Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism.

A nonactivating allosteric modulator of free fatty acid receptor 2 (FFA2R, also called GPCR 43) turns both propionate (an orthosteric FFA2R agonist) and ATP (an agonist for the purinergic P2Y2 receptor), into potent activating ligands that trigger an assembly of the superoxide-generating neutrophil NADPH oxidase. The ATP-induced activation requires the participation of FFA2R, and the signaling is biased toward oxidase activation, leaving the ATP-induced rise in intracellular Ca2+ unaffected. No NADPH oxidase activity was induced by ATP when propionate replaced the allosteric modulator. Signaling downstream of propionate-activated FFA2Rs was insensitive to Gαq inhibition, but the crosstalk activation involving both FFA2R and P2Y2R relied on Gαq signaling. The receptor crosstalk, by which allosterically modulated FFA2Rs communicate with P2Y2Rs and generate NADPH oxidase activating signals downstream of Gαq, represent a novel mechanism by which GPCR activities can be regulated from inside the plasma membrane. Further, the finding that an allosteric FFA2R modulator sensitizes not only the response induced by orthosteric FFA2R agonists, but also the response induced by ATP (P2Y2R-specific agonist) and formyl peptide receptor-specific agonists, violates the receptor restriction characteristics normally defining the selectivity of allosteric GPCR modulators.-Lind, S., Holdfeldt, A., Mårtensson, J., Sundqvist, M., Björkman, L., Forsman, H., Dahlgren, C. Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism.

Functional characterization of purinergic P2Y2 and P2Y12 receptors involved in Japanese flounder (Paralichthys olivaceus) innate immune responses.

G-protein-coupled P2Y receptors activated by extracellular nucleotides play important roles under different physiological and pathophysiological conditions in mammals. To investigate the immunological relevance of P2Y receptors in fish, we identified and characterized the P2Y2 and P2Y12 receptors in Japanese flounder Paralichthys olivaceus. The P. olivaceus P2Y2 and P2Y12 receptors harbor seven transmembrane domains but share only 24% sequence identity. Real-time PCR analysis revealed the constitutive but unequal mRNA expression pattern of P2Y2R and P2Y12R in normal Japanese flounder tissues with the dominant expression of P2Y2R in head kidney and blood and P2Y12R in hepatopancreas. In addition, the expression of P2Y2 and P2Y12 receptors was markedly modulated by PAMPs stimulation and Edwardsiella tarda infection. Furthermore, blockage of P2Y12R potently increased ADP-activated pro-inflammatory cytokine IL-1beta gene expression in the head kidney macrophages (HKMs). Moreover, inhibition of P2Y2 and P2Y12 receptor activity with their respective potent antagonists significantly altered some of the LPS-induced pro-inflammatory cytokine gene expression in the HKMs. However, blockade of P2Y12R did not affect the poly(I:C)-induced pro-inflammatory cytokine gene expression examined in the HKMs. Collectively, we have for the first time reported the role of purinergic P2Y2 and P2Y12 receptors in fish innate immunity. Our findings have also addressed the importance of extracellular ATP and its metabolites in fish innate immune responses.

Homodimer formation by the ATP/UTP receptor P2Y2 via disulfide bridges.

Many class C G-protein coupled receptors (GPCRs) function as homo- or heterodimers and several class A GPCRs have also been shown to form a homodimer. We expressed human P2Y2 receptor (P2Y2R) in cultured cells and compared SDS-PAGE patterns under reducing and non-reducing conditions. Under non-reducing conditions, approximately half of the P2Y2Rs were electrophoresed as a dimer. We then produced Cys to Ser mutants at four sites (Cys25, Cys106, Cys183 and Cys278) in the extracellular domains of P2Y2R and examined the effect on dimer formation and receptor activity. All single mutants formed dimers similarly to the wild-type protein, but C25S, C106S and C183S P2Y2R lost activity, while C278S P2Y2R maintained weak activity. Coexpression with wild-type P2Y2R recovered the activity of the C25S mutant. These results show that Cys106 and Cys183 are required for monomer or homodimer activity; Cys25 is required for monomer activity, but it is not needed in one protomer for homodimer activity; and Cys278 can be replaced in the monomer and homodimer. Approximately, half of C25S/C278S double mutants were electrophoresed as a dimer, similarly to the wild-type and single mutants, and dimers with the wild-type protein were active. These results suggest involvement of Cys106 and Cys183 in disulfide bonding between protomers in homodimer formation.

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor.

The G protein-coupled P2Y2 receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y2R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y2 receptor discovered using the endogenous P2Y2R agonist UTP as the chemical starting point.

Molecular Recognition of Agonists and Antagonists by the Nucleotide-Activated G Protein-Coupled P2Y2 Receptor.

A homology model of the nucleotide-activated P2Y2R was created based on the X-ray structures of the P2Y1 receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design.

Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

BACKGROUND: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. METHODS: Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. RESULTS: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. CONCLUSION: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

A pepducin designed to modulate P2Y2R function interacts with FPR2 in human neutrophils and transfers ATP to an NADPH-oxidase-activating ligand through a receptor cross-talk mechanism.

Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin.

4-Alkyloxyimino derivatives of uridine-5'-triphosphate: distal modification of potent agonists as a strategy for molecular probes of P2Y2, P2Y4, and P2Y6 receptors.

Extended N(4)-(3-arylpropyl)oxy derivatives of uridine-5'-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N(4)-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N(4)-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N(4)-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

Synthesis and P2Y2 receptor agonist activities of uridine 5'-phosphonate analogues.

We explored the influence of modifications of uridine 5'-methylenephosphonate on biological activity at the human P2Y(2) receptor. Key steps in the synthesis of a series of 5-substituted uridine 5'-methylenephosphonates were the reaction of a suitably protected uridine 5'-aldehyde with [(diethoxyphosphinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki-Miyaura coupling. These analogues behaved as selective agonists at the P2Y(2) receptor, with three analogues exhibiting potencies in the submicromolar range. Although maximal activities observed with the phosphonate analogues were much less than observed with UTP, high concentrations of the phosphonates had no effect on the stimulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y(2) receptor.

Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells.

In blood vessels, stimulation of the vascular endothelium by the Ca(2+)-mobilizing agonist ATP initiates a number of cellular events that cause relaxation of the adjacent smooth muscle layer. Although vascular endothelial cells are reported to express several subtypes of purinergic P2Y and P2X receptors, the major isoform(s) responsible for the ATP-induced generation of vasorelaxant signals in human endothelium has not been well characterized. To address this issue, ATP-evoked changes in cytosolic Ca(2+), membrane potential and acute nitric oxide production were measured in isolated human umbilical vein endothelial cells (HUVECs) and profiled using established P2X and P2Y receptor probes. Whereas selective P2X agonist (i.e. α,ß-methyl ATP) and antagonists (i.e. TNP-ATP and PPADS) could neither mimic nor block the observed ATP-evoked cellular responses, the specific P2Y receptor agonist UTP functionally reproduced all the ATP-stimulated effects. Furthermore, both ATP and UTP induced intracellular Ca(2+) mobilization with comparable EC(50) values (i.e. 1-3µM). Collectively, these functional and pharmacological profiles strongly suggest that ATP acts primarily via a P2Y2 receptor sub-type in human endothelial cells. In support, P2Y2 receptor mRNA and protein were readily detected in isolated HUVECs, and siRNA-mediated knockdown of endogenous P2Y2 receptor protein significantly blunted the cytosolic Ca(2+) elevations in response to ATP and UTP, but did not affect the histamine-evoked response. In summary, these results identify the P2Y2 isoform as the major purinergic receptor in human vascular endothelial cells that mediates the cellular actions of ATP linked to vasorelaxation.