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. 2023 Feb 16;13(1):2771.
doi: 10.1038/s41598-023-29639-0.

The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics

Dhanushan Wijayaratna  1   2 , Kasun Ratnayake  1 , Sithurandi Ubeysinghe  1   2 , Dinesh Kankanamge  1   3 , Mithila Tennakoon  1   2 , Ajith Karunarathne  4   5
Affiliations

The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics

Dhanushan Wijayaratna et al. Sci Rep. .

Abstract

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
While GPCR and G proteins stay active, Gi/o-GPCR induced PIP3 subsides. (A) RAW264.7 cells exhibited robust PIP3 production upon α2AR activation, which shows subsequent significant attenuation. Images: RAW264.7 cells expressing Akt-PH-Venus (PIP3 sensor) and α2AR-CFP (not shown). Confocal time-lapse imaging of PIP3 sensor dynamics was performed using 515 nm excitation. α2AR was activated using 100 µM NE at 1 min. Images show PIP3 sensor translocation from the cytosol to the PM upon α2AR activation, which peaks at 300 s, and significantly reverses back to the cytosol in 5–10 min. The corresponding plot shows PIP3 sensor dynamics in the cytosol of the cells. The whisker box plot shows the half-time (t1/2) of PIP3 generation and attenuation (n = 13). (B) RAW264.7 cells expressing α2AR-CFP, Akt-PH-mCh, and Venus-mini-Gi exhibited simultaneous mini-Gi PM-recruitment and PIP3 production (yellow arrows) upon α2AR activation. 100 µM NE was added at 1 min to activate α2AR. Mini-Gi stayed recruited to the PM, while PIP3 showed attenuation. Upon addition of α2AR inhibitor yohimbine (50 µM), both mini-Gi and PIP3 sensors showed complete reversal to the cytosol (blue arrows). Cells were imaged using 515 nm (to monitor mini-Gi), and 594 nm (to capture the PIP3 sensor) excitation. The corresponding plot shows the mini-Gi (green) and PIP3 (red) sensor dynamics in the cytosol of the cells. The whisker box plot shows the half-time (t1/2) of PIP3 generation and attenuation rates (n = 11). Kymographs show PIP3 levels on the plasma membrane. Average curves were plotted using cells from ≥ 3 independent experiments. ‘n’ denotes the number of cells. Error bars represent SEM (standard error). The scale bar = 5 µm. GPCR G protein-coupled receptor, PIP3 Phosphatidylinositol 3,4,5 triphosphate, α2AR Alpha-2-adrenergic receptor, NE Norepinephrine, CFP cyan fluorescence protein, mCh mCherry, PM plasma membrane.
Figure 2
Figure 2
PIP3 attenuation is not a result of substrate (PIP2) depletion. (A) RAW264.7 cells expressing α2AR-CFP, GRPR, Akt-PH-Venus, and mCh-PH exhibited concurrent PIP2 hydrolysis—PIP3 production, and PIP3 attenuation upon simultaneous GRPR and α2AR activation at 1 min using 100 µM NE and 1 µM bombesin, respectively. Cells were imaged using 515 nm (for PIP3 sensor), and 594 nm (for PIP2 sensor). The corresponding plot shows the dynamics of PIP2 hydrolysis (red) and PIP3 production and attenuation (green). The plot compares the normalized PIP2 (red) and PIP3 (green) sensor dynamics at the PM. The two plots show that the changes in the sensor fluorescence in cytosol or the PM can be used to capture the dynamics of PIP2 and PIP3 at the PM (n = 19). (B) RAW264.7 cells expressing α2AR-CFP, GRPR, Akt-PH-Venus, and mCh-PH exhibited PIP3 production and attenuation upon α2AR activation (at 1 min), while PIP2 at the PM stayed intact. The corresponding plot shows the dynamics of PIP3 (green) and PIP2 (red) (n = 16). (C) The whisker box plots show PIP3 generation rates, attenuation rates, and attenuation extents for A and B experiments indicating the influence of PIP2 hydrolysis on PIP3. PIP3 generation rate in cells with PIP2 hydrolysis (+ Bombesin) was significantly lower compared to cells without PIP2 hydrolysis (-Bombesin). However, the rate and extent of PIP3 attenuation under both conditions showed no significant difference. (D) RAW264.7 cells expressing α2AR-CFP and Akt-PH-Venus exhibited PIP3 production and attenuation upon α2AR activation in the presence or absence of phosphatase inhibitor bpV(phen) (5 µM). The corresponding plot shows the dynamics of PIP3 production and attenuation in the presence of bpV(phen) (green) (n = 14), and in the absence of bpV(phen) (red) (n = 12). The whisker box plots show PIP3 generation rates, attenuation rates, and attenuation extents in the presence and absence of bpV(phen). The generation and attenuation rates and attenuation extents showed no significant difference under both conditions. Kymographs show PIP3 levels on the plasma membrane. Statistical comparisons were performed using One-way-ANOVA (p < 0.05). Average curves were plotted using cells from ≥ 3 independent experiments. ‘n’ denotes the number of cells’ data used to plot the average curve. The error bars represent SEM. Scale bar = 5 µm. PIP2 phosphatidylinositol 4,5-bisphosphate, GRPR gastrin-releasing peptide receptor.
Figure 3
Figure 3
Gβγ and PIP3 attenuation (A) RAW264.7 cells expressing α2AR-CFP, Akt-PH-Venus and mCh-Gγ3 exhibited simultaneous mCh-Gγ3 translocation and PIP3 production upon α2AR activation (at 1 min). The translocated mCherry-Gγ3 stayed at IMs (white arrows), while PIP3 level reached an equilibrium after attenuation. In addition to 515 nm excitation for YFP, 594 nm excitation was used to capture mCherry. The corresponding plot shows mCh-Gγ3 (red) and PIP3 (green) dynamics in the cytosol of the cells (n = 20). (B) RAW264.7 cells expressing α2AR-CFP, Akt-PH-Venus and mCh-Gγ3-CC mutant exhibited PIP3 production upon α2AR activation. As expected, the images and the plot show that translocation deficient Gγ mutant (Gγ3-CC) remained at the PM despite receptor activation. PIP3 showed no significant attenuation (n = 16). (C) The whisker box plots show the extents of % PIP3 attenuation in RAW264.7 cells with endogenous Gβγ (Control), or Gγ3 WT, or Gγ3-CC mutant. Extent of PIP3 attenuation was quantified using the increase in the mean cytosolic fluorescence due to PIP3 attenuation. The observed % attenuations were significantly different from each other (p < 0.05). Kymographs show PIP3 levels on the plasma membrane. Averages were plotted using cells from n ≥ 3 independent experiments. ‘n’ denotes the number of cells’ data used to plot the average curve. The error bars represent SEM (standard error of mean). The scale bar = 5 µm. IMs internal membranes.
Figure 4
Figure 4
Despite the source, Gβγ entry induces PIP3 generation and loss results in attenuation. (A) RAW264.7 cells expressing GRPR, YFP-β1, mCh-PH, Gαq-CFP, and Lyn-HTH exhibited robust β1 translocation upon GRPR activation with bombesin (1 µM at 1 min) (n = 8). However, GRPR activation did not induce PIP2 hydrolysis due to the presence of Lyn-HTH. The plot shows the dynamics of β1 translocation (green) and PIP2 (red) in cells. (B) RAW264.7 cells expressing GRPR, Akt-PH-Venus, Gαq-CFP, and Lyn-HTH exhibited PIP3 production and attenuation upon GRPR activation with 1 µM bombesin (n = 8). However, control cells expressing only GRPR and Akt-PH-Venus did not show PIP3 production upon GRPR activation (n = 9). The plot shows PIP3 production and attenuation only in Gαq-expressing cells (green), however not in control cell lacking introduced Gαq. (C) RAW264.7 cells expressing α2AR-CFP, GRPR, Akt-PH-Venus, Gαq-CFP and Lyn-HTH exhibited PIP3 production and attenuation upon α2AR activation. After PIP3 attenuation, 1 µM bombesin was added (at 20 min) to activate GRPR. GRPR activation in these Gαq cells caused disruption of attenuation in the form of PIP3 increase. The plot shows the initial PIP3 generation and attenuation after α2AR activation, and subsequent GRPR-induced PIP3 regeneration in Gαq background (n = 10). Kymographs show PIP3 levels on the plasma membrane. Average curves were plotted using cells from ≥ 3 independent experiments. ‘n’ denotes the number of cells’ data used to plot the average curve. The error bars represent SEM (standard error of mean). The scale bar = 5 µm.
Figure 5
Figure 5
PIP3 generation upon localized GPCR-G protein activation is attenuation-resistant. (A) RAW264.7 cells expressing blue opsin-mTq and Akt-PH-mCh showed attenuation-resistant PIP3 upon localized activation, while global activation induced fast attenuating PIP3. Before activation cells were incubated with 1 µM 11-cis-retinal. Spatially confined blue light pulses (blue boxes) (1 Hz) were used to activate by blue light locally and globally, while confocal imaging of mCherry using 594 nm excitation. Cells activated with localized blue light produced non-attenuating PIP3 response. Cells also showed a directional migration towards the blue light. On the contrary, cells exposed to blue light globally initially showed a robust global PIP3 production. However, PIP3 in these cells showed the attenuation, similar to the attenuation observed upon activation of α2AR. (B) Localized acceleration of GTP hydrolysis on Gα in α2AR activated RAW264.7 cells also showed attenuation-resistant PIP3 generation. Cells expressing α2AR-CFP, Akt-PH-Venus, CRY2-mCh-RGS4Δ, and CIBN-CAAX showed localized PIP3 production when α2AR was activated in a cell after CRY2-mCh-RGS4Δ localized to one side of the cell. A localized blue light pulse was provided to recruit CRY2-mCh-RGS4Δ to one side of the cell. Once RGS4Δ is recruited, (indicated by mCherry fluorescence concentrating to one side of the cell), NE (100 µM) was added to the medium. This resulted in localized PIP3 at the opposite side to the blue pulse (yellow arrow). Cells were imaged using 515 nm (to capture PIP3 sensor), and 594 nm (to capture RGS4Δ). Although images show data from only one cell, experiments were conducted in multiple cells with > 3 independent experiments to test the reproducibility of the results. (C) PIP3-attenuated (post-GPCR activation) RAW264.7 cells show disruption of attenuation upon localized inhibition of  G protein activity. Cells expressed α2AR-CFP, Akt-PH-Venus, CRY2-mCh-RGS4Δ, and CIBN-CAAX. Initially α2AR was activated with 100 µM NE, and PIP3 was produced and significantly attenuated in 10 min. Upon blue light induced recruitment of CRY2-mCh-RGS4Δ to one side of the cell, PIP3 was produced at the opposite side (yellow arrow, 15 and 20 min), indicating attenuation disruption. When the opposite side of the same cell was exposed to blue light (yellow arrow, 25 and 30 min), PIP3 was produced at the opposite side of localized RGS4Δ. Experiments were conducted in multiple cells with > 3 independent experiments to test the reproducibility of the results. The blue box indicates the blue light. The scale bar = 5 µm. mTq mTurquoise, FRAPPA fluorescence recovery after photo-bleaching and photo-activation, RGS4 regulator of G protein signaling 4.
Figure 6
Figure 6
Heterotrimer concentration gradient across the cell breaks PIP3 attenuation. (A) Optical targeting of RGS4Δ to one side of a cell induces localized heterotrimer regeneration. RAW264.7 cells expressing α2AR-CFP, YFP-Gβ1, CRY2-mCh-RGS4Δ, and CIBN-CAAX initially exhibited robust Gβ1 translocation upon α2AR activation (300 s). Then, upon localized blue light-induced recruitment of CRY2-mCh-RGS4Δ resulted in an increase of PM-bound YFP fluorescence at the opposite side, suggesting heterotrimer shuttling from the RGS4Δ-recruited side to the opposite side (400 s, 500 s, and green plot). The corresponding plot shows the dynamics of YFP-β1 with localized inhibition of CRY2-mCh-RGS4Δ. (B) Reversible blue light targeting of RGS4Δ to the PM allows optogenetic control of heterotrimer concentration at the PM. Upon α2AR activation, cells showed YFP-Gβ1 translocation (240 s, grey arrow). Blue light exposure recruited CRY2-mCh-RGS4Δ to the PM, which induced reverse translocation of Gβ1 to the PM (400 s, white arrow).Termination of blue light that resulted in the release of PM-bound CRY2-mCh-RGS4Δ again resulted in YFP-Gβ1 translocation to the IMs (1200 s, grey arrow). Again, we recruited CRY2-mCh-RGS4Δ to the PM using global blue light and it induced reverse translocation of Gβ1 to the PM (1800s, white arrow). Thus ON–OFF blue light allowed reversible control of heterotrimer concentration. The kymograph shows the dynamics of YFP-β1 with global inhibition by CRY2-mCh-RGS4Δ. (C) Fluorescence recovery after subcellular photobleaching (FRAP) in RAW264.7 cells expressing YFP-Gβ1. The corresponding plot shows fluorescence recovery after photobleaching (FRAP) in photobleached PM regions of the cell (green), and fluorescence loss in photobleaching (FLIP) in non-photobleached PM regions. The whisker plot shows the shuttling half times of Gβ1 in FRAP (green) and FLIP (red) (n = 7). Average curves were plotted using cells from ≥ 3 independent experiments. ‘n’ denotes the number of cells’ data used to plot the average curve. The error bars represent SEM (standard error of mean). The scale bar = 5 µm.
Figure 7
Figure 7
Proposed mechanisms for GPCR-G protein-induced PIP3 regulation. Gi/o-coupled GPCR activation induces robust free Gβγ, which stimulates PI3Kγ resulting in PIP3 generation (1). The subsequent partial PIP3 attenuation (2) is due to the translocation of Gβγ away from the plasma membrane to the internal membranes. The steady state of partial PIP3 attenuation is achieved at the equilibrium of Gβγ translocation between the plasma membrane and the internal membranes. Complete inactivation of GPCR or G proteins result in the PIP3 level returning to the basal level.
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