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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.

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Working model. (1) GIV preferentially and avidly binds to inactive GDP-bound Gαi3. Interaction with Gαi3 induces a change in conformation of GIV (dotted arrow). In a quiescent cell, this step is likely to occur predominantly on vesicles near the Golgi where Gαi3 and GIV were previously shown to colocalize. (2) Upon a chemotactic stimulus, Akt signaling is initiated and results in phosphorylation of GIV at S1416. This critical phosphorylation step is facilitated by the favorable conformation of GIV induced by direct interaction with Gαi3. Phosphorylation at S1416 was previously shown to be necessary for GIV's functions during cell migration (Enomoto et al., 2005). (3) Upon phosphorylation, GIV continues to preferentially and avidly bind to Gαi3; however, it selectively loses its affinity for PI4P (Enomoto et al., 2005), a phosphoinositide that is enriched in the Golgi, and redistributes from Golgi membranes to the peripheral actin bed near the PM. (4) Activation of Gαi3 occurs, likely downstream of ligand occupied receptors, and is the key event that mediates simultaneous dissociation of Gβγ and GIV from the Gi3–GIV complexes. (5) Released Gβγ activates PI3K-dependent Akt signaling (Lilly and Devreotes, 1995). (6) Released phospho-GIV enhances the initial Akt signaling (Anai et al., 2005), remodels actin, and promotes migration. (7) This second phase of Akt signal enhancement is critical for formation of the leading edge during polarized cell migration. Cyclical activation and inactivation of Gαi in vivo thus contributes to progressive Akt enhancement, which promotes further cycles of GIV phosphorylation (8 and 2), redistribution of phosphorylated GIV from Golgi to PM/actin (3), and subsequent release from the Gi3–GIV complexes upon activation of Gαi3 (4). This contributes to the previously observed accumulation of phosphorylated GIV at the leading edge (Enomoto et al., 2005) where GIV rapidly remodels actin to generate pseudopods.
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fig9: Working model. (1) GIV preferentially and avidly binds to inactive GDP-bound Gαi3. Interaction with Gαi3 induces a change in conformation of GIV (dotted arrow). In a quiescent cell, this step is likely to occur predominantly on vesicles near the Golgi where Gαi3 and GIV were previously shown to colocalize. (2) Upon a chemotactic stimulus, Akt signaling is initiated and results in phosphorylation of GIV at S1416. This critical phosphorylation step is facilitated by the favorable conformation of GIV induced by direct interaction with Gαi3. Phosphorylation at S1416 was previously shown to be necessary for GIV's functions during cell migration (Enomoto et al., 2005). (3) Upon phosphorylation, GIV continues to preferentially and avidly bind to Gαi3; however, it selectively loses its affinity for PI4P (Enomoto et al., 2005), a phosphoinositide that is enriched in the Golgi, and redistributes from Golgi membranes to the peripheral actin bed near the PM. (4) Activation of Gαi3 occurs, likely downstream of ligand occupied receptors, and is the key event that mediates simultaneous dissociation of Gβγ and GIV from the Gi3–GIV complexes. (5) Released Gβγ activates PI3K-dependent Akt signaling (Lilly and Devreotes, 1995). (6) Released phospho-GIV enhances the initial Akt signaling (Anai et al., 2005), remodels actin, and promotes migration. (7) This second phase of Akt signal enhancement is critical for formation of the leading edge during polarized cell migration. Cyclical activation and inactivation of Gαi in vivo thus contributes to progressive Akt enhancement, which promotes further cycles of GIV phosphorylation (8 and 2), redistribution of phosphorylated GIV from Golgi to PM/actin (3), and subsequent release from the Gi3–GIV complexes upon activation of Gαi3 (4). This contributes to the previously observed accumulation of phosphorylated GIV at the leading edge (Enomoto et al., 2005) where GIV rapidly remodels actin to generate pseudopods.

Mentions: This paper describes a novel mechanism by which Gαi3 serves as a molecular switch that dictates the formation of the leading edge during cell migration via regulation of the distribution, phosphorylation, and functions of GIV. Without Gαi3, Akt amplification downstream of both RTK and GPCR failed to occur, actin remodeling was inhibited, and cells failed to undergo polarized migration after scratch wounding. We also showed that Gαi3 localizes preferentially within pseudopods at the leading edge and that activation of Gαi3 is essential for migration. Collectively, these results support a working model (Fig. 9) of how the switch operates in few key steps: inactive Gi3 heterotrimer interacts avidly with GIV and induces a change in the conformation of GIV. Upon a chemotactic stimulus (when Akt signaling is initiated) the Gαi3-bound conformation of GIV facilitates phosphorylation of GIV at a critical Ser residue that is necessary for its functions at the leading edge. Subsequently, activation of Gαi3 triggers dissociation of the Gi3-GIV macromolecular complex releasing Gβγ-subunits and GIV simultaneously. Released Gβγ participates in localized PI3K-Akt activation (Lilly and Devreotes, 1995), and released GIV amplifies and propagates this Akt signaling (Chen et al., 2003; Postma et al., 2004). Amplification of cellular Akt activity via sequential coupling and uncoupling of Gαi3 and GIV is likely to set up a positive feedback loop that mediates phosphorylation of further GIV molecules to remodel actin and form the leading edge. In this feedback loop, G proteins are presumably directly activated by GPCRs or indirectly transactivated via RTKs.


Activation of Galphai3 triggers cell migration via regulation of GIV.

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

Working model. (1) GIV preferentially and avidly binds to inactive GDP-bound Gαi3. Interaction with Gαi3 induces a change in conformation of GIV (dotted arrow). In a quiescent cell, this step is likely to occur predominantly on vesicles near the Golgi where Gαi3 and GIV were previously shown to colocalize. (2) Upon a chemotactic stimulus, Akt signaling is initiated and results in phosphorylation of GIV at S1416. This critical phosphorylation step is facilitated by the favorable conformation of GIV induced by direct interaction with Gαi3. Phosphorylation at S1416 was previously shown to be necessary for GIV's functions during cell migration (Enomoto et al., 2005). (3) Upon phosphorylation, GIV continues to preferentially and avidly bind to Gαi3; however, it selectively loses its affinity for PI4P (Enomoto et al., 2005), a phosphoinositide that is enriched in the Golgi, and redistributes from Golgi membranes to the peripheral actin bed near the PM. (4) Activation of Gαi3 occurs, likely downstream of ligand occupied receptors, and is the key event that mediates simultaneous dissociation of Gβγ and GIV from the Gi3–GIV complexes. (5) Released Gβγ activates PI3K-dependent Akt signaling (Lilly and Devreotes, 1995). (6) Released phospho-GIV enhances the initial Akt signaling (Anai et al., 2005), remodels actin, and promotes migration. (7) This second phase of Akt signal enhancement is critical for formation of the leading edge during polarized cell migration. Cyclical activation and inactivation of Gαi in vivo thus contributes to progressive Akt enhancement, which promotes further cycles of GIV phosphorylation (8 and 2), redistribution of phosphorylated GIV from Golgi to PM/actin (3), and subsequent release from the Gi3–GIV complexes upon activation of Gαi3 (4). This contributes to the previously observed accumulation of phosphorylated GIV at the leading edge (Enomoto et al., 2005) where GIV rapidly remodels actin to generate pseudopods.
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Related In: Results  -  Collection

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fig9: Working model. (1) GIV preferentially and avidly binds to inactive GDP-bound Gαi3. Interaction with Gαi3 induces a change in conformation of GIV (dotted arrow). In a quiescent cell, this step is likely to occur predominantly on vesicles near the Golgi where Gαi3 and GIV were previously shown to colocalize. (2) Upon a chemotactic stimulus, Akt signaling is initiated and results in phosphorylation of GIV at S1416. This critical phosphorylation step is facilitated by the favorable conformation of GIV induced by direct interaction with Gαi3. Phosphorylation at S1416 was previously shown to be necessary for GIV's functions during cell migration (Enomoto et al., 2005). (3) Upon phosphorylation, GIV continues to preferentially and avidly bind to Gαi3; however, it selectively loses its affinity for PI4P (Enomoto et al., 2005), a phosphoinositide that is enriched in the Golgi, and redistributes from Golgi membranes to the peripheral actin bed near the PM. (4) Activation of Gαi3 occurs, likely downstream of ligand occupied receptors, and is the key event that mediates simultaneous dissociation of Gβγ and GIV from the Gi3–GIV complexes. (5) Released Gβγ activates PI3K-dependent Akt signaling (Lilly and Devreotes, 1995). (6) Released phospho-GIV enhances the initial Akt signaling (Anai et al., 2005), remodels actin, and promotes migration. (7) This second phase of Akt signal enhancement is critical for formation of the leading edge during polarized cell migration. Cyclical activation and inactivation of Gαi in vivo thus contributes to progressive Akt enhancement, which promotes further cycles of GIV phosphorylation (8 and 2), redistribution of phosphorylated GIV from Golgi to PM/actin (3), and subsequent release from the Gi3–GIV complexes upon activation of Gαi3 (4). This contributes to the previously observed accumulation of phosphorylated GIV at the leading edge (Enomoto et al., 2005) where GIV rapidly remodels actin to generate pseudopods.
Mentions: This paper describes a novel mechanism by which Gαi3 serves as a molecular switch that dictates the formation of the leading edge during cell migration via regulation of the distribution, phosphorylation, and functions of GIV. Without Gαi3, Akt amplification downstream of both RTK and GPCR failed to occur, actin remodeling was inhibited, and cells failed to undergo polarized migration after scratch wounding. We also showed that Gαi3 localizes preferentially within pseudopods at the leading edge and that activation of Gαi3 is essential for migration. Collectively, these results support a working model (Fig. 9) of how the switch operates in few key steps: inactive Gi3 heterotrimer interacts avidly with GIV and induces a change in the conformation of GIV. Upon a chemotactic stimulus (when Akt signaling is initiated) the Gαi3-bound conformation of GIV facilitates phosphorylation of GIV at a critical Ser residue that is necessary for its functions at the leading edge. Subsequently, activation of Gαi3 triggers dissociation of the Gi3-GIV macromolecular complex releasing Gβγ-subunits and GIV simultaneously. Released Gβγ participates in localized PI3K-Akt activation (Lilly and Devreotes, 1995), and released GIV amplifies and propagates this Akt signaling (Chen et al., 2003; Postma et al., 2004). Amplification of cellular Akt activity via sequential coupling and uncoupling of Gαi3 and GIV is likely to set up a positive feedback loop that mediates phosphorylation of further GIV molecules to remodel actin and form the leading edge. In this feedback loop, G proteins are presumably directly activated by GPCRs or indirectly transactivated via RTKs.

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