Tania E. Webb

Molecular Cloning of a Novel P2 Purinoceptor from Human Erythroleukemia Cells

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 ± 6 nM), much less for UTP and ADP (~1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Molecular Cloning and Characterization of the Rat P2Y4 Receptor

Abstract: Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full‐length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2‐methylthio‐ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase

1 BIO cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2 The radioligand [35S]‐dATPαS bound with high affinity (Kd = 9.8nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg−1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]‐dATPαS led to the rank order of affinity: dATPαS (Ki 3.4 nM) > 2‐chloroATP (2‐ClATP) (13 nM), ATP (22 nM) > ATPγS (43 nM) > 2‐methylthioATP (2‐MeSATP) (88 nM) > ADP (368 nM) > > UTP, L‐β,γ‐methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid 4‐sodium (PPADS) was able to displace only 20% of [35S]‐dATPαS binding at a concentration of 100 μm 3 2‐ClATP (EC50 = 0.22 μm), 2‐MeSATP (0.54 μm), ADP (7.9 μm) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 μm 4 A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6‐2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y‐type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2‐MeSATP has been shown to increase phospholipase C activity. 5 Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2‐MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y2 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

Nucleotide receptors in the nervous system. An abundant component using diverse transduction mechanisms.

Extracellular nucleotides achieve their role as cell-to-cell communicators by acting at cell surface transmembrane receptors—the P2 receptors. Before molecular cloning led to the isolation of any P2-receptor sequence, a small number of receptor types had been proposed on the basis of pharmacological evidence. The application of molecular biology to this field of receptor research has indicated that a great underestimation of the number of receptor subtypes and of their abundance had occurred. There are now known to be seven characterized P2Y (G protein linked) receptors and the same number again of P2X receptors of the transmitter-gated ion channel type. In this review, we discuss the properties of these cloned receptors, their distribution within the nervous system, and their methods of signal transduction.

Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP.

Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2‐methythio‐ATP and α,β‐methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. Use of the reverse transcriptase‐polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]‐thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

The P2Y6 receptor is a uridine nucleotide‐specific G protein‐linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro‐injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N‐type Ca2+ (ICa(N)) and M‐type K+ (IK(M)) currents. In P2Y6 cRNA‐injected neurones, UDP or UTP produced a voltage‐dependent inhibition of ICa(N) by ∼53% in whole‐cell (disrupted‐patch) mode and by ∼73% in perforated‐patch mode; no inhibition occurred in control cells. Mean IC50 values (whole‐cell) were: UDP, 5.9±0.3 nM; UTP, 20±1 nM. ATP and ADP (1 μM) had no significant effect. Pertussis toxin (PTX) substantially (∼60%) reduced UTP‐mediated inhibition in disrupted patch mode but not in perforated‐patch mode. Uridine nucleotides also inhibited IK(M) in P2Y6 cRNA‐injected cells (by up to 71% at 10 μM UTP; perforated‐patch). Mean IC50 values were: UDP, 30±3 nM; UTP, 115±12 nM. ATP (10 μM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX‐resistant. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Distribution of [35S]dATPαS binding sites in the adult rat neuraxis

Abstract Highly abundant, saturable and specific binding sites for [35S]2′-deoxyadenosine 5′-0-(1-thio) triphosphate ([ 35 S]dATPαS, K d : 9 ± 2nM; B max : 39 ± 8 pmol/mg protein ) are present in adult rat brain membranes and have characteristics consistent with those expected for a P2Y1 receptor. The anatomical distribution of these binding sites in the brain and spinal cord was examined using in vitro autoradiography. The [35S]dATPαS binding sites showed a widespread distribution throughout the brain and spinal cord. They could be displaced by a large excess (100 μM) of 2-methylthioATP (2MeS-ATP) but not by uridine-5′-triphosphate (UTP) or α,β-methyleneATP (α,β-meATP). Within the cortical regions labelling was of equal medium density. However, discrete structures and nuclei within the olfactory bulb, subcortical telencephalon, hippocampal complex, thalamic regions and mesencephalon displayed a variety of densities. Within the spinal cord, gray matter was labelled at a greater density than the funiculi. The present study clarifies the anatomical distribution of P2Y1 and closely related receptors within the central nervous system of rat and extends the evidence that those receptors are abundant and widely distributed within the neuraxis.

Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells.

The expression of the P2 receptors and their functional responses were studied in rat thyroid FRTL‐5 cells. RT‐PCR analysis revealed transcripts for the G protein‐coupled P2Y2, P2Y4 and P2Y6 receptors, and for the transmitter‐gated ion channel P2X3, P2X4 and P2X5 subunits. In Fura‐2‐loaded cells, UTP, ATP, ATPγS or UDP increased [Ca2+]i, and behaved as potent full agonists, while 2‐Methylthio‐ATP (2‐MeSATP), α,β‐methylene‐ATP (α,β‐meATP) and pure ADP were weak agonists. The agonist‐mediated [Ca2+ ]i increases were diminished in Ca2+ ‐free buffer, and by pertussis toxin (PTX) or suramin treatments. ATP, UTP, UDP and ATPγS increased 3H‐thymidine incorporation into DNA and expression of the protooncogenes c‐Fos and c‐Jun, while 2‐MeSATP was ineffective, and α,β‐meATP gave a response only at 100‐μM dose. The ATP‐stimulated expression of c‐Fos and c‐Jun was dependent on Ca2+, and protein kinase C, but not on calmodulin or Ca2+/calmodulin‐dependent protein kinase II. Extracellular signal‐regulated kinases (ERK1 and ERK2) are also involved as the MEK inhibitor, PD98059, reduced both ATP‐evoked 3H‐thymidine incorporation and c‐Fos and c‐Jun expression. These results indicate that multiple P2Y receptor subtypes and at least the P2X5 subtype are functionally expressed in FRTL‐5 cells, and that nucleotides acting via P2 receptors are involved in the regulation of DNA‐synthesis.

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5′‐triphosphate (UTP) or adenosine 5′‐triphosphate (ATP) inhibits the N‐type voltage‐gated calcium current by ∼65%, with an IC50 of 0.5 μM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse‐transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors. None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin. Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides. Isobutylmethylxanthine and 8‐sulphophenyltheophylline prevented the cyclic AMP response, while neither 8‐cyclopentyl‐1,3‐dipropylxanthine nor SCH58261 were effective antagonists. 2‐chloradenosine gave a robust response, but neither 2‐chloro‐N6‐cyclopentyladenosine nor CGS 21680 were agonists. These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.