Expression of multiple P2Y receptors by MDCK-D1 cells: P2Y1 receptor cloning and signaling

2003 ◽  
Vol 59 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Richard J. Hughes ◽  
Brian Torres ◽  
Alexander Zambon ◽  
David Arthur ◽  
Christine Bohmann ◽  
...  
Keyword(s):  
P2y Receptors ◽  
P2y1 Receptor ◽  
Receptor Cloning

Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1566-1566
Author(s):  
Zhongren Ding ◽  
Florin Tuluc ◽  
Kavita R. Bandivadekar ◽  
Lili Zhang ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Human Platelet ◽  
Inositol Phosphate ◽  
Shape Change ◽  
P2y Receptors ◽  
Human Platelets ◽  
Carboxyl Terminus ◽  
P2y1 Receptor ◽  
Functional Responses ◽  
C Terminus

Abstract ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor

2007 ◽  
Vol 409 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Denise Ecke ◽  
Theodor Hanck ◽  
Mohan E. Tulapurkar ◽  
Rainer Schäfer ◽  
Matthias Kassack ◽  
...  
Keyword(s):  
Purinergic Receptor ◽  
Resonance Energy ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Hek293 Cells ◽  
Receptor Subtypes ◽  
P2y1 Receptor ◽  
Ligand Selectivity ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP–P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

Presence of P2X1 Purinoceptors in Human Platelets and Megakaryoblastic Cell Lines

1997 ◽  
Vol 78 (06) ◽  
pp. 1500-1504 ◽  
Author(s):  
Catherine Vial ◽  
Béatrice Hechier ◽  
Catherine Léon ◽  
Jean-Pierre Cazenave ◽  
Christian Gachet
Keyword(s):  
Intracellular Calcium ◽  
G Proteins ◽  
Cell Lines ◽  
Calcium Influx ◽  
Human Platelets ◽  
P2y1 Receptor ◽  
Calcium Response ◽  
Ionotropic Receptors ◽  
External Calcium

SummaryHuman platelets are thought to possess at least two subtypes of purinoceptor, one of which, coupled to G-proteins, could be the P2Y1 receptor (Léon et al. 1997). However, it has been suggested that the unique rapid calcium influx induced by ADP in platelets could involve P2X1 ionotropic receptors (MacKenzie et al. 1996) and the aim of this study was thus to investigate the presence of P2X purinoceptors in platelets and megakaryoblastic cells. Using PCR experiments, we found P2X1 mRNA to be present in human platelets and megakaryoblastic cell lines. In platelets, the selective P2X1 agonist αβMeATP induced a rise in intracellular calcium only in the presence of external calcium and this effect was antagonized by suramin and PPADS. Repeated addition of a�MeATP desensitized the P2X1 purinoceptor but only slightly affected the ADP response, while no calcium response to αβMeATP was observed in megakaryoblastic cells. These results support the existence of functional P2X1 purinoceptors on human platelets and the presence of P2X1 transcripts in megakaryoblastic cell lines.

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

2002 ◽  
Vol 283 (2) ◽  
pp. C569-C578 ◽  
Author(s):  
Alexander A. Mongin ◽  
Harold K. Kimelberg
Keyword(s):  
Amino Acid ◽  
Excitatory Amino Acid ◽  
Atp Release ◽  
Anion Channel ◽  
P2y Receptors ◽  
Cell Swelling ◽  
Channel Blockers ◽  
Medium Osmolarity ◽  
Subsequent Activation

Volume-dependent ATP release and subsequent activation of purinergic P2Y receptors have been implicated as an autocrine mechanism triggering activation of volume-regulated anion channels (VRACs) in hepatoma cells. In the brain ATP is released by both neurons and astrocytes and participates in intercellular communication. We explored whether ATP triggers or modulates the release of excitatory amino acid (EAAs) via VRACs in astrocytes in primary culture. Under basal conditions exogenous ATP (10 μM) activated a small EAA release in 70–80% of the cultures tested. In both moderately (5% reduction of medium osmolarity) and substantially (35% reduction of medium osmolarity) swollen astrocytes, exogenous ATP greatly potentiated EAA release. The effects of ATP were mimicked by P2Y agonists and eliminated by P2Y antagonists or the ATP scavenger apyrase. In contrast, the same pharmacological maneuvers did not inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cells was eliminated by a 5–10% increase in medium osmolarity or by anion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but acts synergistically with cell swelling. Moderate cell swelling and ATP may serve as two cooperative signals in bidirectional neuron-astrocyte communication in vivo.

scholarly journals Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism

2017 ◽  
Vol 596 (15) ◽  
pp. 3245-3269 ◽  
Author(s):  
Vishaal Rajani ◽  
Yong Zhang ◽  
Venkatesh Jalubula ◽  
Vladimir Rancic ◽  
Shahriar SheikhBahaei ◽  
...  
Keyword(s):  
Ventilatory Response ◽  
P2y1 Receptor ◽  
Hypoxic Ventilatory Response ◽  
Bötzinger Complex

Overview of the pharmacology and physiological roles of P2Y receptors

2012 ◽  
Vol 1 (5) ◽  
pp. 581-588 ◽  
Author(s):  
Jean-Marie Boeynaems ◽  
Didier Communi ◽  
Bernard Robaye
Keyword(s):  
P2y Receptors

scholarly journals Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

2006 ◽  
Vol 190 (2) ◽  
pp. 373-384 ◽  
Author(s):  
Shannon M Gifford ◽  
Fu-Xian Yi ◽  
Ian M Bird
Keyword(s):  
Endothelial Cells ◽  
Uterine Artery ◽  
Purinergic Receptor ◽  
Cell Types ◽  
P2y Receptors ◽  
Receptor Expression ◽  
Receptor Subtype ◽  
Initial Transient ◽  
Receptor Coupling ◽  
Specific Variation

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca2+ concentration ([Ca2+]i), but the P-UAEC show a heightened sustained phase. In order to establish whether thiswas due to an altered subclass of purinergic receptor (P2), both the dose dependencyof [Ca2+]i responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca2+ signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca2+ signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca2+is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca2+ entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca2+]i response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca2+signaling.

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