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De- and re-sensitization and trafficking of P2Y nucleotide receptors modulate physiological responses of these receptors. Here, we used the rat brain P2Y1 receptor tagged with green fluorescent protein (P2Y1-GFP receptor) expressed in HEK293 human embryonic kidney cells. Ca2+ release was used as a functional test to investigate ATP-induced receptor de- and re-sensitization. By confocal laser scanning microscopy (CLSM), endocytosis of P2Y1-GFP receptor was visualized in live cells. Stimulation of the cells with ATP induced complete receptor endocytosis within 30 min and appearance of the P2Y1 receptor in small vesicles. Removal of the agonist resulted in reappearance of the receptor after 60 min on the plasma membrane. Exposure of the cells to KN-62 and KN-93, inhibitors of the calmodulin dependent protein kinase II (CaMKII), prevented receptor internalization upon stimulation with ATP. However, the receptor which was still present on the plasma membrane was desensitized, seen by decreased Ca2+ response. The decreased Ca2+ response after 30-min exposure to ATP can be attributed to desensitization and is not as a result of depletion of internal stores, as the cells exposed to ATP for 30 min exhibited a normal Ca2+ response upon stimulation with thrombin. However, okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), did not affect ATP-induced P2Y1 receptor endocytosis, but delayed the reappearance of the P2Y1 receptor on the plasma membrane after ATP withdrawal. Consistently, in okadaic acid-treated cells the ATP-induced Ca2+ response observed after the 30-min exposure to ATP recovered only partially. Thus, CaMKII seems to be involved in P2Y1 receptor internalization, but not desensitization, whereas protein phosphatase 2A might play a role in recycling of the receptor back to the plasma membrane.
Plasma membrane P2 receptors mediate the actions of extracellular nucleotides in cell signalling. These receptors have great clinical potential (Ralevic and Burnstock 1998; Agteresch et al. 1999). P2X receptors are ligand-gated ion channels, whereas P2Y receptors, seven transmembrane domain receptors, are coupled to G proteins. At present, the P2Y receptor family comprises eight cloned and functionally defined subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14), and all these subtypes are found in human tissue (von Kügelgen and Wetter 2000; Nicholas 2001).
P2Y1 receptors are activated most potently by the physiological agonist ATP, ADP and through the selective agonists 2-methylthioadenosine diphosphate (2-Me-S-ADP) and 2-Me-S-ATP. They are coupled both to pertussis toxin-insensitive Gq/G11 and pertussis toxin-sensitive Gi/G0 G proteins (Brown et al. 2000). P2Y receptors activate phospholipase C (PLC), leading to increased levels of inositol 1,4,5-trisphosphate (InsP3), diacylglycerol (DAG), cytosolic free Ca2+ ([Ca2+]i) and stimulation of protein kinase C, which in turn may activate phosphatidylcholine-specific PLC and phospholipase D (Ralevic and Burnstock 1998). Moreover, P2Y receptors were shown to regulate phospholipase A2, adenylyl cyclase, mitogen-activated protein kinase pathway and K+ and Ca2+ influx via voltage-operated channels (Bofill-Cardona et al. 2000; Powell et al. 2000; Wirkner et al. 2004).
After binding the activating ligands, G protein-coupled receptors (GPCRs) undergo a complex series of reactions to turn off the signal transduction. Part of this process is receptor desensitization to attenuate the response to stimulation. This was studied in detail in cell systems expressing β-adrenergic receptors (Ferguson and Caron 1998). After removal or degradation of the agonist, the GPCRs re-sensitize and the receptors regain their ability to respond to the ligands. Prolonged or repetitive stimulation of the cells can also result in a reduction of the number of receptors at the plasma membrane by receptor internalization. Sequestered receptors are either recycled back to the plasma membrane or sorted for degradation into lysosomes (Luzio et al. 2000; Oksche et al. 2000). The latter occurs most likely after prolonged agonist exposure.
Receptor internalization might be involved in processes leading to re-sensitization of the receptors (Ferguson and Caron 1998). Internalized receptors might be connected to further signalling pathways (Pierce et al. 2000). GPCRs that are coupled to adenylyl cyclase are believed to generally follow the scheme of receptor phosphorylation, desensitization, endocytosis and re-sensitization clarified in detail for the β-adrenergic receptors (Ferguson and Caron 1998; Milligan 1999). However, for Gq/PLC-coupled receptors, like the P2Y1 receptor, the mechanisms underlying agonist-induced desensitization and endocytosis are less well understood (Firestein et al. 1996).
We have previously generated an HEK293 cell line stably expressing rat brain P2Y1 receptors and another cell line expressing a chimera of P2Y1 receptor and green fluorescent protein (GFP) at the carboxy terminus. In fura-2AM-loaded cells, the Ca2+ response evoked through receptor activation has been examined. Pharmacological characterization of these heterologously expressed receptors with Ca2+ imaging technique showed a marked increase in high-affinity ligand recognition of adenosine di- and triphosphates in comparison with untransfected cells (Vöhringer et al. 2000; Zündorf et al. 2001). The EC50 values for the agonists 2-Me-S-ADP and 2-Me-S-ATP were 50 and 70 nm, respectively, which were substantially lower than in untransfected cells, where the EC50 values were 450 and 630 nm, respectively (Vöhringer et al. 2000). In the present study, these HEK293 cells transfected with the P2Y1-GFP receptor were used for investigation of receptor trafficking by confocal laser scanning microscopy (CLSM) in live cells with concomitant determination of de- and re-sensitization by measuring the Ca2+ response.
We investigated the effect of inhibition of protein kinase and phosphatase on receptor trafficking. CLSM was employed to visualize the trafficking of the receptor in live cells after stimulation of the cells with ATP. We also used the Ca2+ response to measure the functionality of the receptor on the plasma membrane. It was observed that inhibition of calmodulin dependent kinase II (CaMKII) by KN-62 inhibited internalization of the activated receptor, but did not prevent its functional desensitization. Treatment of the cells with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A), delayed the recycling of the endocytosed receptor back to the plasma membrane and reduced the degree of functional recovery.
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P2Y receptors, which belong to the family of GPCRs, mediate the actions of extracellular nucleoside di- and triphosphates. P2Y receptor regulation is of growing interest, as nucleotides have been shown to be involved in processes leading to protection and degeneration of neural and immune cells (Amadio et al. 2002; Kannan 2002; Volonte et al. 2003; Chorna et al. 2004). Therefore, understanding P2Y receptor regulation should provide clues for pharmacological treatment of various disorders. Previously, we have described the stable expression of a P2Y1 receptor as well as a P2Y1-GFP chimera in HEK293 cells and proved functional coupling to Ca2+ release (Vöhringer et al. 2000; Zündorf et al. 2001). A receptor modification using GFP as a fusion partner is a valuable biochemical tool to monitor localization for investigating receptor internalization and recycling in live cells (Kallal and Benovic 2000). Moreover, HEK293 cells are a most suitable system to study P2Y receptor regulation, because these cells endogenously express the P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 (Van der Weyden et al. 2000; Fischer et al. 2003; Moore et al. 2003).
Agonist-induced internalization has been reported until now only for one other member of the P2Y receptor family, the P2Y2 receptor, using a hemagglutinin A epitope at the N-terminus of the human P2Y2 receptor (Sromek and Harden 1998). Recently the mechanism of endocytosis of the GFP-tagged P2Y2 receptor has been reported, analysing the clathrin and actin cytoskeleton dependence (Tulapurkar et al. 2005). Furthermore, it has been shown that C-terminal phosphorylation of the P2Y2 receptor mediates agonist-induced desensitization (Garrad et al. 1998; Otero et al. 2000), suggesting that several kinases and phosphatases are involved in the regulation of the P2Y2 receptor. Until now nothing is known about the type of kinases, which take part in this process for the P2Y1 receptor. Pharmacological experiments proved that various GPCRs are phosphorylated by CaMKII (Zamani and Bristow 1996), protein kinase A (Post et al. 1996), protein kinase C (Chen and Lin 1999), G protein-coupled receptor kinases (Bünemann and Hosey 1999) or casein kinase 1α (Budd et al. 2000), thereby directly regulating their activity.
We used here the stably transfected HEK293 cells expressing the rP2Y1 receptor tagged with GFP which is potently activated by ATP (Vöhringer et al. 2000; Tulapurkar et al. 2005). With these cells, we were able to both visualize P2Y1 receptor trafficking and detect the functional response by monitoring the agonist-induced Ca2+ release. This enabled us to analyse receptor internalization together with desensitizing events (Garland et al. 1996; Garrad et al. 1998; Szekeres et al. 1998; Ferguson 2001). Thus, we investigated the receptor responsiveness and receptor translocation under the influence of kinase and phosphatase inhibitors.
Stimulation of the cells with ATP induced an elevation of the intracellular Ca2+ level which is caused by P2Y1-GFP receptor-induced Ca2+ release (Vöhringer et al. 2000). With continued stimulation of the cells, the Ca2+ response declined. Removal of the agonist resulted in a return of the intracellular Ca2+ concentration to the basal level. A second ATP stimulus after an interval of 60 min gave a Ca2+ response with 75% recovery of the amplitude. The cells were stimulated for 30 min with 100 μm ATP by two different methods, namely continuous presence of agonist or repeated short applications of the agonist. By both these methods the calcium response that was observed at the end of 30 min stimulation declined to a similarly low value. This indicates that both protocols induce a similar desensitization of the receptor. We also verified that the desensitization profile observed with ATP is similar to that obtained with 2-Me-S-ADP, a highly specific agonist for the P2Y1 receptor. This indicates that the stimulation with ATP, the naturally occurring agonist of the P2Y1 receptor, is not influenced by the presence of the endogenous P2Y2 receptor, which can also be activated by ATP.
The question was whether the decrease in the Ca2+ signal that we obtained after prolonged stimulation is as a result of desensitization of the receptor and not because of depletion of the internal stores. This was verified by stimulation of the cells with the agonist thrombin. We chose thrombin as an agonist, as HEK293 cells endogeneously express PAR receptors [Amadesi et al. (2004) and our own unpublished data]. PAR receptors mediate a rise in intracellular Ca2+ via a mechanism similar to that induced by P2Y receptors. We observed that cells that were pretreated for 30 min with 100 μm ATP exhibited a Ca2+ response upon stimulation with thrombin. The pretreatment with ATP did not affect the amplitude of the thrombin-mediated rise in intracellular Ca2+. Thus, we were able to show that the decrease in Ca2+ response was because of desensitization of the P2Y1 receptor and not as a result of the emptying of the stores.
By CLSM we visualized agonist-induced endocytosis in live cells under the same conditions, using the GFP tag to localize the P2Y1 receptor and LysoTracker to label lysosomes. The inhibitors used did not influence the basal fluorescence observed in the cells. A time-dependent receptor endocytosis was observed. The receptors were not yet co-localized with lysosomes after a 30-min exposure to agonist. Endocytosis was quantified as a decrease in the fluorescence intensity on the plasma membrane and an increase in the fluorescence intensity in the cytoplasm. After removal of the agonist, the receptor reappeared on the plasma membrane after 60 min. At this time point, we observed that the Ca2+ response was back to 75% of the value of the initial response. These results show that the cells are desensitized concomitant with endocytosis of the receptor from the plasma membrane. After removal of the agonist the cells are then re-sensitized together with receptor reappearance.
When the cells were pre-incubated with the CaMKII inhibitor KN-62 and then stimulated with ATP, the de- and re-sensitization pattern of the P2Y1 receptors was similar to that in the absence of the inhibitor. In the presence of KN-62 there was almost complete re-sensitization. The Ca2+ response reached again 111% of the first response obtained upon stimulation with ATP of the KN-62-pretreated cells. This recovery of the Ca2+ response was even higher than in cells that were not pretreated with KN-62.
However, treatment with KN-62 completely inhibited the endocytosis of the receptor. To confirm the specificity of the effects of KN-62 on the endocytosis of the receptor, we repeated the experiments with KN-93, a most recently available and even more potent inhibitor of CaMKII. We observed similar effects. Thus, CaMKII seems to be involved in P2Y1 receptor endocytosis. To further underpin our results we used KN-92, an inactive form of KN-93. The cells that were pretreated with KN-92 exhibited normal endocytosis of the P2Y1 receptor upon challenge with 100 μm ATP. Obviously, P2Y1 receptor desensitization does not depend upon endocytosis.
After treatment of the cells with the protein phosphatase inhibitor okadaic acid, the initial Ca2+ response was slightly reduced, similar to that observed with KN-62. The desensitization proceeded in a manner similar to that seen in untreated cells. However, exposure to okadaic acid greatly inhibited the re-sensitization of the P2Y1 receptor, as the Ca2+ response observed at 60 min after the 30-min long ATP stimulus amounted only to 39% of the initial response.
Okadaic acid did not affect the endocytosis kinetics in P2Y1 receptor trafficking. After removal of the agonist, however, the reappearance of the receptor on the plasma membrane was considerably delayed compared with untreated cells. There was only reduced fluorescence that reappeared on the plasma membrane. Thus, the fact that the Ca2+ response did not return to the initial level seems to be caused by incomplete reappearance of the receptor on the plasma membrane. The complete reappearance of the P2Y1 receptor was apparently suppressed by inhibition of the enzyme PP2A by okadaic acid. This may prevent dephosphorylation of the endocytosed receptor or downstream effectors. Similarly, with β2-adrenergic receptor heterologously expressed in HEK293 cells (Oakley et al. 1999), treatment of the cells with okadaic acid caused a reduced stimulation of adenylate cyclase activity. This was attributed to the reduction in the reappearance of the receptor on the plasma membrane. De-phosphorylation of the receptor seems to be important for the reappearance of the receptor on the plasma membrane (Spampinato et al. 2002).
In summary, our results indicate that endocytosis of the P2Y1 receptor is controlled by the activity of CaMKII. Inhibition of CaMKII suppresses endocytosis of the receptor. However, the receptor nevertheless undergoes desensitization. This prevents continued activation of the cell because of lingering ligand in the extracellular compartment. Inhibition of the activity of okadaic acid-sensitive phosphatase does not affect internalization and desensitization but delays the reappearance of the P2Y1 receptor on the plasma membrane. With partial reappearance of the P2Y1 receptor back on the plasma membrane, there is only partial recovery of the ATP-induced Ca2+ response in the cells. These observations underline that phosporylation and de-phosporylation of the P2Y1 receptor play important roles in the functionality of this receptor.