José L. Boyer

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

Second messenger cascade specificity and pharmacological selectivity of the human P2Y1-purinoceptor

1 . The coding sequence of the P2Y1‐purinoceptor was cloned from a human genomic library. 2 . The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1‐purinoceptor and is ≥95% homologous to the recently cloned rat, mouse, and bovine P2Y1‐purinoceptors. 3 . The human P2Y1‐purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1‐purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in 1321N1 cells infected with the control virus, this agonist markedly stimulated inositol phosphate accumulation in cells infected with the P2Y1‐purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1‐receptor‐expressing 1321N1 cells. 4 . The pharmacological selectivity of 18 purinoceptor agonists was established for the expressed human P2Y1‐purinoceptor. 2MeSATP was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, α,β‐MeATP had no effect and 3′‐NH2‐3′‐deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1‐purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1‐purinoceptor. 5 . C6 glioma cells express a P2Y‐purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1‐purinoceptor in C6 cells conferred 2MeSATP‐stimulated inositol lipid hydrolysis to these cells. The phospholipase C‐activating human P2Y1‐purinoceptor could be delineated from the endogenous P2Y‐purinoceptor of C6 glioma cells by use of the P2‐purinoceptor antagonist, PPADS, which blocks the P2Y1‐purinoceptor but does not block the endogenous P2Y‐ purinoceptor of C6 cells. P2‐purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y‐purinoceptors.

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate.

The antagonist activity of N6‐methyl 2′‐deoxyadenosine 3′,5′‐bisphosphate (N6MABP) has been examined at the phospholipase C‐coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP‐stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′‐bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pKB=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors

1 Stimulation of P2Y‐purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y‐purinoceptors on C6 rat glioma cells are not coupled to phospholipase C., but rather, inhibit adenylyl cyclase. 2 In this study we investigated the pharmacological selectivity of the P2‐purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6‐azophenyl 2′,4′‐disulphonic acid (PPADS) for phospholipase C‐ and adenylyl cyclase‐coupled P2Y‐purinoceptors. 3 In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 ± 0.2 and 7.6 ± 0.1, respectively), whereas PPADS at concentrations up to 100 μm had no effect. 4 In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 μm was a competitive antagonist of P2Y‐purinoceptor‐stimulated phospholipase C activity (pKB = 5.9 ± 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration‐effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5 Turkey erythrocytes also express a phospholipase C‐coupled β‐adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y‐purinoceptor‐mediated response had only minimal effects on the activation of phospholipase C by β‐adrenoceptors. In contrast, suramin and reactive blue 2 produced a non‐competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this β‐adrenoceptor agonist. 6 The differential effect of PPADS on P2Y‐purinoceptors of C6 glioma cells and turkey erythrocytes adds further support to the idea that different P2Y‐purinoceptor subtypes mediate coupling to adenylyl cyclic and phospholipase C.

2‐Chloro N6‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate is a selective high affinity P2Y1 receptor antagonist

We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y1 receptor and that modification of the ribose in these analogues is tolerated in the P2Y1 receptor binding pharmacophore. Here we delineate the pharmacological activity of one such non‐nucleotide molecule, 2‐chloro N6‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)‐conformation by a cyclopropane moiety. MRS2279 antagonized 2MeSADP‐stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pKB=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y1 receptor (pKB=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y1 receptor since MRS2279 had no effect on activation of the human P2Y2, P2Y4, P2Y6, or P2Y11 receptors by their cognate agonists. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. In contrast, the P2Y1 receptor is known to be obligatory in the process of ADP‐induced platelet aggregation, and MRS2279 competitively inhibited ADP‐promoted platelet aggregation with an apparent affnity (pKB=8.05) similar to that observed at the human P2Y1 receptor heterologously expressed in 1321N1 cells. Taken together these results illustrate selective high affinity antagonism of the P2Y1 receptor by a non‐nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.

Structure–activity relationships of dinucleotides: Potent and selective agonists of P2Y receptors

Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y1 while uridine containing dinucleotides have some level of agonist response on P2Y2 and P2Y6. The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

Functional expression of a cDNA encoding a human ecto‐ATPase

The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto‐ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established. Based on the sequence of CD39L1 we isolated from mRNA from human ECV‐304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto‐ATPase. Stable expression of isolated sequence in NIH‐3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto‐ATPase. The human ecto‐ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca2+‐ or Mg2+‐dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394±62 μM and 107±7 nmol Pi min−1 106 cells−1 for the hydrolysis of ATP, and 102±33 μM and 4±0.4 nmol Pi min−1 106 cells−1 for the hydrolysis of ADP, respectively. In conclusion, we report here the cloning and functional expression of a human ecto‐ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto‐ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.

Fidelity in functional coupling of the rat P2Y1 receptor to phospholipase C.

1 The rat homologue of the P2Y1 receptor has been heterologously expressed in 1321N1 human astrocytoma cells and in C6 rat glioma cells. 2 As has been shown previously for the turkey and human P2Y1 receptors, the rat P2Y1 receptor expressed in either cell type responded to 2MeSATP with increases in inositol phosphate accumulation that were competitively blocked by the antagonist PPADS. Neither of the wild type cell lines exhibited inositol phosphate responses to P2Y1 receptor agonists. 3 Expression of the rat P2Y1 receptor did not confer a capacity of 2MeSATP to inhibit adenylyl cyclase activity in 1321N1 cells. Moreover, the inhibition of adenylyl cyclase mediated by an endogenous P2Y receptor of C6 glioma cells was not enhanced by expression of the rat P2Y1 receptor. The P2Y receptor‐mediated inhibition of adenylyl cyclase in C6 glioma cells expressing both the endogenous P2Y receptor and the rat P2Y1 receptor remained unaffected by PPADS. 4 Since the P2Y receptor responsible for inhibition of adenylyl cyclase in C6 glioma cells does not share the pharmacological or functional properties of the P2Y1 receptor, even when both receptors originate from the same species and are simultaneously expressed in the same cell line, it is concluded that the P2Y1 receptor is distinct from an endogenous P2Y receptor in C6 cells that couples to inhibition of adenylyl cyclase.

Identification of potent P2Y-purinoceptor agonists that are derivatives of adenosine 5'-monophosphate.

1 A series of chain‐extended 2‐thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C‐linked P2Y‐purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase‐linked P2Y‐purinoceptor of C6 rat glioma cells, and the cloned human P2U‐receptor stably expressed in 1321N1 human astrocytoma cells. 2 Although adenosine monophosphate itself was not an agonist in the two P2Y‐purinoceptor test systems, eleven different 2‐thioether‐substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2‐hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2‐hexylthio ATP in the same system. 2‐hexylthio adenosine was inactive. 3 Monophosphate analogues that were the most potent activators of the C6 cell P2Y‐purinoceptor were also the most potent activators of the turkey erythrocyte P2Y‐purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4 Although 2‐thioether derivatives of adenosine monophosphate were potent P2Y‐purinoceptor agonists no effect of these analogues on the human P2U‐purinoceptor were observed. 5 These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y‐purinoceptors, and provide insight for strategies for development of novel P2Y‐purinoceptor agonists of high potency and selectivity.

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal‐5′‐phosphate‐6‐phenylazo‐2′,4′‐disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6‐phenylazo and 6‐naphthylazo derivatives were also evaluated. Among the 6‐phenylazo derivatives, 5′‐methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea‐pig taenia coli P2Y1 receptors, in guinea‐pig vas deferens and bladder P2X1 receptors, and in ion flux experiments by using recombinant rat P2X2 receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X1 receptors in differentiated HL‐60 cell membranes was carried out by using [35S]ATP‐γ‐S. A 2′‐chloro‐5′‐sulfo analog of PPADS (C14H12O9N3ClPSNa), a vinyl phosphonate derivative (C15H12O11N3PS2Na3), and a naphthylazo derivative (C18H14O12N3PS2Na2), were particularly potent in binding to human P2X1 receptors. The potencies of phosphate derivatives at P2Y1 receptors were generally similar to PPADS itself, except for the p‐carboxyphenylazo phosphate derivative C15H13O8N3PNa and its m‐chloro analog C15H12O8N3ClPNa, which were selective for P2X vs. P2Y1 receptors. C15H12O8N3ClPNa was very potent at rat P2X2 receptors with an IC50 value of 0.82 μM. Among the phosphonate derivatives, [4‐formyl‐3‐hydroxy‐2‐methyl‐6‐(2‐chloro‐5‐sulfonylphenylazo)‐pyrid‐5‐yl]methylphosphonic acid (C14H12O8N3ClPSNa) showed high potency at P2Y1 receptors with an IC50 of 7.23 μM. The corresponding 2,5‐disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y1 receptors, whereas at recombinant P2X2 receptors had an IC50 value of 1.1 μM. An ethyl phosphonate derivative (C15H15O11N3PS2Na3), whereas inactive at turkey erythrocyte P2Y1 receptors, was particularly potent at recombinant P2X2 receptors. Drug Dev. Res. 45:52–66, 1998. Published 1998 Wiley‐Liss, Inc.