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.

P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes

2000 ◽  
Vol 278 (6) ◽  
pp. H1751-H1761 ◽  
Author(s):  
Vincent Sauzeau ◽  
Hélène le Jeune ◽  
Chrystelle Cario-Toumaniantz ◽  
Nathalie Vaillant ◽  
Alain-Pierre Gadeau ◽  
...  
Keyword(s):  
Rho Kinase ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Receptor Subtypes ◽  
Actin Stress Fiber ◽  
P2y Receptor ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Vascular Myocytes ◽  
Stimulation Of

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca2+ sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y1, P2Y2, P2Y4, or P2Y6 receptor subtypes. Our data demonstrate that P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.

Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia

2001 ◽  
Vol 281 (4) ◽  
pp. G1059-G1067 ◽  
Author(s):  
Jonathan A. Dranoff ◽  
Anatoly I. Masyuk ◽  
Emma A. Kruglov ◽  
Nicholas F. LaRusso ◽  
Michael H. Nathanson
Keyword(s):  
Bile Duct ◽  
Intrahepatic Bile Duct ◽  
P2y Receptors ◽  
Bile Secretion ◽  
Extracellular Nucleotides ◽  
Apical Plasma Membrane ◽  
Receptor Subtypes ◽  
P2y Receptor ◽  
Atp Receptors ◽  
And Function

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+ (Ca[Formula: see text]) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4 receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca[Formula: see text] that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5′-triphosphate, UTP, and UDP each increased Ca[Formula: see text]. Basolateral addition of adenosine 5′- O-(3-thiotriphosphate) (ATP-γ-S), but not ATP, to the perifusing bath increased Ca[Formula: see text]. IBDU perfusion with ATP-γ-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.

Polarized expression of human P2Y receptors in epithelial cells from kidney, lung, and colon

2005 ◽  
Vol 288 (3) ◽  
pp. C624-C632 ◽  
Author(s):  
Samuel C. Wolff ◽  
Ai-Dong Qi ◽  
T. Kendall Harden ◽  
Robert A. Nicholas
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Confocal Microscopy ◽  
Cell Function ◽  
Mdck Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
P2y Receptors ◽  
Extracellular Nucleotides ◽  
Receptor Subtypes

Eight human G protein-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current ( Isc) or Ca2+ mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y1, P2Y11, P2Y12, and P2Y14 receptors reside at the basolateral membrane, P2Y2, P2Y4, and P2Y6 receptors are expressed at the apical membrane, and the P2Y13 receptor is unsorted. Biotinylation studies and Isc measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the Gq-coupled P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) in lung and colonic epithelial cells (16HBE14o− and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells.

Mechanisms of P2X7 receptor-mediated ERK1/2 phosphorylation in human astrocytoma cells

2003 ◽  
Vol 284 (2) ◽  
pp. C571-C581 ◽  
Author(s):  
Fernand-Pierre Gendron ◽  
Joseph T. Neary ◽  
Patty M. Theiss ◽  
Grace Y. Sun ◽  
Fernando A. Gonzalez ◽  
...  
Keyword(s):  
P2y Receptors ◽  
P2x Receptors ◽  
Extracellular Nucleotides ◽  
Phosphatidylinositol 3 Kinase ◽  
Brain Functions ◽  
Astrocytoma Cells ◽  
Human Astrocytoma ◽  
G Protein Coupled ◽  
Human Astrocytoma Cells ◽  
Protein Kinase Cδ

Astrocytes are involved in normal and pathological brain functions, where they become activated and undergo reactive gliosis. Astrocytes have been shown to respond to extracellular nucleotides via the activation of P2 receptors, either G protein-coupled P2Y receptors or P2X receptors that are ligand-gated ion channels. In this study, we have examined the manner in which activation of the P2X7 nucleotide receptor, an extracellular ATP-gated ion channel expressed in astrocytes, can lead to the phosphorylation of ERK1/2. Results showed that the P2X7 receptor agonist 2′,3′- O-(4-benzoyl)benzoyl-ATP induced ERK1/2 phosphorylation in human astrocytoma cells overexpressing the recombinant rat P2X7 receptor (rP2X7-R), a response that was inhibited by the P2X7 receptor antagonist, oxidized ATP. Other results suggest that rP2X7-R-mediated ERK1/2 phosphorylation was linked to the phosphorylation of the proline-rich/Ca2+-activated tyrosine kinase Pyk2, c-Src, phosphatidylinositol 3′-kinase, and protein kinase Cδ activities and was dependent on the presence of extracellular Ca2+. These results support the hypothesis that the P2X7 receptor and its signaling pathways play a role in astrocyte-mediated inflammation and neurodegenerative disease.

Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells

1996 ◽  
Vol 271 (3) ◽  
pp. F610-F618 ◽  
Author(s):  
B. L. Firestein ◽  
M. Xing ◽  
R. J. Hughes ◽  
C. U. Corvera ◽  
P. A. Insel
Keyword(s):  
Signaling Pathways ◽  
Purinergic Receptors ◽  
Mdck Cells ◽  
Purinergic Receptor ◽  
Receptor Occupancy ◽  
P2y Receptors ◽  
Epithelial Cell Line ◽  
Receptor Subtypes ◽  
Renal Epithelial Cell ◽  
Renal Epithelium

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.

scholarly journals Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 622
Author(s):  
Magdiel Martínez ◽  
Namyr A. Martínez ◽  
Jorge D. Miranda ◽  
Héctor M. Maldonado ◽  
Walter I. Silva Ortiz
Keyword(s):  
Cell Viability ◽  
Mechanical Injury ◽  
P2y2 Receptor ◽  
Caspase 9 ◽  
Post Injury ◽  
Caveolin 1 ◽  
Receptor Coupling ◽  
Downstream Signaling ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

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.

scholarly journals Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

2018 ◽  
Vol 115 (47) ◽  
pp. 12051-12056 ◽  
Author(s):  
Akil A. Puckerin ◽  
Donald D. Chang ◽  
Zunaira Shuja ◽  
Papiya Choudhury ◽  
Joachim Scholz ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Wild Type ◽  
Voltage Dependent ◽  
Somatosensory Neurons ◽  
Potential Applications ◽  
Voltage Dependent Calcium Channels ◽  
Potential Therapeutics

Genetically encoded inhibitors for voltage-dependent Ca2+ (CaV) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca2+ (CaV1/CaV2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary CaVβ-subunits. To investigate latent CaVβ-independent components of inhibition, we coexpressed each RGK individually with CaV1 (CaV1.2/CaV1.3) or CaV2 (CaV2.1/CaV2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β2a or a mutant version (β2a,TM) that does not bind RGKs. All four RGKs strongly inhibited CaV1/CaV2 channels reconstituted with WT β2a. By contrast, when channels were reconstituted with β2a,TM, Rem inhibited only CaV1.2, Rad selectively inhibited CaV1.2 and CaV2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT CaVβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted CaV1.2 while Rad[R208A/L235A] inhibited CaV1.2/CaV2.2 but not CaV1.3/CaV2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous CaV1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for CaV1.2 and CaV1.2/CaV2.2 channels. Thus, we have exploited latent β-binding–independent Rem and Rad inhibition of specific CaV1/CaV2 channels to develop selective GECCIs with properties unmatched by current small-molecule CaV channel blockers.

Abstract 340: Recombinant Soluble Apyrase Inhibits Vascular Smooth Muscle Cell Migration

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Oluwaseun Adeola ◽  
Yan Ji ◽  
Phillip Fish ◽  
Tammy Strawn ◽  
Gary A Weisman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Purinergic Receptor ◽  
Neointimal Hyperplasia ◽  
Receptor Activation ◽  
Vehicle Control ◽  
Extracellular Nucleotides ◽  
Lower Chamber ◽  
Upper Chamber

Background: Purinergic receptor activation by extracellular nucleotides is involved in thrombosis and neointimal hyperplasia that accompany atherosclerosis and postangioplasty restenosis. Human apyrases [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases)] are membrane bound enzymes that hydrolyze extracellular nucleotides, thereby inhibiting purinergic receptor activation. CD39, the first identified human apyrase, is constitutively expressed on endothelial cell (EC) and vascular smooth muscle cell (VSMC) surfaces. APT102, a recombinant soluble form of CD39L3, has been shown to reduce platelet activation through its ADPase activity, but its effects on VSMC and EC function are yet to be established. We tested the hypothesis that APT102 will inhibit migration of VSMCs and ECs. Methods: We studied cell migration using a modified Boyden chamber assay in which 5x10 4 cells suspended in 0.2% FBS/DMEMF12 were added to the upper chamber of transwells separated from the lower chamber medium by a microporous membrane through which VSMCs and ECs can migrate. APT102 (100 nM) or vehicle control was added to the upper chamber; lower chamber contained 2.5% FBS/DMEMF12 and either ATP (10 μM) or vehicle control. Transwells were incubated at 37 0 C for 6 h, after which cells that migrated through pores and adhered to the lower chamber side of the membrane were fixed, stained and counted. Results: ATP (10μM) significantly enhanced migration of both VSMCs and ECs. APT102 significantly inhibited VSMC migration and completely abrogated the pro-migratory effect of ATP. In contrast, APT102 had no inhibitory effect on EC migration, either spontaneous or ATP-enhanced. Conclusion: APT102 inhibits VSMC but not EC migration. These results suggest that pharmacological targeting of extracellular nucleotides may provide a safe and effective therapeutic strategy to inhibit neointimal hyperplasia and restenosis after angioplasty, without delaying endothelial cell recovery, which is a significant limitation of drug-eluting stents. Further studies are needed to clarify the mechanism(s) underlying the differential effect of extracellular nucleotide degradation by APT102 on VSMC and EC migration.

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