of purified WT, FA (GEF-deficient), or RL (SH2-deficient) mutants of His-GIV-CT (aa 1660–1870; lanes 1–3, inputs). Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. As anticipated, GST-pY EGFR-T (lane 5) but not GST-EGFR-T (lane 4) directly binds His-GIV-CT WT. The FA (lane 6) but not RL (lane 7) mutant of GIV-CT binds GST-pY EGFR-T. (b) WT, FA, and RL mutants of His-GIV-CT were incubated with 5 μg of GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by IB for GIV-CT using anti-His mAb. His-GIV-CT WT (lane 3) and RL mutant (lane 9) bound GST-Gαi3 equally, and, as anticipated, the FA mutant did not (lane 6). (c) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of WT (∆), FA (Δ), and RL mutant (•) His-GIV-CT. Both WT and RL mutants increased the steady-state GTPase activity of His-Gαi3 efficiently and equally in a dose-dependent manner, whereas the FA mutant did not. The basal steady-state GTPase activity of His-Gαi3 is 0.021 ± 0.04 mol Pi mol Gαi3−1 min−1 and the results are displayed as percentage of basal activity. (d) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro with recombinant EGFR kinase (GST-pY EGFR-T; lanes 2 and 4–7) or mock treated (GST-EGFR-T; lanes 1 and 3) and subsequently used in pull-down assays with purified His-Gαi3 alone (lanes 1 and 2), His-GIV-CT alone (lanes 3 and 4), or a mix of His-Gαi3 and WT, FA, or RL mutants of His-GIV-CT as indicated. Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. Gαi3 binds GST-pY EGFR in the presence of WT (lane 5) but not FA (lane 6) or RL (lane 7). (e) HeLa GIV-WT, HeLa GIV-FA, and HeLa GIV-RL cells stably expressing the indicated siRNA-resistant GIV constructs were depleted of endogenous GIV by siRNA, serum starved for 16 h, and then stimulated with EGF for the indicated durations before lysis. Equal aliquots of lysates (bottom) were incubated with anti-EGFR mAb (#225 IgG). Immune complexes (top) were analyzed for total (t-EGFR) and Gαi3 by IB. (f) Equal aliquots of lysates (bottom) from starved or EGF-treated HeLa GIV-WT and GIV-RL cells were incubated with anti-Gαi:GTP mAb. Immune complexes (top) were analyzed for Gαi3 by IB. (g) HeLa-GIV-WT and GIV-RL cells were depleted of endogenous GIV as in e, serum starved (0.2% FBS), and subsequently stimulated with EGF and analyzed for cAMP by RIA (see Materials and Methods). Results are displayed as fold change in cAMP (y-axis) in each cell line normalized to their respective starved state. (h) HeLa-GIV-WT and GIV-RL cells were stimulated with EGF before lysis as in e. Top, equal aliquots of whole-cell lysates were analyzed for phosphorylated CREB (pCREB) and tubulin by IB. Bottom, bar graphs display the quantification of pCREB/tubulin by band densitometry. All values are normalized to the starved HeLa-GIV-WT cells. Results are shown as mean ± SEM; n = 3.

Mentions: Next we investigated the functional relationship between GIV's SH2-like domain and its previously defined GEF motif (Garcia-Marcos et al., 2009), which binds and activates Gαi. To determine whether an intact GEF motif is required for GIV's SH2 domain to bind phosphotyrosines, we used His-GIV-CT WT or mutant proteins that are either GEF-deficient (FA) or SH2-deficient (RL) in binding assays with in vitro–phosphorylated, GST-tagged pY1148 EGFR tail. We found that the WT GIV-CT and the FA mutant bound phosphorylated EGFR tail to an equal extent, whereas the RL mutant did not (Figure 4a), indicating that in the absence of an intact GEF motif, GIV's SH2-like domain is sufficient to recognize and bind phosphotyrosines on the EGFR tail. To whether if an intact SH2-like domain is required for GIV's GEF motif to bind and activate Gαi, we used the His-GIV-CT proteins in binding assays with GDP-loaded GST-Gαi3 (Figure 4b) and in steady-state GTPase assays with His-Gαi3 (Figure 4c). We found that the WT GIV-CT and the RL mutant bound (Figure 4b) and activated (Figure 4c) Gαi3 to an equal extent, whereas the FA mutant did not, demonstrating that the SH2-like domain is not required for GIV to bind and activate Gαi3. To determine the contribution of GIV's SH2-like domain and its GEF motif in the formation of Gαi-GIV-EGFR ternary complexes, we carried out binding assays with three recombinant proteins—His-Gαi3, His-GIV-CT, and in vitro–phosphorylated GST-pY1148 EGFR tail. We found that Gαi3 bound phosphorylated EGFR tail exclusively in the presence of WT GIV-CT (Figure 4d), indicating that the Gαi3-EGFR interaction is indirect and requires GIV-CT. This interaction was abolished when the WT GIV-CT protein was replaced by either the SH2-deficient or the GEF-deficient mutant (Figure 4d), indicating that both the SH2-like domain and the GEF motif are required for GIV to facilitate the interaction between Gαi3 and EGFR and to trigger the assembly of Gαi3-GIV-EGFR ternary complexes in vitro.


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)

GIV's SH2 and GEF domains are required for the recruitment of Gαi3 to ligand-activated EGFR and activation of G protein after EGF stimulation. (a) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro using 5 ng of recombinant EGFR kinase (GST-pY EGFR-T; lanes 5–7) or mock treated (GST-EGFR-T; lane 4) and subsequently used in pull-down assays with equal amounts of purified WT, FA (GEF-deficient), or RL (SH2-deficient) mutants of His-GIV-CT (aa 1660–1870; lanes 1–3, inputs). Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. As anticipated, GST-pY EGFR-T (lane 5) but not GST-EGFR-T (lane 4) directly binds His-GIV-CT WT. The FA (lane 6) but not RL (lane 7) mutant of GIV-CT binds GST-pY EGFR-T. (b) WT, FA, and RL mutants of His-GIV-CT were incubated with 5 μg of GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by IB for GIV-CT using anti-His mAb. His-GIV-CT WT (lane 3) and RL mutant (lane 9) bound GST-Gαi3 equally, and, as anticipated, the FA mutant did not (lane 6). (c) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of WT (∆), FA (Δ), and RL mutant (•) His-GIV-CT. Both WT and RL mutants increased the steady-state GTPase activity of His-Gαi3 efficiently and equally in a dose-dependent manner, whereas the FA mutant did not. The basal steady-state GTPase activity of His-Gαi3 is 0.021 ± 0.04 mol Pi mol Gαi3−1 min−1 and the results are displayed as percentage of basal activity. (d) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro with recombinant EGFR kinase (GST-pY EGFR-T; lanes 2 and 4–7) or mock treated (GST-EGFR-T; lanes 1 and 3) and subsequently used in pull-down assays with purified His-Gαi3 alone (lanes 1 and 2), His-GIV-CT alone (lanes 3 and 4), or a mix of His-Gαi3 and WT, FA, or RL mutants of His-GIV-CT as indicated. Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. Gαi3 binds GST-pY EGFR in the presence of WT (lane 5) but not FA (lane 6) or RL (lane 7). (e) HeLa GIV-WT, HeLa GIV-FA, and HeLa GIV-RL cells stably expressing the indicated siRNA-resistant GIV constructs were depleted of endogenous GIV by siRNA, serum starved for 16 h, and then stimulated with EGF for the indicated durations before lysis. Equal aliquots of lysates (bottom) were incubated with anti-EGFR mAb (#225 IgG). Immune complexes (top) were analyzed for total (t-EGFR) and Gαi3 by IB. (f) Equal aliquots of lysates (bottom) from starved or EGF-treated HeLa GIV-WT and GIV-RL cells were incubated with anti-Gαi:GTP mAb. Immune complexes (top) were analyzed for Gαi3 by IB. (g) HeLa-GIV-WT and GIV-RL cells were depleted of endogenous GIV as in e, serum starved (0.2% FBS), and subsequently stimulated with EGF and analyzed for cAMP by RIA (see Materials and Methods). Results are displayed as fold change in cAMP (y-axis) in each cell line normalized to their respective starved state. (h) HeLa-GIV-WT and GIV-RL cells were stimulated with EGF before lysis as in e. Top, equal aliquots of whole-cell lysates were analyzed for phosphorylated CREB (pCREB) and tubulin by IB. Bottom, bar graphs display the quantification of pCREB/tubulin by band densitometry. All values are normalized to the starved HeLa-GIV-WT cells. Results are shown as mean ± SEM; n = 3.
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Figure 4: GIV's SH2 and GEF domains are required for the recruitment of Gαi3 to ligand-activated EGFR and activation of G protein after EGF stimulation. (a) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro using 5 ng of recombinant EGFR kinase (GST-pY EGFR-T; lanes 5–7) or mock treated (GST-EGFR-T; lane 4) and subsequently used in pull-down assays with equal amounts of purified WT, FA (GEF-deficient), or RL (SH2-deficient) mutants of His-GIV-CT (aa 1660–1870; lanes 1–3, inputs). Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. As anticipated, GST-pY EGFR-T (lane 5) but not GST-EGFR-T (lane 4) directly binds His-GIV-CT WT. The FA (lane 6) but not RL (lane 7) mutant of GIV-CT binds GST-pY EGFR-T. (b) WT, FA, and RL mutants of His-GIV-CT were incubated with 5 μg of GST-Gαi3 or GST preloaded with GDP immobilized on glutathione beads. Bound proteins were analyzed by IB for GIV-CT using anti-His mAb. His-GIV-CT WT (lane 3) and RL mutant (lane 9) bound GST-Gαi3 equally, and, as anticipated, the FA mutant did not (lane 6). (c) The amount of GTP hydrolyzed in 10 min by His-Gαi3 was determined in the presence of the indicated amounts of WT (∆), FA (Δ), and RL mutant (•) His-GIV-CT. Both WT and RL mutants increased the steady-state GTPase activity of His-Gαi3 efficiently and equally in a dose-dependent manner, whereas the FA mutant did not. The basal steady-state GTPase activity of His-Gαi3 is 0.021 ± 0.04 mol Pi mol Gαi3−1 min−1 and the results are displayed as percentage of basal activity. (d) Equal aliquots (15 μg) of GST-EGFR-T (aa 1064–1210) were either phosphorylated in vitro with recombinant EGFR kinase (GST-pY EGFR-T; lanes 2 and 4–7) or mock treated (GST-EGFR-T; lanes 1 and 3) and subsequently used in pull-down assays with purified His-Gαi3 alone (lanes 1 and 2), His-GIV-CT alone (lanes 3 and 4), or a mix of His-Gαi3 and WT, FA, or RL mutants of His-GIV-CT as indicated. Bound proteins were analyzed for His-GIV-CT and phosphorylation of GST-EGFR-T by IB. Gαi3 binds GST-pY EGFR in the presence of WT (lane 5) but not FA (lane 6) or RL (lane 7). (e) HeLa GIV-WT, HeLa GIV-FA, and HeLa GIV-RL cells stably expressing the indicated siRNA-resistant GIV constructs were depleted of endogenous GIV by siRNA, serum starved for 16 h, and then stimulated with EGF for the indicated durations before lysis. Equal aliquots of lysates (bottom) were incubated with anti-EGFR mAb (#225 IgG). Immune complexes (top) were analyzed for total (t-EGFR) and Gαi3 by IB. (f) Equal aliquots of lysates (bottom) from starved or EGF-treated HeLa GIV-WT and GIV-RL cells were incubated with anti-Gαi:GTP mAb. Immune complexes (top) were analyzed for Gαi3 by IB. (g) HeLa-GIV-WT and GIV-RL cells were depleted of endogenous GIV as in e, serum starved (0.2% FBS), and subsequently stimulated with EGF and analyzed for cAMP by RIA (see Materials and Methods). Results are displayed as fold change in cAMP (y-axis) in each cell line normalized to their respective starved state. (h) HeLa-GIV-WT and GIV-RL cells were stimulated with EGF before lysis as in e. Top, equal aliquots of whole-cell lysates were analyzed for phosphorylated CREB (pCREB) and tubulin by IB. Bottom, bar graphs display the quantification of pCREB/tubulin by band densitometry. All values are normalized to the starved HeLa-GIV-WT cells. Results are shown as mean ± SEM; n = 3.
Mentions: Next we investigated the functional relationship between GIV's SH2-like domain and its previously defined GEF motif (Garcia-Marcos et al., 2009), which binds and activates Gαi. To determine whether an intact GEF motif is required for GIV's SH2 domain to bind phosphotyrosines, we used His-GIV-CT WT or mutant proteins that are either GEF-deficient (FA) or SH2-deficient (RL) in binding assays with in vitro–phosphorylated, GST-tagged pY1148 EGFR tail. We found that the WT GIV-CT and the FA mutant bound phosphorylated EGFR tail to an equal extent, whereas the RL mutant did not (Figure 4a), indicating that in the absence of an intact GEF motif, GIV's SH2-like domain is sufficient to recognize and bind phosphotyrosines on the EGFR tail. To whether if an intact SH2-like domain is required for GIV's GEF motif to bind and activate Gαi, we used the His-GIV-CT proteins in binding assays with GDP-loaded GST-Gαi3 (Figure 4b) and in steady-state GTPase assays with His-Gαi3 (Figure 4c). We found that the WT GIV-CT and the RL mutant bound (Figure 4b) and activated (Figure 4c) Gαi3 to an equal extent, whereas the FA mutant did not, demonstrating that the SH2-like domain is not required for GIV to bind and activate Gαi3. To determine the contribution of GIV's SH2-like domain and its GEF motif in the formation of Gαi-GIV-EGFR ternary complexes, we carried out binding assays with three recombinant proteins—His-Gαi3, His-GIV-CT, and in vitro–phosphorylated GST-pY1148 EGFR tail. We found that Gαi3 bound phosphorylated EGFR tail exclusively in the presence of WT GIV-CT (Figure 4d), indicating that the Gαi3-EGFR interaction is indirect and requires GIV-CT. This interaction was abolished when the WT GIV-CT protein was replaced by either the SH2-deficient or the GEF-deficient mutant (Figure 4d), indicating that both the SH2-like domain and the GEF motif are required for GIV to facilitate the interaction between Gαi3 and EGFR and to trigger the assembly of Gαi3-GIV-EGFR ternary complexes in vitro.

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