Limits...
Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Show MeSH

Related in: MedlinePlus

Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF4−. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF4− loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF4−, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2483528&req=5

fig6: Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF4−. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF4− loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF4−, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.

Mentions: Next, we asked whether the interaction between Gαi3 and GIV is activity dependent and found that the inactive GST-Gαi3G203A mutant consistently bound ∼10–15-fold more GIV than the active Gαi3Q204L mutant in in vitro binding assays using HeLa cell lysates or TnT (in vitro–translated) GIV (Fig. 6 A). We confirmed these results by preloading GST-Gαi3 with GDP alone or in the presence of AlF4−, which mimics the active state (Coleman et al., 1994). In the presence of GDP alone, GST-Gαi3 bound ∼10–30% of the total GIV, and addition of AlF4− virtually abolished the binding (Fig. 6 B). In contrast, GST-Gαs bound ∼100–300-fold less than GST-Gαi3 (Fig. 6 C), which is consistent with our finding that Gαs-depletion had no influence on cell migration (Fig. 1 B), Akt activation (Fig. 4 A), or actin stress fiber formation (not depicted). These results demonstrate that GIV binding is specific for Gαi3 and is greatly reduced upon activation of the G protein. Because inactive GDP-bound Gαi3 interacts with both Gβγ and GIV, we asked whether the α-subunit can interact with GIV when it is assembled into an intact heterotrimer. That this is the case is suggested by the fact that the Gβ subunit could be coimmunoprecipitated from brain lysates with GIV antibodies and that this interaction was abolished in the presence of GDP and AlF4− (Fig. 6 D).


Activation of Galphai3 triggers cell migration via regulation of GIV.

Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG - J. Cell Biol. (2008)

Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF4−. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF4− loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF4−, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483528&req=5

fig6: Activation status of Gαi3 regulates its interaction with GIV. (A and B) GIV binds preferentially to the inactive (Gai3G203A) form of Gai3. (A, Top) In vitro binding assays were performed using GST-Gαi3 (wt or mutants) and either HeLa cell lysates (GIV (HeLa)) or in vitro–translated GIV (GIV (TnT)). Bead-bound fractions were separated by SDS-PAGE. Bound GIV was analyzed either by immunoblotting or autoradiography (TnT). (B, Top) In vitro binding assay performed as in A with GST-Gαi3wt after GDP loading in the absence or presence of AlF4−. (A and B, Bottom) Ponceau S-staining of PVDF membranes to show equal loading of GST proteins. (C) GIV preferentially binds to Gαi3. In vitro binding assays were performed as in Bwith GST-Gαi3 wt or Gαs in the presence of GDP ± AlF4− loading to compare the relative binding of Gαs and Gαi3 to GIV. Gβ-subunits were used as a positive control. Bound GIV from HeLa cell lysates was visualized by immunoblotting (GIV (Low exp), low exposure; GIV (High exp), high exposure) and autoradiography (TnT), respectively. (D) GIV interacts with the inactive Gαβγ heterotrimer in vivo. GIV was immunoprecipitated from rat brain lysate in the absence (lanes 1–3) or presence (lanes 4–6) of GDP + AlF4−, and immune complexes were probed for GIV and Gβ subunits using anti–pan Gβ.
Mentions: Next, we asked whether the interaction between Gαi3 and GIV is activity dependent and found that the inactive GST-Gαi3G203A mutant consistently bound ∼10–15-fold more GIV than the active Gαi3Q204L mutant in in vitro binding assays using HeLa cell lysates or TnT (in vitro–translated) GIV (Fig. 6 A). We confirmed these results by preloading GST-Gαi3 with GDP alone or in the presence of AlF4−, which mimics the active state (Coleman et al., 1994). In the presence of GDP alone, GST-Gαi3 bound ∼10–30% of the total GIV, and addition of AlF4− virtually abolished the binding (Fig. 6 B). In contrast, GST-Gαs bound ∼100–300-fold less than GST-Gαi3 (Fig. 6 C), which is consistent with our finding that Gαs-depletion had no influence on cell migration (Fig. 1 B), Akt activation (Fig. 4 A), or actin stress fiber formation (not depicted). These results demonstrate that GIV binding is specific for Gαi3 and is greatly reduced upon activation of the G protein. Because inactive GDP-bound Gαi3 interacts with both Gβγ and GIV, we asked whether the α-subunit can interact with GIV when it is assembled into an intact heterotrimer. That this is the case is suggested by the fact that the Gβ subunit could be coimmunoprecipitated from brain lysates with GIV antibodies and that this interaction was abolished in the presence of GDP and AlF4− (Fig. 6 D).

Bottom Line: We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate.A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane.Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Show MeSH
Related in: MedlinePlus