P2Y GPCR Assays

Background of P2Y Receptors

P2Y purinergic receptors are a family of extracellular G protein-coupled receptors for purine and pyrimidine nucleotides, such as ADP, ATP, and UTP, participating in purinergic signaling.

Structure of human P2Y<sub>1</sub> receptor showing 7 transmembrane domains and intracellular domains for binding G proteins and protein kinases. Fig.1 Structure of human P2Y₁ receptor showing 7 transmembrane domains and intracellular domains for binding G proteins and protein kinases. (Zhang, 2015)

Distribution and Function of P2Y Receptors

P2Y receptors are mainly distributed in the placenta, prostate, brain, gastrointestinal tract, skeletal muscle, lung, heart, spleen, macrophages, lymphocytes, liver, and kidney. P2Y₁ receptors participate in endothelial cell migration, mitogenic effects, smooth muscle relaxation, induction of platelet aggregation, regulation of EGF activity, and neuroprotection. P2Y₂ receptors are involved in vasodilation, migration of vascular smooth muscle cells, orientation of neutrophil chemotaxis, and inhibition of apoptosis. P2Y₄ receptors play a role in the activation of luminal K⁺ secretion, regulation of rod bipolar cell activity, adipogenesis and osteogenesis, etc. In addition, P2Y₂ and P2Y₄ receptors mediate the chloride epithelial transport in the jejunum. P2Y₆ receptors are responsible for the contraction of smooth muscle, DNA synthesis, IL-8 release, Cl-secretion, tumor cell proliferation, regulation of insulin and glucagon secretion, etc. P2Y₁₁ receptors play a role in the maturation and migration of DC cells, platelet release of nitric oxide, inhibition of neutrophil apoptosis, and chemotaxis of granulocytes. P2Y₁₂ receptors are important in maintaining platelet aggregation and promoting thrombus growth and stabilization, as well as chemotaxis and response to arterial injury and thrombosis. P2Y₁₃ receptors inhibit ATP release from erythrocytes, insulin secretion, and CFA-induced hyperalgesia. P2Y₁₄ receptors are involved in chemotaxis, neuroimmune function, DC cell activation, etc.

Subtypes and Mechanisms of P2Y Receptors

The family of P2Y receptors in humans contains 8 subtypes of G protein-coupled receptors, including P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, P2Y₁₃, and P2Y₁₄ receptors. They promote signal transduction primarily through heterotrimeric G proteins and ERK signaling pathways and regulation of ion channels, and kinases.

Receptor	Gene	Mechanism	Agonists	Antagonists
P2Y₁ receptor	P2RY1	P2Y₁ receptor couples with G_q/G₁₁ protein to stimulate phospholipase C The activation of phospholipase C leads to the mobilization of calcium from IP3-sensitive intracellular stores P2Y₁ receptor couples with G_i/G_o protein to regulate potassium channel in CHO cells	[³H]2MeSADP MRS2365 2-Cl-ADP(α-BH₃) compound 3a ADPβS Ap5a 2',3'-ddATP ATP ADP	[³²P]MRS2500 [³H]MRS2279 MRS2500 BMS compound 4c Pfizer compound 67 MRS2279
P2Y₂ receptor	P2RY2	Activation of the P2Y₂ receptor leads to stimulation of chloride secretion through G_q/G₁₁, phospholipase C activation, and then Na⁺ transport inhibition P2Y² receptor couples to Gⁱ/G^o protein through integrin, causing Rac activation, vitronectin up-regulation, and increasing cell migration P2Y² receptor couples to G¹² protein, causing Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation, and chemotaxis towards UTP With nerve growth factor (NGF), the P2Y² receptor interacts with tyrosine kinase A (TrkA) through Src, activating ERK1/2 phosphorylation	MRS2698 uridine triphosphate 4-thio-UTP diquafosol PSB1114 ATP	AR-C126313 reactive blue-2 AR-C118925XX PSB-416 suramin
P2Y₄ receptor	P2RY4	P2Y₄ receptor couples with G_q/G₁₁ protein to stimulate phospholipase C P2Y₄ receptor activation stimulates phospholipase A₂, and COX-1 P2Y₄ receptor binds UTP, activating ERK1/2 phosphorylation	MRS4062 MRS2927 (N)methanocarba-UTP diquafosol uridine triphosphate denufosol	ATP PPADS PSB-16133 Reactive blue-2
P2Y₆ receptor	P2RY6	P2Y₆ receptor couples with G_q/G₁₁ protein to stimulate phospholipase C P2Y₆ receptor couples to G₁₂/G₁₃ protein, activating Pho and c-Jun kinase increasing the expression of fibrogenic genes and Ang-converting enzyme (ACE)	Rp-5-OMe-UDPαB MRS2957 MRS2693 MRS4162 UDP-β-S 3-phenacyl-UDP INS48823	Reactive blue-2 PPADS suramin TIM-38 MRS2578 MRS2567
P2Y₁₁ receptor	P2RY11	P2Y₁₁ receptor couples with G_q/G₁₁ protein to stimulate phospholipase C P2Y₁₁ receptor couples with G_s protein to stimulate adenylyl cyclase	AR-C67085 NF546 ATPγS uridine triphosphate ATP NAADP NAD	NF157 NF340 suramin reactive blue-2
P2Y₁₂ receptor	P2RY12	P2Y₁₂ receptor couples with G_i/G_o protein to inhibit adenylyl cyclase P2Y₁₂ receptor amplifies platelet responses through inhibition of cyclic AMP production, VASP dephosphorylation, and activation of PI 3-K and small GTPase Rap1b P2Y₁₂ receptor increases Ca²⁺, especially in microglial and satellite glial cells from trigeminal ganglia	2MeSADP ADP ADPβS [³H]2MeSADP	PSB-0739 ticagrelor [³H]PSB-0413 AR-C67085 compound 20o AZD1283 clopidogrel compound 4
P2Y₁₃ receptor	P2RY13	P2Y₁₃ receptor couples with G_i/G_o protein to inhibit adenylyl cyclase The P2Y₁₃ receptor increases Ca²⁺, especially in microglial and satellite glial cells from trigeminal ganglia The P2Y₁₃ receptor mediates intracellular calcium responses to BzATP in cerebellar astrocytes	[³³P]2MeSADP 2MeSADP 2MeSADTP ADP ADPβS ATPγS ATP	cangrelor Ap4A MRS2603 MRS2211 reactive blue-2 suramin 2MeSAMP PPADS
P2Y₁₄ receptor	P2RY14	P2Y₁₄ receptor couples with G_i/G_o protein to inhibit adenylyl cyclase and stimulates phospholipase C P2Y₁₄ receptor couples to Gα_i, activating PLCβ P2Y₁₄ receptor couples to Gα_o, mediating calcium influx in endothelial cells P2Y₁₄ receptor binds to UDP-glucose in primary glial cells for the signal transduction	α.β-methylene-2-thio-UDP MRS4183 MRS2905 2-thio-UDP UDP-glucuronic acid MRS2802 UDP UDP-galactose MRS2690 UDP-glucose UDP N-acetyl-glucosamine	PPTN MRS4174 MRS4625 MRS4458 MRS4478

Assay List of P2Y Receptors

Creative Biolabs can provide a range of assays of P2Y receptors. You can choose the assay in the list or contact us for more information:

P2RY1 P2RY2 P2RY4 P2RY6 P2RY12

Assay No.	Assay Name	Host Cell	Assay Type	Datasheet
IP1 Assay
S01YF-1122-KX845	Magic™ Human P2RY1 In Vitro IP1 Assay	1321N1	IP1 Assay

Assay No.	Assay Name	Host Cell	Assay Type	Datasheet
IP1 Assay
S01YF-1122-KX848	Magic™ Human P2RY2 In Vitro IP1 Assay	1321N1	IP1 Assay

Assay No.	Assay Name	Host Cell	Assay Type	Datasheet
IP1 Assay
S01YF-1122-KX849	Magic™ Human P2RY4 In Vitro IP1 Assay	1321N1	IP1 Assay

Assay No.	Assay Name	Host Cell	Assay Type
IP1 Assay
S01YF-1122-KX851	Magic™ Human P2RY6 In Vitro IP1 Assay	1321N1	IP1 Assay
S01YF-1122-KX852	Magic™ Mouse P2RY6 In Vitro IP1 Assay	1321N1	IP1 Assay

Assay No.	Assay Name	Host Cell	Assay Type
cAMP Assay
S01YF-0722-KX51	Magic™ Human P2RY12 In Vitro Agonist & Antagonist cAMP Assay, CHO	CHO	cAMP Assay
Calcium Flux Assay
S01YF-1122-KX847	Magic™ Human P2RY12 In Vitro Calcium Flux Assay	CHO-K1-Gqi5	Calcium Flux Assay

Published Data

Paper Title	Identification of P2Y receptors involved in oleamide-suppressing inflammatory responses in murine microglia and human dendritic cells
Journal	Scientific reports
Published	2019
Abstract	The Previous study has found that oleamide is an endocannabinoid from fermented dairy products, which plays a role as a neuroprotective compound suppressing microglial inflammation. The suppressive effect of oleamide may be exerted via GPCR downstream signaling. Thus, the study aimed to explain the mechanism underlying the anti-inflammatory activity of the oleamide by screening the GPCRs using a transforming growth factor-α shedding assay system identified P2Y₁, P2Y₄, P2Y₆, P2Y₁₀, and P2Y₁₁ as candidates for the oleamide target. They also observed a relationship between the P2Y₁ agonistic activities and the suppressive activities of oleamide and its analogs. The study indicated that oleamide may exert as an antagonist for the P2Y₁ receptor, which plays a role in the suppression of microglial inflammatory responses.
Result	The study has found that the suppressive effect of oleamide on microglial tumor necrosis factor-α (TNF-α) production was canceled by inhibitors of GPCR downstream signaling instead of a CB2 antagonist, which suggested that GPCRs are involved in the anti-inflammatory effects of oleamide. The result of the transforming growth factor-α shedding assay has demonstrated that the P2Y₁ and P2Y₁₀ agonists suppressed microglial TNF-α production, whereas a pan P2 receptor antagonist canceled the suppressive effect. The relationship between the P2Y₁ agonistic activities and the suppressive activities of oleamide and its analogs, such as trans-OAD (tOAD), oleoylethylamide(OEtA), oleic acid, and palmitoylethanolamide (PEA). The analogs of oleamide displayed lower agonistic activity against P2Y₁, P2Y₄, and P2Y₁₁ than oleamide, while oleoylethanolamide (OEA) had agonist activity. In summary, P2Y GPCRs are the potential targets of oleamide, and P2Y₁ is important in the suppression of microglial inflammatory responses by oleamide. Fig.2 Improvement in the TGF-α shedding response to OAD in HEK293 cells expressing P2Y₁, P2Y₄, P2Y₆, P2Y₁₀, P2Y₁₁, CB1, and CB2. (Kita, 2019)

References

Zhang, D.; et al. Two disparate ligand-binding sites in the human P2Y₁ receptor. Nature. 2015, 520(7547): 317-321.
Kita, M.; et al. Identification of P2Y receptors involved in oleamide-suppressing inflammatory responses in murine microglia and human dendritic cells. Scientific reports. 2019, 9(1): 1-10.

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