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First published online 16 October 2007
doi: 10.1242/jcs.010348

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Regulation of P2X₄ receptors by lysosomal targeting, glycan protection and exocytosis

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

View larger version (63K): [in this window] [in a new window]   Fig. 1. Endogenous P2X4 receptors are localized to lysosomes in macrophages, microglia and endothelial cells. (A) Confocal images showing the subcellular distribution of endogenous P2X4 receptors in primary mouse peritoneal macrophages, cultured rat brain microglia and rat brain endothelial cells (RBECs). Cells were fixed, permeabilized and stained with antibody against P2X4 (green) and colabelled with antibodies against LAMP-1 or EEA-1 (red). Bars, 10 µm. Lower panels show high-magnification images of intracellular structures colabelled with antibodies against P2X4 and LAMP-1 or EEA-1. Bars, 1 µm. (B) Cell lysates from wild-type NRK cells, NRK cells transfected with vector encoding P2X4, mouse peritoneal macrophages, rat brain microglia and RBECs were analyzed by SDS-PAGE and immunoblotted using the antibody against P2X4. (C) Comparison of staining with antibody against P2X4 and immunoblotting of J774 and primary peritoneal macrophages. Bar, 20 µm. (D) Confocal images of live J774 cells transfected with vector encoding P2X4-GFP (green) and incubated with 50 nM Lysotracker (red) for 30 minutes. Bar, 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Targeting of P2X4 receptors to lysosomes mediated by N-terminal dileucine and C-terminal tyrosine-based motifs. (A) Confocal images of wild-type and mutant P2X4 receptors containing an extracellular AU5 epitope expressed in NRK cells. Surface proteins were labelled by incubating live cells for 30 minutes with extracellular antibody against AU5 at 37°C. Cells were then fixed, permeabilized and total receptors labelled with antibody against P2X4. Bars, 10 µm. (B) Histogram showing the ratio of labelled to total fluorescence normalized to the P2X4 (AU5) receptor. n=22–39 per construct. For P2X4 (AU5), only cells showing some detectable surface labelling were analyzed and so the ratio is likely to be an overestimate relative to the mutant receptors. (C) Graph showing the effects of mutating dileucine and tyrosine motifs on the internalization of P2X4 receptors. NRK cells expressing vectors encoding P2X4 dileucine and tyrosine mutant proteins were incubated with extracellular antibody against AU5 at 37°C for 30 minutes. Cells were fixed, surface receptors detected by secondary antibody, permeabilized and internalized receptors detected with a different secondary antibody. n=21–43 cells per construct. (D) Surface expression of P2X4 receptors with mutations in dileucine and tyrosine motifs was measured by biotinylating surface proteins, purifying with streptavidin beads and probing with antibody against P2X4 by western blot analysis. The `Total' gel shows dilutions of total cell lysate for each construct. (E) Effect of dileucine and tyrosine mutations on the lysosomal distribution of P2X4 receptors. GFP-tagged constructs (green) were expressed in NRK cells and were stained with antibody against LAMP-1 (red). The histogram shows the values of Pearson's correlation coefficient calculated using Zeiss LSM software. n=16–28 per construct. Experiments were repeated three times and the data shown are from one representative experiment; error bars indicate means+s.e.m. (***=P<0.001). Bars, 10 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 3. N-glycosylation protects P2X4 receptors from degradation in lysosomal compartments. (A) NRK cells transfected with vectors encoding either P2X4 or GFP-tagged M4 muscarinic receptors were incubated with 5 µg/ml of CHX for the indicated durations. Thp-1 cells were similarly treated with CHX. Cell lysates were then analysed by SDS-PAGE and immunoblotted with antibodies against either GFP or P2X4. (B) Lysates from NRK cells transfected with vector encoding P2X4 were grown in the presence or absence of DMJ to prevent complex glycosylation and were treated with either 500 U Endo H or 12 U N-glycosidase F for 5 hours. (C) A DMJ-treated cell lysate was incubated with 125 U Endo H for 30 minutes to reveal six different glycosylation states. (D) NRK cells treated for 24 hours with DMJ (in the presence of 5 µg/ml of CHX) and Thp-1 cells treated for 3 days with DMJ were incubated with 50 mU/ml Endo H for the indicated number of hours. Cell lysates were then analyzed by immunoblotting with an antibody against P2X4. (E) A similar experiment was performed with DMJ-treated RAW264.7 cells, and the lysates were analyzed by immunoblotting with antibodies against P2X4 and LAMP-1.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Trafficking of P2X4 receptors to phagosomes. (A) Primary macrophages were incubated with latex beads or zymosan particles for 30 minutes, followed by a chase for 60 minutes, or incubated for 90 minutes with E. coli expressing GFP. The cells were fixed, permeabilized and stained with antibodies against P2X4 (green) and LAMP-1 (red). (B) Confocal images of RAW264.7 cells expressing P2X4-GFP (green) incubated with latex beads for 30 minutes, followed by a 1 hour chase, fixed, permeabilized and stained with antibody against LAMP-1 (red). Bars, 10 µm. (C) Detection of P2X4 and LAMP-1 in latex bead phagosomes isolated by sucrose density-gradient flotation from bone-marrow-derived macrophages by immunoblotting (`Phag') and dilutions of total cell lysates (`Total').

View larger version (47K): [in this window] [in a new window]   Fig. 5. Lysosome exocytosis upregulates P2X4 receptors at the plasma membrane. (A) NRK cells transfected with vector encoding P2X4 (AU5) receptors were incubated with 5 µM ionomycin for 10 minutes in normal extracellular solution (NES) at 37°C. Next, cells were washed and returned to NES lacking ionomycin for a further 45 minutes at 37°C, and the solution was then assayed for -Hex activity. The experiment was repeated three times and the data shown are from one representative experiment. Shown are means+s.d., n=3 (***=P<0.001). Alongside are shown confocal images of cells incubated with ionomycin for 10 minutes in NES at 37°C and then surface labelled with antibodies against AU5 or LAMP-1 (LYC16 clone) at 12°C for 30 minutes. Bars, 10 µm. Between 50 and 70 cells were analysed for each condition. The experiment was repeated three times and the data shown are from one representative experiment. Shown are means+s.e.m., n=3 (***=P<0.001). (B) A similar experiment was carried out with cultured rat brain microglia, but surface expression of endogenous P2X4 was measured by biotinylation at 12°C followed by immunoblotting with antibody against P2X4. `Total' gels show dilutions of total cell lysates. (C) Time course of lysosomal enzyme release induced by 50 mM methylamine (MA) in peritoneal macrophages and HEK 293 cells. Cells were incubated in NES, and -Hex activity was measured at different time points (mean±s.d., n=3). (D) Surface expression of P2X4 and LAMP-1 in peritoneal macrophages following 15 minutes incubation with 50 mM MA. Surface receptors were detected by biotinylation at 12°C followed by immunoblotting. Samples from the total cell lysates show that the overall expression of P2X4 and LAMP-1 was unchanged by treatment with MA.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Lysosome exocytosis increases P2X4-mediated currents. (A) Whole-cell currents measured using the perforated patch-clamp technique following application of 30 µM ATP or BzATP at a holding potential of –60 mV in LPS-primed mouse peritoneal macrophages. 3 µM ivermectin was applied 3 minutes before and during ATP application. The histogram shows the normalized mean peak current amplitude +s.e.m. for 30 µM ATP and BzATP (n=4–10). (B) ATP-evoked inward currents before and after a 30 minute pre-incubation with 50 mM methylamine (MA) at 37°C. Pre-incubation with MA caused an approximately fivefold increase in the peak amplitude of ATP-evoked inward currents, whereas MA had no effect when co-applied with ATP. Following MA preincubation, currents remained ivermectin sensitive and were potentiated a further threefold. Pre-incubation with 10 µg/ml brefeldin A for 1 hour before and during incubation with MA did not inhibit MA-induced potentiation of ATP-evoked currents. The histogram shows the normalized mean peak current amplitude plus s.e.m.; n=8–14. (C) Currents evoked by 30 µM ATP or BzATP following treatment with MA. (D) MA pre-treatment did not increase the amplitude of currents evoked by 30 µM ATP in HEK 293 cells transfected with P2X4. The data shown are the mean+s.e.m.; n=5. (A-D: *P<0.05; **P<0.01; ***P<0.001; n.s., not significant.) Patch-clamp measurements were performed at RT.