Reconstruction of the P2X(2) receptor reveals a vase-shaped structure with lateral tunnels above the membrane.

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.

Detection of P2Y(14) protein in platelets and investigation of the role of P2Y(14) in platelet function in comparison with the EP(3) receptor.

mRNA encoding the recently discovered P2Y(14) receptor has been reported in platelets, but the presence of P2Y(14) receptor protein and its functionality have not been studied. If P2Y(14) is expressed along with P2Y(1) and P2Y(12) receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y(14) receptor in platelets and its role in platelet function. The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP(3) receptor agonist. Expression of P2Y(14) receptor was investigated by immunoblotting and confocal microscopy. Platelet aggregation in platelet-rich plasma (PRP) and whole blood was measured using light absorbance and platelet counting. VASP phosphorylation was investigated using flow cytometry. Immunoblotting provided evidence for P2Y(14) receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 muM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists. P2Y(14) did not substitute for P2Y(12) in experiments using the P2Y(12) antagonist AR-C69931. No effect of UDP-glucose was seen on adenylate cyclase activity as measured by VASP phosphorylation. In contrast, sulprostone acting via the EP(3) receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP(3) also partially substituted for P2Y(12) receptor. We have demonstrated the presence of P2Y(14) receptor protein in platelets, but no contribution of this receptor to several measures of platelet function has been observed. Further studies are necessary to determine whether the P2Y(14) receptor in platelets has any functionality.

Purification and aqueous phase atomic force microscopic observation of recombinant P2X2 receptor.

Recombinant P2X2 receptor was observed by atomic force microscope in the aqueous phase. The P2X2 receptor was expressed in an insect cell line, and recombinant proteins were prepared under native conditions. The membrane fractions were extracted, and histidine-tagged receptor protein was purified from the fractions by column chromatography. When the purified protein fraction was diluted with water and served for atomic force microscopy, dispersed particles of about 3 nm in height were observed. In the presence of 1 mM ATP, the assembly-like images of the particles were obtained. More densely assembled images of the particles were achieved when the protein was dissolved in a Tris buffer containing 1 mM ATP. Under this condition, imaging of the surface of the particles exhibited a circular structure with a diameter of about 10 nm having a pore-like structure. These results suggest that atomic force microscopy provides structural information about P2X2 receptor in aqueous phase.

Purification and functional reconstitution of the human P2Y12 receptor.

The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y12-R. The G protein selectivity of the P2Y12-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. These results illustrate that the signaling properties of the P2Y12-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.

Molecular characterization of the zebrafish P2X receptor subunit gene family.

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.

P2Y(13): identification and characterization of a novel Galphai-coupled ADP receptor from human and mouse.

We have identified an orphan G protein-coupled receptor, SP174, that shares a high degree of homology with the recently described ADP receptor P2Y(12). mRNA for SP174 is abundant in the brain and in cells of the immune system. In the present study, we demonstrate that SP174 is also a receptor for ADP, which is coupled to Galphai. ADP potently stimulates SP174 with an EC(50) of 60 nM, and other related nucleotides are active as well, with a rank order of potency 2-methylthio-ADP tetrasodium = adenosine 5'-O-2-(thio)diphosphate = 2-methylthio-ATP tetrasodium > ADP > AP3A >ATP > IDP. This pharmacological profile is similar to that for P2Y(12). We have also identified the murine homolog of SP174, which exhibits 75% homology to the human receptor. ADP is also a potent agonist at the murine receptor, and its pharmacological profile is similar to its human counterpart, but ADP and related nucleotides are more potent at the murine receptor than the human receptor. In keeping with the general nomenclature for the purinergic receptors, we propose designating this novel receptor P2Y(13).

Stimulation of Ca(2+) influx through ATP receptors on rat brain synaptosomes: identification of functional P2X(7) receptor subtypes.

1. ATP receptors of the P2X class have previously been identified on autonomic nerve endings and on a limited population of CNS neurons. 2. In the present study P2X receptors on mammalian cortical synaptosomes have been identified by a variety of functional and biochemical studies. In choline buffer ATP analogues caused concentration/time dependent Ca(2+) influx. Relative to the effects caused by ATP, benzoylbenzoyl ATP (BzATP) was about seven times more active than ATP while 2-me-S-ATP and ATPgammaS were much less active. alpha,beta-me- ATP and beta,gamma-me-ATP were virtually inactive. In sucrose buffer, relative to choline buffer, the activity of BzATP was more than doubled while activity in sodium buffer was reduced. Moreover, the P2X antagonists PPADS or Brilliant Blue G both significantly attenuated influx. These observations suggest the presence of P2X receptors on synaptosomes which subserve Ca(2+) influx. This activity profile of the ATP analogues and the response to blocking agents are characteristic of responses of P2X(7) receptors. 3. Influx was unaffected by the VSCC inhibitors omega-CTx-MVIIC and (-) 202 - 791, indicating that ATP induced Ca(2+) influx occurred primarily through P2X receptors. 4. P2X(7) receptor protein was identified by Western blotting and immunohistochemical staining. Purified preparations were devoid of significant concentrations of GFAP or the microglial marker OX-42 but contained greatly enriched amounts of syntaxin and SNAP 25. 5. The various pharmacological and biochemical studies were all consistent with the presence of functional P2X(7) receptors.

Heteromeric association creates a P2Y-like adenosine receptor.

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G(i/o) protein-coupled A1 adenosine receptor (A1R) and Gq protein-coupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A1R agonist and antagonist binding to cell membranes were reduced by coexpression of A1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5'-O-(2-thiotriphosphate) (ADPbetaS) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1R coexpressed cells showed an ADPbetaS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADPbetaS binds A1R and inhibits adenylyl cyclase activity via G(i/o) proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1R with P2Y1R generates A1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.

An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction.

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).

Novel modified adenosine 5'-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y(1) receptor: Biotinylated analogue potentially suitable for specific P2Y(1) receptor isolation.

Rat brain P2Y(1) (rP2Y(1)) receptor-transfected human embryonic kidney cells (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (Vöhringer C, Schäfer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for further studying novel adenine nucleotide analogues (Halbfinger et al. J Med Chem 1999;42:5325-37) and for the biochemical characterization of the P2Y(1) receptor. By measurement of intracellular Ca(2+) release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC(50) values of 1.3, 5, and 60 nM were determined, markedly lower than the value for ATP (130 nM). The EC(50) for 2-BuSADP was 1.1 nM. The corresponding 8-substituted ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuSATP > 8-BuNHATP approximately 8-BuOATP). AMP induced intracellular Ca(2+) release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC(50) = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC(50) = 6 nM), revealing that even more bulky substituents linked to the C-2 position bind with high affinity at the P2Y(1) receptor. This biotinylated probe was successfully used for the enrichment of the P2Y(1) receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y(1) receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y(1) receptor represent a powerful model system for pharmacological characterization of the P2Y(1) receptor, circumventing problems associated with natural systems. They provide a means for the development of P2Y(1) ligands of high potency and a good source for obtaining purified P2Y(1) receptor.

A primitive ATP receptor from the little skate Raja erinacea.

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.

Molecular cloning, functional characterization and possible cooperativity between the murine P2X4 and P2X4a receptors.

We have cloned and functionally characterised the mouse orthologue of the P2X4 receptor, mP2X4, and a splice variant of this receptor, mP2X4a. mP2X4 is 388 amino acids in length and shares 94% and 87% identity with the rat and human P2X4 receptors, respectively, while mP2X4a is 361 amino acids in length and lacks a 27-amino acid region in the extracellular domain corresponding to exon 6 of the known P2X receptor gene structures. When expressed in Xenopus laevis oocytes, mP2X4 produces a rapid inward current in response to ATP with an EC50 of 1.68+/-0.2 microM, consistent with the affinity of the rat and human P2X4 receptors for ATP. This agonist response is potentiated by the P2X receptor antagonists suramin, Reactive blue 2 and, over a limited concentration range, by PPADS. Although mP2X4a forms a poorly functional homomeric receptor, it appears able to interact with the full-length mP2X4 subunit to result in a functional channel with a reduced affinity for ATP. These results suggest a possible role for splice variants of P2X receptors in the formation of functional heteromeric ion channels.

Molecular assembly of the extracellular domain of P2X2, an ATP-gated ion channel.

We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X2, in a bacterial expression system. The hexahistidine-tagged protein was purified by immobilized metal affinity chromatography and refolded by sulfitolysis and dialysis. We demonstrate that P2X2-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [alpha-32P]ATP has been covalently cross-linked by UV irradiation to the P2X2-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X2-ECD subunits is appreciably stronger than 3.4 microM (0.1 mg/ml), implying that the extracellular domain of P2X2 is primarly responsible for tetramerization of whole P2X2 and thus probably plays a role in determining homo- and heteromerization specificity of P2X channel subunits.

Immunoaffinity method to identify aggregin, a putative ADP-receptor in human blood platelets.

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses.

Receptors responsive to extracellular pyrimidine nucleotides.

Extracellular adenine nucleotides have long been known to mediate pharmacological responses via several subtypes of P2 purinoceptors. More recently, however, similar responses to uracil nucleotides have been demonstrated. Here, Didier Communi and Jean-Marie Boeynaems discuss the evidence to suggest that distinct pyrimidinoceptors do indeed exist, although they do not appear to constitute a separate receptor family of their own.

Homologous desensitization of ATP-stimulated mitogenesis: mechanism involves desensitization of arachidonic acid release and cAMP elevation but not the activation of protein kinase A.

Prolonged incubation of quiescent 3T3, 3T6, and A431 cells with the P2Y purinoceptor agonists ATP, ADP, or AMPPNP reduced the mitogenic responses of target cells to a further challenge by these agonists, as measured by [3H]thymidine incorporation. The mitogenic desensitization was agonist-specific, for no effect was seen on DNA synthesis stimulated by epidermal growth factor, insulin, bombesin, 12-O-tetradecanoyl-phorbol-12 acetate (TPA), or adenosine. The desensitization was completely reversible, since after a 24 hr incubation in the absence of ATP, the cells responded fully to the mitogenic action of ATP. The presence of a low level of cycloheximide blocked recovery, suggesting that down-regulation of the P2Y receptor may have occurred during desensitization. In Swiss 3T3 cells, stimulation of DNA synthesis occurs predominantly by activation of arachidonic acid release, followed by its oxidation to prostaglandin E2 and stimulation of adenylyl cyclase. Interestingly, prolonged preincubation with ATP produced a similar degree of desensitization of DNA synthesis and of ATP-dependent arachidonic acid release and cAMP accumulation. Furthermore, this was true for both wild type cells and mutants with a defective cAMP-dependent protein kinase (PKA). We conclude that homologous desensitization is likely due to uncoupling of the P2Y purinoceptor from phospholipase A2, and this process does not require activation of protein kinase A.

Inhibition of ADP-induced platelet responses by covalent modification of aggregin, a putative ADP receptor, by 8-(4-bromo-2,3-dioxobutylthio)ADP.

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.