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. 2016 Mar;147(3):255-71.
doi: 10.1085/jgp.201511477.

Contributions of Protein Kinases and β-Arrestin to Termination of Protease-Activated Receptor 2 Signaling

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Free PMC article

Contributions of Protein Kinases and β-Arrestin to Termination of Protease-Activated Receptor 2 Signaling

Seung-Ryoung Jung et al. J Gen Physiol. .
Free PMC article

Abstract

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2.

Figures

Figure 1.
Figure 1.
Intracellular Ca2+ concentration rises transiently during the activation of PAR2. (A and B) PDECs with endogenous PAR2 (A) and tsA201 cells with overexpressed PAR2-GFP (B) were preloaded with Ca2+-sensitive fura-2 AM dye and treated with 100 nM trypsin or 100 µM AP (sequence SLIGKT), respectively. Ca2+ is expressed as fluorescence excitation ratios (F340/F380) of the dye. The traces are means of seven PDECs and five tsA201 cells each, respectively. The dashed lines indicate the basal Ca2+ level before stimulation, and error bars (SEM) are gray.
Figure 2.
Figure 2.
Monitoring of PIP2 and DAG during activation of overexpressed PAR2. (A) tsA201 cells were transfected with PH-RFP (PIP2 probe) for visualization of PIP2 depletion and C1-citrine (DAG probe) for estimation of DAG production during PAR2 activation. At rest, PIP2 and DAG probes are located at the PM and the cytoplasm, respectively (insets). After the addition of 100 µM AP, the two probes translocated toward the other compartment. Fluorescence intensity of both probes in a cytoplasmic region of interest was measured and normalized (Norm. cyt. intensity). (B) DAG and PIP2 probe translocation rates during receptor activation and desensitization. For A and B, n = 8 cells. (C and D) Concentration–response curves for PIP2 depletion (C) and DAG production (D). The peak values were measured at the indicated AP concentrations. Each point is the mean of three to nine cells. Error bars are SEM.
Figure 3.
Figure 3.
Stimulation of PKC stops signaling from PAR2 without recruiting β-arrestin. Cells were treated with 100 nM PMA to activated PKC or 100 nM BIS I to block PKC. (A) PKC activator (PMA) stops PIP2 depletion and Ca2+ signaling from PAR2. Probes are PH-RFP for PIP2 (n = 4) and fura-2 for intracellular Ca2+ (n = 3). (B) PKC inhibitor (BIS I, n = 11) and GRK2/3 inhibitor (compound101 [Cmpd101]) potentiate the PIP2 depletion compared with 100 nM BIS V (n = 7) or control without the blockers (n = 17). AP was 10 µM. (C) 100 µM AP recruits β-arrestin (open circles, n = 8) and PMA does not (closed circles, n = 4). Error bars are SEM.
Figure 4.
Figure 4.
β-Arrestin locks in the desensitized state of PAR2. (A) PIP2 loss and recovery during maintained AP was monitored by the PIP2 probe in the cells transfected with scrambled siRNA (Scrmbld) or knocked down with β-arrestin 1/2 siRNA (β-arr 1/2). (B) Rate of translocation of the PIP2 probe measured in A. **, P < 0.01. (C) Amounts of β-arrestin in dishes of cells transfected with β-arrestin 1/2 siRNA (green) compared with cells transfected with scrambled siRNA. The amount of protein from Western blots was normalized to the value for cells transfected with scrambled siRNA. ***, P < 0.001. (D) Calyculin A restores PIP2 recovery in β-arrestin 1/2 knockdown cells (red symbols). For comparison, the data from A are plotted as dashed lines (scrambled, black; and β-arrestin 1/2 siRNA, green). (E) Rate recovery with (red) or without (green) calyculin A. The dotted line indicates the value for scrambled siRNA. (F) Overexpressing β-arrestin 2–YFP (yellow) does not speed up PIP2 recovery (red). (G and H) Two different AP concentrations were used, 10 and 100 µM. Rates of PIP2 translocation (G) and β-arrestin 2–YFP translocation (H). Error bars are SEM.
Figure 5.
Figure 5.
Counting PAR2 molecules. (A) TIRF microscopy of GFP-tagged PAR2 receptors expressed at a low density to observe single PAR2-GFP molecules. Although the cells were fixed with 4% paraformaldehyde, most PAR2-GFPs were still mobile. We chose immobilized molecules to analyze single GFP bleaching steps. (B) Photobleaching of single GFP molecules. (B, a) Time course of intensity during one photobleaching event. (b) Intensity histogram for the fluorescence of the basal and first levels for 21 GFP molecules in different regions of three cells. The single-step size was estimated by fitting two Gaussian curves, giving an intensity difference between the two peaks of 198 a.u. The background intensity after GFP photobleaching was subtracted. (C) TIRF image of a single cell overexpressing a typical amount of PAR2-GFP using the same microscope but with 20-fold lower gain (see section Counting single receptor molecules at the PM). (D) Frequency distribution of PAR2-GFP density in 147 overexpressing cells using TIRF microscopy from three independent experiments. (E) Frequency distribution of PAR2-GFP fluorescence in 187 overexpressing cells observed by conventional confocal microscopy. As seen in the insert image, most of the PAR2 was at the PM. (F) Comparison of the frequency distributions for confocal intensity (black line) and PAR2 molecular density (green bars) made by scaling the two curves for optimal register. Vertical dotted lines define the range of GFP fluorescence of the cells selected for our confocal experiments in Figs. 2, 3, 4, and 10.
Figure 6.
Figure 6.
Schematic reaction diagram of the model including PAR2 signaling, desensitization, and internalization. Reactions in the dashed box were taken from the previous model for muscarinic receptor signaling (Falkenburger et al., 2013) except for the Ca2+-dependent PLC activation and β-arrestin–dependent PIP5K activity. After binding of ligand (L) to PAR2 (R), heterotrimeric Gαqβγ protein (Gq) binds to the receptor forming RLGq* (PAR2 bound to both ligand and Gqβγ). The soluble ligand can dissociate from the RLG state reversibly to yield RG. RLGq* generates active Gαq (Gq*), which in turn activates PLC to hydrolyze PIP2 into IP3 and DAG. The DAG-bound PKC is the activated form (active PKC) that catalyzes reaction 3. Phosphorylation of ligand-bound receptor (reaction 1) is mediated in parallel by two enzymes, GRK and active PKC. Based on literature, phosphorylated and ligand-bound receptors recruit two types of β-arrestins (β-arrestin 1 and 2; Ricks and Trejo, 2009). We assumed that translocation and binding of the arrestins to RLP can be described as a one-step reaction. To describe intracellular Ca2+ dynamics, the model considers only Ca2+ release from ER via IP3 receptors and uptake by SERCA pumps. Phosphatidylinositol 4-kinase (4K) and phosphatidylinositol 4-phosphate 5-kinase (5K) phosphorylate PI and PI(4)P, and 5-phosphatase (5P) and 4-phosphatase (4P) dephosphorylate PI(4,5)P2 (or PIP2) and PI(4)P (or PIP), respectively.
Figure 7.
Figure 7.
Simulation showing the predicted time courses of several intracellular signals triggered by AP and the distribution of PAR2 among different states. For PIP2 recovery at the PM (PM PIP2), the model accelerated PI4 and PIP5 kinases, with the maximal rate constant for PIP5K being regulated by the amount of β-arrestin. The PM PIP2 scale was inverted for easier comparison with the PH-domain signals from this study. The simulated concentration of AP was 100 µM. Parameters and equations for the underlying reactions are listed in Tables 1 and 2.
Figure 8.
Figure 8.
Simulated time courses of PIP2 depletion and β-arrestin recruitment. (A and B) Depletion of PM PIP2 by different concentrations of AP (A) replotted as a dose versus peak response curve (black squares; B) in simulation. For easier comparison with the experimental data, the y axes of simulated data in A and C were inverted. The peak response at various concentrations of AP was normalized with the value at 300 µM. Experimental results in B (Fig. 2 C), D (Fig. 2 C), F (Fig. 4 H), and H (Fig. 2 C) are overlaid for comparison (red symbols). (C and D) PM PIP2 levels during 100 µM AP treatment for different surface densities of PAR2 receptors. (E and F) β-Arrestin translocation time course and rates at different AP concentrations. (G and H) β-Arrestin translocation time course and rates at different PAR2 densities. Concentrations and densities used in the simulations are as follows: (A, B, E, and F) 0.1, 3, 10, 30, 100, 300, and 1,000 µM AP; (C and D) 5, 50, 500, 1,500, 5,000, and 10,000 µm−2; and (G and H) 5, 50, 500, 1,500, 5,000, and 10,000 µm−2.
Figure 9.
Figure 9.
Simulated effects of serine/threonine phosphatases in β-arrestin knockdown cells. AP was set at 100 µM. (A) PM PIP2 time course with 100% or only 30% of control β-arrestin. (B) PM PIP2 time course with 30% β-arrestin with and without inhibition of phosphatase. Calyculin A was simulated by setting the reverse rate constants in reactions 1 and 3 to zero (30% β-arrestin + calyculin A). Compare with experiments in Fig. 4 D. (C) Simulated PM PIP2 time course with 30% β-arrestin and with β-arrestin–dependent activity of PIP5K. The blue line indicates the PIP2 hump generated by reduced PIP5K activity. The black dashed line indicates control without β-arrestin knockdown.
Figure 10.
Figure 10.
A role for PIP5K in the delayed PIP2 hump with β-arrestin knockdown cells. (A) Recruitment of PIP5K using the rapamycin system. Cells were transfected with β-arrestin 1/2 siRNA and then transfected with PIP5K-FKBP-CFP, Lyn11-FRB-CFP, PAR2-GFP, and PH-RFP. The kinase PIP5K-FKBP-CFP translocated from cytoplasm to PM during treatment with 5 µM rapamycin and AP (red symbols; n = 6). However, the kinase did not translocate during treatment with AP alone (black symbols; n = 6). (B) PIP5K translocation to the PM restores recovery of PIP2 in β-arrestin knockdown cells. Symbols indicate the effect of AP without (black) or with (red) PIP5K recruitment using rapamycin (for both cases, n = 6). Error bars are SEM.

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