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Gαi3-Dependent Inhibition of JNK Activity on Intracellular Membranes.

Bastin G, Yang JY, Heximer SP - Front Bioeng Biotechnol (2015)

Bottom Line: The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane.Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell.This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Heart and Stroke, Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto , Toronto, ON , Canada.

ABSTRACT
Heterotrimeric G-protein signaling has been shown to modulate a wide variety of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) family. The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane. Using a unique set of G-protein signaling tools developed in our laboratory, we show that subcellular domain-specific JNK activity is inhibited by the activation of Gαi3, the Gαi isoform found predominantly within intracellular membranes, such as the endoplasmic reticulum (ER)-Golgi interface, and their associated vesicle pools. Regulators of intracellular Gαi3, including activator of G-protein signaling 3 (AGS3) and the regulator of G-protein signaling protein 4 (RGS4), have a marked impact on the regulation of JNK activity. Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell. This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

RGS4 Shows preferential co-localization with Gαi3RC. HEK293 cells were co-transfected with RGS4-YFP (yellow), and either Gαi3 (WT)- or Gαi3 (RC)-CFP (blue) to assess the extent of co-localization using spinning disk confocal microscopy. The merged view is a composite two-channel view of cells expressing the two indicated constructs. Data are representative of at least 100 dual-transfected cells. Arrows indicate co-localization on intracellular endosomal structures between RGS4 and Gαi3. Scale bars represent 1 μm.
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Figure 1: RGS4 Shows preferential co-localization with Gαi3RC. HEK293 cells were co-transfected with RGS4-YFP (yellow), and either Gαi3 (WT)- or Gαi3 (RC)-CFP (blue) to assess the extent of co-localization using spinning disk confocal microscopy. The merged view is a composite two-channel view of cells expressing the two indicated constructs. Data are representative of at least 100 dual-transfected cells. Arrows indicate co-localization on intracellular endosomal structures between RGS4 and Gαi3. Scale bars represent 1 μm.

Mentions: Our previous work with the RGS4 protein showed that there exists at least two distinct membrane-bound pools of RGS4 within mammalian cells (Bastin et al., 2012). These pools consisted of the known pool at the plasma membrane and a newly appreciated pool that targeted intracellular membranes, such as endosomes, Golgi, and other punctate structures (Bastin and Heximer, 2013). The demonstration that differential palmitoylation of cysteine residues in the amino-terminus of RGS4 could alter its distribution between these two membrane pools guided efforts to demonstrate potential functional differences between RGS4 at these different locations in the cell. While it was relatively straightforward to show that prevention of RGS4 trafficking to the plasma membrane via mutation of Cys12, the palmitoylation site adjacent to its membrane-targeting amphipathic helix, could inhibit the ability of RGS4 to inhibit Gq-mediated signaling from the plasma membrane, it was more complicated to demonstrate a functional consequence of the Cys2 mutation that prevented RGS4 localization to the intracellular membrane pool. Our attention turned to regulation of intracellular Gαi3 after we discovered overlapping expression of RGS4-YFP-containing punctae with those targeted by CFP-tagged Gαi3 (Figure 1). Notably, the extent of co-localization on intracellular punctae was typically greater (larger number of punctae/cell) between RGS4 and the constitutively active Gαi3 (R178C; RC) compared to Gαi3 (WT). A significant plasma membrane signal was also present for Gαi3, suggesting that like RGS4, Gαi3 may also traffic between the plasma membrane and the intracellular membrane compartments. These data suggested that intracellular RGS4 and Gαi3 may target some of the same intracellular domains to co-ordinately regulate specific intracellular signaling pathways. Consistent with previous reports, Gαi3 and AGS3 were also found together on intracellular membrane structures in our expression system (Figure 2). Notably, in the presence of AGS3, we observed dramatically reduced co-localization of RGS4 and WT Gαi3 compared to Gαi3 (RC) (marked by arrowheads in Figures 3A,B). Together, these data suggested that Gαi3 may shuttle between AGS3-containing (GDP-bound Gαi3) and RGS4-containing (GTP-bound Gαi3) compartments depending on its state of activation. These data simply reflect the preferences of the RGS box for GTP-bound Gαi3 and AGS3 GPR/GoLoco motifs for inactive/GDP-bound Gαi3; however, preliminary evidence to argue against this notion comes from experiments showing the catalytically dead RGS4 (EN-AA) mutant had similar co-localization with Gαi3 (RC)-containing punctae as wild-type RGS4. Moreover, the co-expression of Gαi3 with either RGS4 or AGS3 did not alter their localization in any discernable manner. These data suggest that these proteins traffic together on similar endosome-like structures where they may be co-localized, without necessarily interacting stably with one another. Such a system would allow RGS4 to fine tune the levels of Gαi3 activity, while they are in the same compartment and then pass off inactive Gαi3-GDP to another membrane compartment (presumably an AGS3-containing one), where Gαi3 could be primed for reactivation.


Gαi3-Dependent Inhibition of JNK Activity on Intracellular Membranes.

Bastin G, Yang JY, Heximer SP - Front Bioeng Biotechnol (2015)

RGS4 Shows preferential co-localization with Gαi3RC. HEK293 cells were co-transfected with RGS4-YFP (yellow), and either Gαi3 (WT)- or Gαi3 (RC)-CFP (blue) to assess the extent of co-localization using spinning disk confocal microscopy. The merged view is a composite two-channel view of cells expressing the two indicated constructs. Data are representative of at least 100 dual-transfected cells. Arrows indicate co-localization on intracellular endosomal structures between RGS4 and Gαi3. Scale bars represent 1 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4555961&req=5

Figure 1: RGS4 Shows preferential co-localization with Gαi3RC. HEK293 cells were co-transfected with RGS4-YFP (yellow), and either Gαi3 (WT)- or Gαi3 (RC)-CFP (blue) to assess the extent of co-localization using spinning disk confocal microscopy. The merged view is a composite two-channel view of cells expressing the two indicated constructs. Data are representative of at least 100 dual-transfected cells. Arrows indicate co-localization on intracellular endosomal structures between RGS4 and Gαi3. Scale bars represent 1 μm.
Mentions: Our previous work with the RGS4 protein showed that there exists at least two distinct membrane-bound pools of RGS4 within mammalian cells (Bastin et al., 2012). These pools consisted of the known pool at the plasma membrane and a newly appreciated pool that targeted intracellular membranes, such as endosomes, Golgi, and other punctate structures (Bastin and Heximer, 2013). The demonstration that differential palmitoylation of cysteine residues in the amino-terminus of RGS4 could alter its distribution between these two membrane pools guided efforts to demonstrate potential functional differences between RGS4 at these different locations in the cell. While it was relatively straightforward to show that prevention of RGS4 trafficking to the plasma membrane via mutation of Cys12, the palmitoylation site adjacent to its membrane-targeting amphipathic helix, could inhibit the ability of RGS4 to inhibit Gq-mediated signaling from the plasma membrane, it was more complicated to demonstrate a functional consequence of the Cys2 mutation that prevented RGS4 localization to the intracellular membrane pool. Our attention turned to regulation of intracellular Gαi3 after we discovered overlapping expression of RGS4-YFP-containing punctae with those targeted by CFP-tagged Gαi3 (Figure 1). Notably, the extent of co-localization on intracellular punctae was typically greater (larger number of punctae/cell) between RGS4 and the constitutively active Gαi3 (R178C; RC) compared to Gαi3 (WT). A significant plasma membrane signal was also present for Gαi3, suggesting that like RGS4, Gαi3 may also traffic between the plasma membrane and the intracellular membrane compartments. These data suggested that intracellular RGS4 and Gαi3 may target some of the same intracellular domains to co-ordinately regulate specific intracellular signaling pathways. Consistent with previous reports, Gαi3 and AGS3 were also found together on intracellular membrane structures in our expression system (Figure 2). Notably, in the presence of AGS3, we observed dramatically reduced co-localization of RGS4 and WT Gαi3 compared to Gαi3 (RC) (marked by arrowheads in Figures 3A,B). Together, these data suggested that Gαi3 may shuttle between AGS3-containing (GDP-bound Gαi3) and RGS4-containing (GTP-bound Gαi3) compartments depending on its state of activation. These data simply reflect the preferences of the RGS box for GTP-bound Gαi3 and AGS3 GPR/GoLoco motifs for inactive/GDP-bound Gαi3; however, preliminary evidence to argue against this notion comes from experiments showing the catalytically dead RGS4 (EN-AA) mutant had similar co-localization with Gαi3 (RC)-containing punctae as wild-type RGS4. Moreover, the co-expression of Gαi3 with either RGS4 or AGS3 did not alter their localization in any discernable manner. These data suggest that these proteins traffic together on similar endosome-like structures where they may be co-localized, without necessarily interacting stably with one another. Such a system would allow RGS4 to fine tune the levels of Gαi3 activity, while they are in the same compartment and then pass off inactive Gαi3-GDP to another membrane compartment (presumably an AGS3-containing one), where Gαi3 could be primed for reactivation.

Bottom Line: The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane.Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell.This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Heart and Stroke, Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto , Toronto, ON , Canada.

ABSTRACT
Heterotrimeric G-protein signaling has been shown to modulate a wide variety of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) family. The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane. Using a unique set of G-protein signaling tools developed in our laboratory, we show that subcellular domain-specific JNK activity is inhibited by the activation of Gαi3, the Gαi isoform found predominantly within intracellular membranes, such as the endoplasmic reticulum (ER)-Golgi interface, and their associated vesicle pools. Regulators of intracellular Gαi3, including activator of G-protein signaling 3 (AGS3) and the regulator of G-protein signaling protein 4 (RGS4), have a marked impact on the regulation of JNK activity. Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell. This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells.

No MeSH data available.


Related in: MedlinePlus