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Gβγ subunits inhibit Epac-induced melanoma cell migration.

Baljinnyam E, Umemura M, De Lorenzo MS, Xie LH, Nowycky M, Iwatsubo M, Chen S, Goydos JS, Iwatsubo K - BMC Cancer (2011)

Bottom Line: We next examined the effect of mSIRK on Epac-induced Ca 2+ response.In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration.These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark 07103, USA.

ABSTRACT

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.

Methods: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.

Results: The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

Conclusion: We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

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Ca 2+ signal cross talk between Epac and Gβγ. Activation of GPCR releases two signaling molecules, Gα and Gβγ. Gα activates cAMP production, leading to Ca 2+ release from IP3 receptor in the ER via Epac/PLC/IP3 pathway. Ca 2+ release from IP3 receptor induces cell migration. On the other hand, Gβγ stimulates Ca 2+ influx from the extracellular space, leading to activation of calmodulin and the following inactivation of IP3 receptor. Finally, Gβγ inhibits Epac-induced Ca 2+ release from the ER, leading to inhibition of cell migration.
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Figure 5: Ca 2+ signal cross talk between Epac and Gβγ. Activation of GPCR releases two signaling molecules, Gα and Gβγ. Gα activates cAMP production, leading to Ca 2+ release from IP3 receptor in the ER via Epac/PLC/IP3 pathway. Ca 2+ release from IP3 receptor induces cell migration. On the other hand, Gβγ stimulates Ca 2+ influx from the extracellular space, leading to activation of calmodulin and the following inactivation of IP3 receptor. Finally, Gβγ inhibits Epac-induced Ca 2+ release from the ER, leading to inhibition of cell migration.

Mentions: In the present study, we demonstrated that Gβγ interferes with the Ca 2+ signaling evoked by Epac, leading to an inhibition of Epac-induced cell migration. We found that Gβγ activates Ca 2+ entry from the extracellular space, which inhibits Epac-induced cytosolic Ca 2+ elevation, and cell migration. Calmodulin is presumably involved in these Gβγ's effects. Since Gβγ is a common molecule located downstream of various GPCRs, our findings would provide novel insights in terms of Ca 2+ signaling, cell migration, and cross talk of intracellular signaling in melanoma (Figure 5).


Gβγ subunits inhibit Epac-induced melanoma cell migration.

Baljinnyam E, Umemura M, De Lorenzo MS, Xie LH, Nowycky M, Iwatsubo M, Chen S, Goydos JS, Iwatsubo K - BMC Cancer (2011)

Ca 2+ signal cross talk between Epac and Gβγ. Activation of GPCR releases two signaling molecules, Gα and Gβγ. Gα activates cAMP production, leading to Ca 2+ release from IP3 receptor in the ER via Epac/PLC/IP3 pathway. Ca 2+ release from IP3 receptor induces cell migration. On the other hand, Gβγ stimulates Ca 2+ influx from the extracellular space, leading to activation of calmodulin and the following inactivation of IP3 receptor. Finally, Gβγ inhibits Epac-induced Ca 2+ release from the ER, leading to inhibition of cell migration.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141774&req=5

Figure 5: Ca 2+ signal cross talk between Epac and Gβγ. Activation of GPCR releases two signaling molecules, Gα and Gβγ. Gα activates cAMP production, leading to Ca 2+ release from IP3 receptor in the ER via Epac/PLC/IP3 pathway. Ca 2+ release from IP3 receptor induces cell migration. On the other hand, Gβγ stimulates Ca 2+ influx from the extracellular space, leading to activation of calmodulin and the following inactivation of IP3 receptor. Finally, Gβγ inhibits Epac-induced Ca 2+ release from the ER, leading to inhibition of cell migration.
Mentions: In the present study, we demonstrated that Gβγ interferes with the Ca 2+ signaling evoked by Epac, leading to an inhibition of Epac-induced cell migration. We found that Gβγ activates Ca 2+ entry from the extracellular space, which inhibits Epac-induced cytosolic Ca 2+ elevation, and cell migration. Calmodulin is presumably involved in these Gβγ's effects. Since Gβγ is a common molecule located downstream of various GPCRs, our findings would provide novel insights in terms of Ca 2+ signaling, cell migration, and cross talk of intracellular signaling in melanoma (Figure 5).

Bottom Line: We next examined the effect of mSIRK on Epac-induced Ca 2+ response.In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration.These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology and Molecular Medicine, New Jersey Medical School-University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark 07103, USA.

ABSTRACT

Background: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.

Methods: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.

Results: The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.

Conclusion: We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

Show MeSH
Related in: MedlinePlus