Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes

Leukotriene E₄ (LTE₄), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y₁₂ is required for LTE₄ mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y₁₂ to cysteinyl leukotrienes. Models of human CysLT₁, CysLT₂ and P2Y₁₂ overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT₁ and CysLT₂ but failed to induce any calcium response in cells expressing P2Y₁₂ or P2Y₁₂+ Gα₁₆. In contrast, selective P2Y₁₂ agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y₁₂+ Gα₁₆. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y₁₂ when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y₁₂ to analyse potential signalling and cell activation through P2Y₁₂ receptor or receptor heterodimers but no specific LTE₄ responses were observed. These results show that LTE₄ as well as other cysLTs do not activate intracellular signalling acting through P2Y₁₂ and suggest that another LTE₄ specific receptor has yet to be identified.     

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y₂ receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y₂ receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y₂ nucleotide receptors from metabolically [³²P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [³²P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y₂ receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y₂ receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y₂ receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y₂ nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)     

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y₂ receptors in both 1321N1 cell transfectants expressing the P2Y₂ receptor and human HT-29 epithelial cells expressing endogenous P2Y₂ receptors with an EC₅₀ of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC₅₀ for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y₂ nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y₂ receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,₂ receptor. In these cells, potent P2Y₂ receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca²⁺]_i, after addition of desensitizing concentrations of UTP, indicating that P2Y₂ receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca²⁺]_i induced by UTP in 1321N1 cell transfectants expressing the P2Y₂ receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y₂ receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y₂ receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y₂ receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y₂ nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y₂ receptor agonists.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

P2Y2 Nucleotide Receptor-Mediated Responses in Brain Cells

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G_q protein-coupled P2Y₂ nucleotide receptor subtype (P2Y₂R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y₂R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y₂R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y₂R activation increases astrocyte migration. P2Y₂R activation by UTP increases the expression in astrocytes of α_Vβ_3/5 integrins that bind directly to the P2Y₂R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y₂R, an interaction required for G_o and G₁₂ protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y₂R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ_1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y₂Rs in neurons. Data with rPCNs suggest that P2Y₂R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y₂Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y₂Rs in glial cells can promote neuroprotective responses, suggesting that P2Y₂Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.     

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y₂ receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y₂ receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y₂ receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y₂ receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y₂ receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y₂ receptors. While ATP and UTP activated the murine and human P2Y₂ receptors with similar potencies (EC₅₀ values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC₅₀ = 9.1-21.2 M) than UTP (IC₅₀ = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.     

UTP is not a biased agonist at human P2Y11 receptors

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y₁₁ receptor, as it increased cytosolic [Ca²⁺], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y₁₁ receptor in greater detail. Intracellular Ca²⁺ was monitored in 1321N1 cells stably expressing human P2Y₁₁ receptors using the Ca²⁺-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca²⁺, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca²⁺ in 1321N1 cells stably expressing the human P2Y₂ receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y₁₁ receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y₁₁ receptor, nor does UTP act as an antagonist.     

P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca²⁺]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca²⁺]i was significantly blocked by MRS2211 (a selective P2Y₁₃ receptor antagonist), but was unaffected by MRS2179 (a selective P2Y₁ receptor antagonist) or MRS2395 (a selective P2Y₁₂ receptor antagonist), which suggest that P2Y₁₃ receptor may be responsible for ADP-evoked Ca²⁺ mobilization in cultured microglia. P2Y₁₃-evoked Ca²⁺ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca²⁺ (by EGTA) also can obvious suppress the Ca²⁺ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y₁₃ receptor-evoked Ca²⁺ mobilization. However, P2Y₁₃ receptor-evoked Ca²⁺ response was not impaired after CdCl₂ and verapamil administration, which suggest that voltage-operated Ca²⁺ channels may be not related with P2Y₁₃-evoked Ca²⁺ response. In addition, Ca²⁺ mobilization induced by ADP was abolished by different store-operated Ca²⁺ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y₁₃ receptor might be involved in the effect of ADP on [Ca²⁺]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y₁₃ receptor activation causes Ca²⁺ release from Ca²⁺ store, which leads to the opening of SOCs.     

Alanine‐(87)‐threonine polymorphism impairs signaling and internalization of the human P2Y11 receptor,when co‐expressed with the P2Y1 receptor

The P2Y₁₁ nucleotide receptor detects high extracellular ATP concentrations. Mutations of the human P2RY₁₁ gene can play a role in brain autoimmune responses, and the P2Y₁₁ receptor alanine‐87‐threonine (A87T) polymorphism has been suggested to affect immune‐system functions. We investigated receptor functionality of the P2Y₁₁A87T mutant using HEK293 and 1321N1 astrocytoma cells. In HEK293 cells, the P2Y₁₁ receptor agonist 3′‐O‐(4‐benzoylbenzoyl)adenosine 5′‐triphosphate (BzATP) was completely inactive in evoking intracellular calcium release while the potency of ATP was reduced. ATP was also less potent in triggering cAMP generation. However, 1321N1 astrocytoma cells, which lack any endogenous P2Y₁ receptors, did not display a reduction. Only when 1321N1 cells were co‐transfected with P2Y₁₁A87T and P2Y₁ receptors, the calcium responses to the P2Y₁₁ receptor‐specific agonist BzATP were reduced. It is already known that P2Y₁ and P2Y₁₁ receptors interact. We thus conclude that the physiological impact of A87T mutation of the P2Y₁₁ receptor derives from detrimental effects on P2Y₁–P2Y₁₁ receptor interaction. We additionally investigated alanine‐87‐serine and alanine‐87‐tyrosine P2Y₁₁ receptor mutants. Both mutations rescue the response to BzATP in HEK293 cells, thus ruling out polarity of amino acid‐87 to be the molecular basis for altered receptor characteristics. We further found that the P2Y₁₁A87T receptor shows complete loss of nucleotide‐induced internalization in HEK293 cells. Thus, we demonstrate impaired signaling of the P2Y₁₁ A87T‐mutated receptors when co‐operating with P2Y₁ receptors.

     

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y₁₂-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y₁₂, have been deorphanized. The P2Y₁₂-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y₁₂ in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y₁₂-like receptor members.     

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y₆. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y₆ receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y₆. Interestingly, UDP induced clathrin-dependent P2Y₆ internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y₆ was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y₆ internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y₆ receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y₆ signaling.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

The P2Y2 Nucleotide Receptor Mediates the Proliferation and Migration of Human Hepatocellular Carcinoma Cells Induced by ATP

ATP is an abundant biochemical component of the tumor microenvironment and a physiologic ligand for the P2Y₂ nucleotide receptor (P2Y₂R). In this study, we investigated the effect of ATP on the cellular behavior of human hepatocellular carcinoma (HCC) cells and the role of P2Y₂R in ATP action and aimed to find a new therapeutic target against HCC. The experiments were performed in native isolated human HCC cells, normal hepatocytes, human HCC cell lines, and nude mice. We found that the mRNA and protein expression levels of P2Y₂R in native human HCC cells and the human HCC cell lines HepG2 and BEL-7404 were enhanced markedly compared with human normal hepatocytes and the normal hepatocyte line LO2, respectively. ATP induced intracellular Ca²⁺ increases in HCC cells and promoted the proliferation and migration of HCC cells and the growth of HCC in nude mice. The P2Y receptor antagonist suramin, P2Y₂R-specific shRNA, the store-operated calcium channel inhibitors 2-aminoethoxydiphenyl borate (2-APB) and 1-(β-3-(4-methoxy-phenyl) propoxyl-4-methoxyphenethyl)1H-imidazole-hydrochloride ({"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365), and stromal interaction molecule (STIM1)-specific shRNA inhibited the action of ATP on HCC cells. In conclusion, P2Y₂R mediated the action of ATP on the cellular behavior of HCC cells through store-operated calcium channel-mediated Ca²⁺ signaling, and targeting P2Y₂R may be a promising therapeutic strategy against human HCC.     

Differential expression of P2Y receptors in the rat cochlea during development

Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₂, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the driving force for sound transduction. In addition, early postnatal P2Y₂ and P2Y₄ protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca²⁺-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance of extracellular nucleotide and nucleoside signaling to hearing.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y₁₄ receptor (P2Y₁₄R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y₁R, P2Y₁₂R, and P2Y₁₃R, were similar, and P2Y₁₁R and P2Y₄R were highly expressed at 5.8- and 3.8-fold of P2Y₁R, respectively. Least expressed P2Y₂R was 40-fold lower than P2Y₁R, and P2Y₆R and P2Y₁₄R were ≤50 % of P2Y₁R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y₁₁R and P2Y₄R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y₄R agonist UTP nor P2Y₁₁R agonists ATPγS and NF546 had a substantial effect. P2Y₁R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y₁R potency, and the effect of P2Y₆R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y₁₄R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y₁₄R antagonist, specific P2Y₁₄R siRNA and pertussis toxin, suggesting a role of P2Y₁₄R activation in promoting human mast cell degranulation.     

The Roles of P2Y2 Purinergic Receptors in Osteoblasts and Mechanotransduction

We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y₂ purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y₂ receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y₂ knockout mice. We found that bones from P2Y₂ knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y₂ receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y₂ knockout mice. We found that P2Y₂ receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y₂ knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y₂ knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y₂ knock out osteoblasts. Our findings suggest that P2Y₂ receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y₂ knockout mice.     

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y<Subscript>13</Subscript>-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca²⁺ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y₁₂ and P2Y₁₃ subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC₅₀ values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y₁ receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y₁₂ receptor, suggesting that were mediated by P2Y₁₃-like receptors.¹These authors equally contributed to this work.