Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis.
ABSTRACT: BACKGROUND AND PURPOSE: The P2Y(11) receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P(alpha)-substituted ATP derivatives (ATP-alpha-S isomers). These compounds are suitable candidates for the development of selective P2Y(11) receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y(11) receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y(11) receptor. EXPERIMENTAL APPROACH: Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y(11) receptor. KEY RESULTS: After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-alpha-S isomers at the P2Y(11) receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants. CONCLUSIONS AND IMPLICATIONS: Our results show that the stereospecificity of the P2Y(11) receptor for P(alpha)-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.
Project description:BACKGROUND AND PURPOSE: We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor. EXPERIMENTAL APPROACH: The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca2+]i). In a series of ATP-alpha-B and ATP-alpha-S analogues, a non-bridging oxygen atom of Palpha was substituted by BH3 or sulphur, respectively, introducing a chiral center at Palpha. The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. KEY RESULTS: The (B) isomers (ATP-alpha-B Sp isomers and ATP-alpha-S Rp isomers) of all derivatives tested were more potent at the P2Y11 receptor than the corresponding (A) isomers (ATP-alpha-B Rp isomers and ATP-alpha-S Sp isomers) and the parent compounds. This characteristic of the P2Y11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y1 receptor, at which the (A) isomers are more active. CONCLUSIONS AND IMPLICATIONS: The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y11 and the P2Y1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-alpha-B diastereoisomers are not active at the P2Y2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y2 and P2Y11 receptor, in physiological or pathophysiological responses of cells.
Project description:To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs.Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists.MECs expressed the MEC proteins ?-smooth muscle actin, vimentin, ?-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), ?,? methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATP?S increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective.MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).
Project description:The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca(2+)-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca(2+) concentration ([Ca(2+)](i)) from 150 to 300 nM in single cells. The change in [Ca(2+)](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTP?S, ATP?S, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, ??-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP(3) cascade-mediated Ca(2+) release, indicating that the nucleotide-induced Ca(2+) signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca(2+) signal and suppressed the Ca(2+) signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATP?S (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca(2+) signalling of the hCMEC/D3 cells.
Project description:The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels.Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons.In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed.Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.
Project description:The P2Y(12) receptor, a Gi protein-coupled receptor, plays a central role in platelet activation. In this study, we did a mutational analysis of residues possibly involved in the ligand interactions with the human P2Y(12) receptor. Mutant receptors were stably expressed in CHO-K1 cells with an HA-tag at the N-terminus. Expression of wild-type and mutant receptors was confirmed by detecting the HA-tag on the cell membrane. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7, which are homologous to residues important for P2Y(1) receptor activation and ligand recognition, were replaced by site-directed mutagenesis. ADP-induced inhibition of forskolin-stimulated cAMP levels in the presence or absence of antagonist AR-C69931MX were investigated for each of the mutant receptors. F104S and S288P significantly increased agonist-induced receptor function without affecting the antagonism by AR-C69931MX. Arg256 in TM6 and Arg 265 in extracellular loop 3 (EL3) are more important for antagonist recognition than effect on agonist-mediated receptor function. Compared to wild-type P2Y(12) receptor, mutations in Arg 256 or/and Arg 265 significantly increased the sensitivity to antagonist AR-C69931MX. Our study shows that the cytosolic side of TM3 and the exofacial side of TM5 are critical for P2Y(12) receptor function, which is different from P2Y(1). Arg 256 in TM6 and Arg265 in EL3 appear to play a role in antagonist recognition rather than effects on agonist-induced receptor function.
Project description:Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2-YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally co-transfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin-2-YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.
Project description:PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (-)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (-)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.
Project description:Purinergic P2Y 2 receptors, G-protein coupled receptors that primarily couple with G?q/11-proteins, are activated equipotently by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate. Evidence suggests that P2Y 2 agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y 2 agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y 2 agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y 2 agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other G?q/11-coupled human P2Y (hP2Y) receptors and one representative G?i/o-coupled hP2Y12 receptor. Its P2Y 2 agonistic effect on mouse P2Y 2 receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca2+ mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y2 activation in various cardiovascular diseases.
Project description:Cholesterol is a major component of the membrane and a key regulator of many ion channels. Multiple studies showed that cholesterol regulates ion channels in a stereospecific manner, with cholesterol but not its chiral isomers having a functional effect. This stereospecificity has been universally attributed to the specificity of cholesterol binding, with the assumption that only native cholesterol binds to the channels whereas its isomers do not. In this study, we challenge this paradigm by docking analyses of cholesterol and its chiral isomers to five ion channels whose response to cholesterol was shown to be stereospecific, Kir2.2, KirBac1.1, TRPV1, GABAA and BK. The analysis is performed using AutoDock Vina to predict the binding poses and energies of the sterols to the channels and identify amino acids interacting with the sterol molecules. We found that for every ion channel tested herein all three sterols showed similar binding poses and significant overlap in the set of the amino acids that comprise the predicted binding sites, along with similar energetic favorability to these overlapping sites. We also found, however, that specific orientations of the three sterols within the binding sites of the channels are distinct, so that a subset of the interacting amino acids is unique to each sterol. We propose therefore, that contrary to previous thought, stereospecific effects of cholesterol should be attributed not to the lack of binding of the stereoisomers but to specific, unique interactions between the cholesterol molecule and the residues within the binding sites of the channels.
Project description:Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y(14) receptor (P2Y(14)R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y(1)R, P2Y(12)R, and P2Y(13)R, were similar, and P2Y(11)R and P2Y(4)R were highly expressed at 5.8- and 3.8-fold of P2Y(1)R, respectively. Least expressed P2Y(2)R was 40-fold lower than P2Y(1)R, and P2Y(6)R and P2Y(14)R were ?50 % of P2Y(1)R. None of the native P2YR agonists alone induced ?-hexosaminidase (?-Hex) release, but some nucleotides significantly enhanced ?-Hex release induced by C3a or antigen, with a rank efficacy order of ATP?>?UDPG???ADP?>>?UDP, UTP. Although P2Y(11)R and P2Y(4)R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y(4)R agonist UTP nor P2Y(11)R agonists ATP?S and NF546 had a substantial effect. P2Y(1)R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y(1)R potency, and the effect of P2Y(6)R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y(14)R, MRS2690, enhanced C3a-induced ?-Hex release, which was inhibited by a P2Y(14)R antagonist, specific P2Y(14)R siRNA and pertussis toxin, suggesting a role of P2Y(14)R activation in promoting human mast cell degranulation.