Differential Regulation of the Uridine Nucleotide-activated P2Y4 and P2Y6 Receptors: SER-333 AND SER-334 IN THE CARBOXYL TERMINUS ARE INVOLVED IN AGONIST-DEPENDENT PHOSPHORYLATION DESENSITIZATION AND INTERNALIZATION OF THE P2Y4 RECEPTOR

Agonist-promoted regulation of the uridine nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor (P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human astrocytoma cells with the cognate agonist UTP resulted in rapid desensitization of the inositol phosphate response and a 50% loss of cell surface receptors. In contrast, incubation of P2Y6-R-expressing cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the medium of UTP-pretreated cells resulted in rapid and complete recovery of surface P2Y4-R even after 12 h of agonist treatment. Although extended incubation with UDP also caused a loss of surface P2Y6-R, rapid recovery of surface P2Y6-R did not occur following removal of agonist. Pharmacological studies indicated that neither protein kinase C nor other Ca(2+)-activated kinases were involved in agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R. Mutational analyses were carried out to identify domains involved in agonist-dependent regulation of P2Y4-R. Sequential truncation of the carboxyl-terminal domain revealed that sequence between amino acids 332 and 343 was necessary for UTP-promoted desensitization and internalization. Further mutational analyses of the three serines in this domain confirmed that Ser-333 and Ser-334 play a major role in these agonist-promoted changes in P2Y4-R. Experiments were carried out with [(32)P]P(i)-labeled cells to ascertain the role of phosphorylation in regulation of P2Y4-R. Incubation with UTP for 2 min caused a marked increase in phosphorylation of both the wild-type P2Y4-R and the P2Y4-343 truncation mutant. In contrast, no UTP-promoted phosphorylation of the P2Y4-332 truncation mutant was observed. Taken together, these results demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl terminus of P2Y4-R are important for UTP-dependent phosphorylation, desensitization, and loss of surface receptors

Activation of Human Phospholipase C-η2 by Gβγ †

Phospholipase C-η2 (PLC-η2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667–676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128–29134]. In this study, we investigated the direct regulation of PLC-η2 by Gβγ subunits of heterotrimeric G proteins. Coexpression of PLC-η2 with Gβ1γ2, as well as with certain other Gβγ dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gβ1γ2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-η2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-η2 and its potential direct activation by Gβγ, a construct of PLC-η2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-η2 was quantified after reconstitution with PtdIns(4,5)P2-containing phospholipid vesicles, and values for Km (14.4 µM) and Vmax [12.6 µmol min−1 (mg of protein)−1] were similar to activities previously observed with purified PLC-β or PLC-ε isozymes. Moreover, purified Gβ1γ2 stimulated the activity of purified PLC-η2 in a concentration-dependent manner similar to that observed with purified PLC-β2. Activation was dependent on the presence of free Gβ1γ2 since its sequestration in the presence of Gαi1 or GRK2-ct reversed Gβ1γ2-promoted activation. The PH domain of PLC-η2 is not required for Gβ1γ2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gβ1γ2. Taken together, these studies illustrate that PLC-η2 is a direct downstream effector of Gβγ and, therefore, of receptor-activated heterotrimeric G proteins

Human P2Y 14 Receptor Agonists: Truncation of the Hexose Moiety of Uridine-5′-Diphosphoglucose and Its Replacement with Alkyl and Aryl Groups

Uridine-5′-diphosphoglucose (UDPG) activates the P2Y14 receptor, a neuroimmune system GPCR. P2Y14 receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine-5′-diphosphate (UDP), a full agonist human P2Y14 receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y14 vs. the P2Y6 receptor, such as 2-thio-UDP 4 (EC50 1.92 nM at P2Y14, 224-fold selectivity vs. P2Y6) and its β-propyloxy ester 18. EC50 of β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. β-t-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y14 receptor agonist activity. α,β-Methylene and difluoromethylene groups were well tolerated at the P2Y14 receptor and are expected to provide enhanced stability in biological systems. α,β-Methylene-2-thio-UDP 11 (EC50 0.92 nM) was 2160-fold selective versus P2Y6. Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y14 receptor

Protein Kinase C-Promoted Inhibition of G␣ 11 -Stimulated Phospholipase C-␤ Activity

ABSTRACT The effects of protein kinase C (PKC) activation on inositol lipid signaling were examined. Using the turkey erythrocyte model of receptor-regulated phosphoinositide hydrolysis, we developed a membrane reconstitution assay to study directly the effects of activation of PKC on the activities of G␣ 11 , independent of potential effects on the receptor or on PLC-␤. Membranes isolated from erythrocytes pretreated with 4␤-phorbol-12␤-myristate-13␣-acetate (PMA) exhibited a decreased capacity for G␣ 11 -mediated activation of purified, reconstituted PLC-␤1. This inhibitory effect was dependent on both the time and concentration of PMA incubation and occurred as a decrease in the efficacy of GTP␥S for activation of PLC-␤1, both in the presence and absence of agonist; no change in the apparent affinity for the guanine nucleotide occurred. Similar inhibitory effects were observed after treatment with the PKC activator phorbol-12,13-dibutyrate but not after treatment with an inactive phorbol ester. The inhibitory effects of PMA were prevented by coaddition of the PKC inhibitor bisindolylmaleimide. Although the effects of PKC could be localized to the membrane, no phosphorylation of G␣ 11 occurred either in vitro in the presence of purified PKC or in intact erythrocytes after PMA treatment. These results support the hypothesis that a signaling protein other than G␣ 11 is the target for PKC and that PKCpromoted phosphorylation of this protein results in a phosphorylation-dependent suppression of G␣ 11 -mediated PLC-␤1 activation. Various receptors transduce signals through heterotrimeric G proteins of the G q family, resulting in activation of phospholipase C (PLC)-␤ isoenzymes and subsequent cleavage of membrane phosphatidylinositol(4,5)P 2 [PtdIns(4,5)P 2 ] to the second messengers inositol(1,4,5)P 3 [Ins(1,4,5)P 3 ] and diacylglycerol Agonist-induced desensitization is an important regulatory process in inositol lipid signaling The turkey erythrocyte is a well-characterized model of receptor-promoted inositol phospholipid signaling and is particularly useful because the three primary proteins in the pathway, i.e., the turkey P2Y 1 recepto

Constitutive Release of ATP and Evidence for Major Contribution of Ecto-nucleotide Pyrophosphatase and Nucleoside Diphosphokinase to Extracellular Nucleotide Concentrations

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase

Synthesis of extended uridine phosphonates derived from an allosteric P2Y2 receptor ligand

In this study we report the synthesis of C5/C6-fused uridine phosphonates that are structurally related to earlier reported allosteric P2Y2 receptor ligands. A silyl-Hilbert-Johnson reaction of six quinazoline-2,4-(1H,3H)-dione-like base moieties with a suitable ribofuranosephosphonate afforded the desired analogues after full deprotection. In contrast to the parent 5-(4-fluoropheny)uridine phosphonate, the present extended-base uridine phosphonates essentially failed to modulate the P2Y2 receptor

Identification of an Ecto-nucleoside Diphosphokinase and Its Contribution to Interconversion of P2 Receptor Agonists

The P2Y4 receptor is selectively activated by UTP. Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 human astrocytoma cells stably expressing the P2Y4 receptor, combined addition of these nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]UDP or [3H]ADP under conditions in which cellular release of ATP occurred or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-phosphate from [gamma-33P]ATP to nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP also were determined for 1321N1 cells. All four nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-nucleoside diphosphokinase and ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-nucleoside diphosphokinase together with previous observations that stress-induced release of ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of adenine and uridine nucleotides. Since the P2Y receptor class of signaling proteins differs remarkably in their respective specificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine and uridine nucleotides plays a key role in defining activities in nucleotide-mediated signaling

Direct Demonstration of Mechanically Induced Release of Cellular UTP and Its Implication for Uridine Nucleotide Receptor Activation

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors