G<sub>i</sub>-Dependent Cell Signaling Responses of the Human P2Y<sub>14</sub>Receptor in Model Cell Systems

Fricks, Ingrid; Carter, Rhonda L.; Lazarowski, Eduardo R.; Harden, T. Kendall

doi:10.1124/jpet.109.150730

Cited by 50 publications

(61 citation statements)

References 39 publications

(39 reference statements)

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“…The potency (Table 1) and maximal effect (Table 2) of UDP for inhibition of forskolin-stimulated cyclic AMP accumulation were very similar to the potency and maximal effect of UDP-glucose. As was previously observed with UDP-glucose (Fricks et al, 2009), UDP had no effect on cyclic AMP accumulation in P2Y 14 -HEK293 cells in which GPCR-promoted activation of G i was inhibited by preincubation of cells with pertussis toxin (Fig. 1C).…”

Section: Resultssupporting

confidence: 60%

“…However, most studies of P2Y 14 -R-promoted signaling have relied on overexpression of either G␣ 16 or a chimera of G␣ q (G␣ q/i ) that engineers coupling of this G i -coupled receptor to activation of phospholipase C (Chambers et al, 2000;Freeman et al, 2001;Fricks et al, 2008). Using C6 rat glioma cells stably expressing the human P2Y 14 -R, we recently illustrated that adenylyl cyclase activity in both intact cells and isolated membranes was inhibited by UDP-glucose in a concentration-and pertussis toxin-sensitive manner (Fricks et al, 2009). Based on these and similar results obtained with hP2Y 14 -HEK293 cells, we reasoned that cells displaying P2Y 14 -R-promoted inhibition of adenylyl cyclase provide a more physiologically relevant system for quantification of P2Y 14 -R-dependent signaling over an engineered signaling system that depends on overexpression of a G protein that activates phospholipase C. Given our recent observation of activities of UDP at the P2Y 14 -R transiently expressed with an unnatural chimeric G protein, G␣ q/i , in COS-7 cells, we believed it important to quantify the relative actions of UDP and UDP-glucose for promotion of a native cell signaling response in cell lines stably expressing the human P2Y 14 -R.…”

Section: Resultsmentioning

confidence: 99%

“…Cell Lines Stably Expressing P2Y 14 -R. Human embryonic kidney 293 cells stably expressing the hP2Y 14 -R (P2Y 14 -HEK293 cells) and C6 rat glioma cells stably expressing the hP2Y 14 -R (P2Y 14 -C6 cells) were generated as described previously (Fricks et al, 2009). Chinese hamster ovary cells stably expressing the hP2Y 14 -R (P2Y 14 -CHO cells) were produced using similar methods.…”

Section: Methodsmentioning

confidence: 99%

“…MAP Kinase Activation Assays. Phosphorylation of ERK1/2 was quantified as we have described in detail (Fricks et al, 2009). In brief, wild-type HEK293 or P2Y 14 -HEK293 cells were serum-starved for 18 h before addition of either UDP or UDP-glucose for 15 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

Carter¹,

Fricks²,

Barrett³

et al. 2009

Mol Pharmacol

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106

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The P2Y 14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y 14 receptor, allowing facile examination of its coupling to native G i family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330: [162][163][164][165][166][167][168] 2009). In the current study, we examined P2Y 14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y 14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC 50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y 14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC 50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y 14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y 14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y 14 receptor over the UDP-activated P2Y 6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y 14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y 14 receptor.The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, that primarily signal through G q -activated signaling pathways and a subgroup of three receptors, the P2Y 12 , P2Y 13 , and P2Y 14 receptors, that primarily signal by activating heterotrimeric G proteins of the G i family (Abbracchio et al., 2006;Burnstock, 2007). The human P2Y 1 , P2Y 11 , P2Y 12 , and P2Y 13 receptors are activated by adenine nucleotides. The human P2Y 4 and P2Y 6 receptors are activated by uridine nucleotides, and the P2Y 2 receptor is activated by both ATP and UTP.

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Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

Carter¹,

Fricks²,

Barrett³

et al. 2009

Mol Pharmacol

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106

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“…On a speculative basis, UDP-Nacetylglucosamine and potentially UDP-galactose and UDPglucuronic acid may contribute to P2Y 14 R-dependent lung phenotypes. However, given the low P2Y 14 R agonist potency exhibited by these UDP-sugars, relative to UDP-glucose [13,54], UDP-glucose predictably is the most important autocrine/ paracrine regulator of P2Y 14 R-mediated lung inflammation. Recent studies examining P2Y 14 R-dependent second messenger formation in model cell lines revealed that UDP is a very potent agonist of this receptor [15].…”

Section: Discussionmentioning

confidence: 99%

UDP-glucose promotes neutrophil recruitment in the lung

Sesma

Weitzer

Livraghi‐Butrico

et al. 2016

Purinergic Signalling

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In addition to their role in glycosylation reactions, UDP-sugars are released from cells and activate widely distributed cell surface P2Y 14 receptors (P2Y 14 R). However, the physiological/pathophysiological consequences of UDPsugar release are incompletely defined. Here, we report that UDP-glucose levels are abnormally elevated in lung secretions from patients with cystic fibrosis (CF) as well as in a mouse model of CF-like disease, the βENaC transgenic (Tg) mouse. Instillation of UDP-glucose into wild-type mouse tracheas resulted in enhanced neutrophil lung recruitment, and this effect was nearly abolished when UDP-glucose was coinstilled with the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid]. Importantly, administration of PPTN to βENaC-Tg mice reduced neutrophil lung inflammation. These results suggest that UDP-glucose released into the airways acts as a local mediator of neutrophil inflammation.

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P2Y₁₄ receptor is functionally expressed in satellite glial cells and mediates interleukin‐1β and chemokine CCL2 secretion

Lin

Liu

Zhang

et al. 2019

Journal Cellular Physiology

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Satellite glial cells (SGCs) activation in the trigeminal ganglia (TG) is critical in various abnormal orofacial sensation in nerve injury and inflammatory conditions. SGCs express several subtypes of P2 purinergic receptors contributing to the initiation and maintenance of neuropathic pain. The P2Y 14 receptor, a G-protein-coupled receptor activated by uridine diphosphate (UDP)-glucose and other UDP sugars, mediates various physiologic events such as immune, inflammation, and pain. However, the expression, distribution, and function of P2Y 14 receptor in SGCs remains largely unexplored. Our study reported the expression and functional identification of P2Y 14 receptor in SGCs. SGCs were isolated from TG of rat, and the P2Y 14 receptor expression was examined using immunofluorescence technique. Cell proliferation and viability were examined via cell counting kit-8 experiment. Immunofluorescence demonstrated the presence of P2Y 14 receptor in SGCs. Immunofluorescence and western blot showed that UDP-glucose treatment upregulated glial fibrillary acid protein, a common marker for glial activation. Extracellular UDP-glucose enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which were both abolished by the P2Y 14 receptor inhibitor (PPTN). Furthermore, quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay demonstrated that extracellular UDPglucose significantly enhanced interleukin-1β (IL-1β) and chemokine CCL2 (CCL2) release, which was abolished by PPTN and significantly decreased by inhibitors of MEK/ERK (U0126) and p38 (SB202190). Our findings directly proved the functional presence of P2Y 14 receptor in SGCs. It was also verified that P2Y 14 receptor activation was involved in activating SGCs, phosphorylating MAPKs, and promoting the secretion of IL-1β and CCL2 via ERK and p38 pathway.

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G_i-Dependent Cell Signaling Responses of the Human P2Y₁₄Receptor in Model Cell Systems

Abstract: Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins.

Cited by 50 publications

References 39 publications

Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

UDP-glucose promotes neutrophil recruitment in the lung

P2Y₁₄ receptor is functionally expressed in satellite glial cells and mediates interleukin‐1β and chemokine CCL2 secretion

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Gi-Dependent Cell Signaling Responses of the Human P2Y14Receptor in Model Cell Systems

Abstract: Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins.

Cited by 50 publications

References 39 publications

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

UDP-glucose promotes neutrophil recruitment in the lung

P2Y14 receptor is functionally expressed in satellite glial cells and mediates interleukin‐1β and chemokine CCL2 secretion

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Resources

About

G_i-Dependent Cell Signaling Responses of the Human P2Y₁₄Receptor in Model Cell Systems

Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

Quantification of G_i-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y₁₄ Receptor

P2Y₁₄ receptor is functionally expressed in satellite glial cells and mediates interleukin‐1β and chemokine CCL2 secretion