1321N1 Cells Research Articles

Structural Basis of Agonist-induced Desensitization and Sequestration of the P2Y2 Nucleotide Receptor: CONSEQUENCES OF TRUNCATION OF THE C TERMINUS

Molecular determinants of P2Y2 receptor desensitization and sequestration have been investigated. Wild-type P2Y2 receptors and a series of five C-terminal truncation mutants of the receptor were epitope-tagged and stably expressed in 1321N1 cells. These constructs were used to assess the importance of the intracellular C terminus on 1) UTP-stimulated increases in intracellular calcium concentration, 2) homologous desensitization of the receptor, and 3) agonist-induced decreases in cell-surface density (receptor sequestration) of epitope-tagged receptors using fluorescence-activated cell sorting. The potency and efficacy of UTP were similar for the wild-type and all mutant P2Y2 receptors. Truncation of 18 or more amino acids from the C terminus increased by approximately 30-fold the concentration of UTP necessary to desensitize the receptor. Both the rate and magnitude of UTP-induced receptor sequestration were decreased with progressively larger truncations of the C terminus. Furthermore, the recovery from sequestration was slower for the most extensively truncated receptor. Complete desensitization was obtained with >50% of the original receptor complement remaining on the cell surface. Protein kinase C activation, which desensitizes the P2Y2 receptor, had no effect on sequestration, consistent with the ideas that desensitization and sequestration are discrete events and that agonist occupancy is required for receptor sequestration.

A new factor derived from 1321N1 human astrocytoma cells causes differentiation of PC-12 cells mediated through mitogen-activated protein kinase cascade

Glial cells play an important role in maintaining neural function. In the present study, we examined the effects of a factor derived from human astrocytoma cells (1321N1) on differentiation of rat pheochromocytoma cells (PC-12). The conditioned medium which had been used for culture of 1321N1 cells caused the differentiation of PC-12 cells, suggesting that 1321N1 cells release a neurotrophic factor. The factor was apparently distinct from well-known neurotrophic factors, such as nerve growth factor (NGF), since it was resistant to boiling and trypsin treatment. The molecular size of the factor was assumed to be below 1000 through dialysis and ultrafiltration experiments. Furthermore, PC-12 cells were differentiated synergistically by the combined addition of NGF and the conditioned medium of 1321N1 cells. Partially purified fraction of the factor by Sephadex G-15 gel filtration column caused the prolonged activation of mitogen-activated protein kinase (MAPK). The differentiation of PC-12 cells induced by the fraction or NGF disappeared after the treatment with PD98059, a specific inhibitor of MAPK kinase (MEK), suggesting the involvement of MAPK in the differentiation. These results suggest that the new low-molecular factor derived from glial cells causes differentiation of PC-12 cells mediated through an activation of MAPK.

A Comparison of the Acute and Chronic Effects of Antidepressants in Cultured C6 and 1321N1 Cells

The cytotoxicities of the antidepressants amitriptyline, imipramine (both tricyclic), fluoxetine (a selective serotonin re-uptake inhibitor) and tranylcypromine (a monoamine oxidase inhibitor) were compared in vitro in rat (C6) glioma and human (1321N1) astrocytoma cell lines. Differences in toxicity were determined after acute (24-hour) and chronic (7-day) administration and assessed by using the neutral red uptake (NRU) assay, the MTT assay, increased expression of glial fibrillary acidic protein (GFAp), and reactive morphology criteria. The relative toxicities (EC50 [concentration causing an effect in 50% of cells] value range) were fluoxetine > amitriptyline > imipramine > tranylcypromine for all the tests employed, in both cell lines and at both exposure times. There was a high and significant positive correlation between the different cell types, at both exposure times, with both the NRU and MTT assays. Increases in MTT reduction, NRU, and GFAp expression associated with cell activation were noted in C6 cells after exposure for 24 hours, but decreased after exposure for 7 days. For 1321N1 cells, increases in NRU were only observed after exposure for 24 hours. Reactive-type changes in morphology were seen after exposure to all the antidepressants, in both the C6 and 1321N1 cell lines. The data show that low concentrations of antidepressants induce metabolic changes in the astrocyte cell lines, with some significant differences in the patterns of toxicity of the tested substances.

Cystic Fibrosis Transmembrane Regulator-independent Release of ATP: ITS IMPLICATIONS FOR THE REGULATION OF P2Y2RECEPTORS IN AIRWAY EPITHELIA

The cystic fibrosis (CF) transmembrane regulator (CFTR) is a cyclic AMP-dependent Cl- channel that is defective in CF cells. It has been hypothesized that CFTR exhibits an ATP release function that controls the airway surface ATP concentrations. In airway epithelial cells, CFTR-independent Ca2+-activated Cl- conductance is regulated by the P2Y2 receptor. Thus, ATP may function as an autocrine signaling factor promoting Cl- secretion in normal but not CF epithelia if ATP release is defective. We have tested for CFTR-dependent ATP release using four independent detection systems. First, a luciferase assay detected no differences in ATP concentrations in the medium from control versus cyclic AMP-stimulated primary normal human nasal epithelial (HNE) cells. A marked accumulation of extracellular ATP resulted from mechanical stimulation effected by a medium displacement. Second, high pressure liquid chromatography analysis of 3H-labeled species released from [3H]adenine-loaded HNE cells revealed no differences between basal and cyclic AMP-stimulated cells. Mechanical stimulation of HNE cells again resulted in enhanced accumulation of extracellular [3H]ATP and [3H]ADP. Third, when measuring ATP concentrations via nucleoside diphosphokinase-catalyzed phosphorylation of [alpha-33P]dADP, equivalent formation of [33P]dATP was observed in the media of control and cyclic AMP-stimulated HNE cells and nasal epithelial cells from wild-type and CF mice. Mechanically stimulated [33P]dATP formation was similar in both cell types. Fourth, 1321N1 cells stably expressing the human P2Y2 receptor were used as a reporter system for detection of ATP via P2Y2 receptor-promoted formation of [3H]inositol phosphates. Basal [3H]inositol phosphate accumulation was of the same magnitude in control and CFTR-transduced cells, and no change was observed following addition of forskolin and isoproterenol. In both cell types, mechanical stimulation resulted in hexokinase-attenuable [3H]inositol phosphate formation. In summary, our data suggest that ATP release may be triggered by mechanical stimulation of cell surfaces. No evidence was found supporting a role for CFTR in the release of ATP.

Peroxynitrite modulates tyrosine phosphorylation and phosphoinositide signalling in human neuroblastoma SH-SY5Y cells: attenuated effects in human 1321N1 astrocytoma cells.

Peroxynitrite may contribute to oxidative stress involving neurodegeneration in several disorders, including Alzheimer's disease. As with other reactive oxygen species, peroxynitrite might affect neuronal signalling systems, actions that could contribute to adaptive or deleterious cellular outcomes, but such effects have not previously been studied. To address this issue directly, peroxynitrite (50-500 microM) was administered to human neuroblastoma SH-SY5Y cells to assess its effects on protein tyrosine nitration, phosphoinositide signalling and protein tyrosine phosphorylation. Peroxynitrite rapidly increased the nitrotyrosine immunoreactivity of numerous proteins, primarily in the cytosol. Peroxynitrite inhibited, in a concentration-dependent manner, phosphoinositide hydrolysis stimulated by activation of muscarinic receptors with carbachol and the inhibition was greater after the depletion of cellular glutathione. In comparison, muscarinic receptor-stimulated phosphoinositide hydrolysis in human astrocytoma 1321N1 cells was less vulnerable to inhibition by peroxynitrite either without or with prior depletion of glutathione. There was a large, rapid and reversible increase in the tyrosine phosphorylation of the p120 Src substrate in peroxynitrite-treated SH-SY5Y cells, a response that was potentiated by glutathione depletion; in contrast, peroxynitrite decreased the tyrosine phosphorylation of focal adhesion kinase and paxillin. Tyrosine phosphorylation of p120 in 1321N1 astrocytoma cells was less sensitive to modulation by peroxynitrite. Thus alterations in phosphoinositide signalling and protein tyrosine phosphorylation were greater in neuroblastoma than astrocytoma cells, and modulation of these signalling processes probably contributes to neuronal mechanisms of the response to peroxynitrite.

Requirement for Rho-mediated Myosin Light Chain Phosphorylation in Thrombin-stimulated Cell Rounding and Its Dissociation from Mitogenesis

Thrombin treatment causes a dose-dependent rounding of 1321N1 astrocytoma cells. This cytoskeletal response is rapid, peaking 2 h after thrombin stimulation, and reverses by 50% after 24 h. The thrombin receptor peptide SFLLRNP also induces cell rounding, whereas other G protein-linked receptor agonists such as carbachol, lysophosphatidic acid, or bradykinin fail to do so. Results of studies using pharmacological inhibitors do not support a requirement for phosphatidylinositol 3-kinase, mitogen-activated protein kinase, or Ca2+ mobilization in this response. Inhibition of protein kinase C or tyrosine kinase produces minimal blockade. Pertussis toxin treatment is also without effect. However, thrombin-induced rounding is fully blocked by the C3 toxin from Clostridium botulinum, which specifically ADP-ribosylates and inactivates the small G protein Rho. Thrombin also leads to a rapid, 2.4-fold increase in 32P incorporation into myosin light chain while carbachol does not. Myosin phosphorylation, like cell rounding is inhibited by inactivation of Rho with C3 exoenzyme, suggesting that myosin phosphorylation is necessary for this cytoskeletal response. This is supported by the observation that thrombin-induced rounding is also blocked by the myosin light chain kinase inhibitor KT5926. However, treatment with KT5926 fails to inhibit mitogenesis. Thus, cell rounding is not prerequisite to thrombin-induced DNA synthesis. We conclude that stimulation of the heterotrimeric G protein-coupled thrombin receptor in 1321N1 cells activates Rho-dependent pathways for both DNA synthesis and cell rounding, the cytoskeletal response being mediated in part through increases in myosin phosphorylation.

The 5 α-reductase in the central nervous system: expression and modes of control

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5 α-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5 α-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5 α-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5 α-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5 α-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.

Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells.

The compartmentation of inositol phospholipids was examined by using a combination of radiolabelling approaches in intact and permeabilized 1321N1 astrocytoma cells. A 'chase' protocol was developed with whole cells in which phosphoinositide (PI) pools were labelled to steady state with [3H]inositol and the cellular [3H]inositol pool was then diluted selectively with non-radioactive inositol. In these cells muscarinic-receptor-stimulated phospholipase C (PLC) hydrolysed [3H]PI at approx. 1-2%/min. However, after the chase procedure the relative specific radioactivity of [3H]Ins(1,3,4)P3, a rapidly metabolized and sensitive marker of PLC activity, decreased only after more than 5 min and over a time course similar to that during which the labelling of each [3H]PtdIns, [3H]PtdInsP and [3H]PtdInsP2 declined by at least 50%. These results demonstrate a large receptor-responsive [3H]PI pool that is accessed by stimulated PLC without apparent metabolic compartmentation, despite its probable distribution between different membrane fractions. Support for this was obtained in intact cells by using an acute [3H]inositol labelling method in which increases in the specific radioactivity of [3H]inositol phosphates stimulated by carbachol occurred only in parallel with similar increases in the labelling of the bulk of cellular [3H]PI. In [3H]inositol-prelabelled cells permeabilized to deplete cytosolic proteins, carbachol and guanosine 5'-[gamma-thio]triphosphate stimulated the endogenous PLC to degrade only approx. 5% of [3H]PI. This was increased to approx. 30% in the presence of exogenous PtdIns transfer protein, which, at a concentration approx. 5-10% of that in 1321N1 cell cytosol, was sufficient to support PLC activity comparable with that observed in response to carbachol in whole cells. These and earlier results in 1321N1 cells suggest a model of integrated PI pools involving an obligatory role for lipid transport. Given the multifunctional capacity of PI in cellular signalling mechanisms, this model has important implications, particularly for the hypothesis that the ability of Li+ ions to influence these selectively might account for its therapeutic actions.

Histamine-induced calcium mobilization in single cultured cells expressing histamine H1 receptors: a relationship between its sensitivity and the density of H1 receptors.

Stimulation of histamine H1 receptors initiates the hydrolysis of phosphatidylinositides and results in the production of inositol (1, 4,5)-triphosphate and intracellular Ca2+ mobilization. Although the mechanism for signal transduction via the H1 recptor has been extensively investigated, little is known about the correlation between the sensitivity of histamine-induced Ca2+ mobilization and the density of H1 receptors in cultured cells. Cytosolic free Ca2+ concentration ([Ca2+]i) after stimulation by histamine was monitored in single CHO and rat C6-glioma cells stably expressed with H1 receptors and astrocytoma 1321N1 cells using the Ca2+-sensitive dye Indo-1 and dynamic single cell imaging techniques (ACAS 570 laser cytometer). Both of the H1 receptor-expressed CHO cells and C6-glioma cells were over 10 times more sensitive to histamine than astrocytoma 1321N1 cells in which H1 receptors were naturally present. The density of H1 receptors in the transfected cells was also more than 10-fold that of 1321N1 cells. In addition, inhibition of intracellular Ca2+-ATPase by thapsigargin elicited an increase in [Ca2+]i in H1 receptor-overexpessed cells and astrocytoma 1321N1 cells with similar sensitivity. These data suggest that the sensitivity of Ca2+ mobilization by histamine in these cells was correlatively augmented with the increase in the density of H1 receptors.

Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1

The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such as p-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptor p-aminophenyl-alpha-D-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation.

Structure and function of P2Y2 nucleotide receptors in cystic fibrosis (CF) epithelium.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane-conductance regulator (CFTR), a cAMP-dependent chloride transporter expressed in epithelial tissue. A novel treatment being developed for CF utilizes inhaled nucleotides, particularly uridine 5′-triphosphate (UTP), to activate calcium-dependent chloride channels in airway epithelial cells. This therapy is based on earlier studies from the laboratory of Dr. Richard C. Boucher at the University of North Carolina-Chapel Hill, which have shown that luminal administration of UTP1 and the Na+ channel blocker amiloride,2 together overcome the ion transport defects in CF airway epithelial cells by promoting increased Cl− secretion and decreased Na+ absorption.3 UTP stimulates Cl− secretion in epithelial cells by activating a P2Y2 (formerly named P2U) nucleotide receptor subtype4,5 that is coupled to phospholipase C via Gqα protein resulting in inositol 1,4,5 trisphosphate (IP3)-dependent increases in the cytoplasmic calcium concentration ([Ca2+]i) that serve to activate calciumdependent chloride channels.6 Evidence that activation of this non-CFTR Cl− secretory conductance may be beneficial in CF therapy was provided by studies with CFTR knockout mice in which the presence of a large, basal, non-CFTR Cl− conductance was associated with the lack of respiratory disease in this species.4 Early clinical studies involving administration of aerosolized UTP and amiloride indicate that this therapy has a positive effect on mucociliary clearance in CF nasal epithelia.7 However, little is known about the mechanisms of nucleotide regulation of epithelial ion transport. It is clear that fundamental information concerning P2Y2 receptor biology in epithelia will be useful in optimizing nucleotide therapy for CF. We have utilized the CFTR knockout mouse and its normal counterpart to isolate cultures of gallbladder epithelial cells that form tight junctions and represent a good model for studying the roles of P2Y2 receptors in mediating signal transduction pathways leading to increased Cl− secretion. In addition, we have cloned the human airway epithelial cell P2Y2 receptor cDNA and expressed the receptor in a heterologous cell system, human 1321N1 astrocytoma cells, that lack any endogenous nucleotide receptors.8 In the 1321 N1 cell transfectants, the human airway P2Y2 receptor is distinguished pharmacologically by its ability to be activated equipotently by the purine nucleotide ATP and the pyrimidine nucleotide, UTP. The investigation of the molecular and pharmacological properties of the P2Y2 receptor expressed in 1321N1 cells is being undertaken to develop methods to maximize calcium-dependent chloride conductance in CF epithelial cells through stimulation of endogenous P2Y2 receptors.

Crosstalk between normal and tumoral brain cells. Effect on sex steroid metabolism.

The present article shows for the first time that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5 alpha-reduced metabolites dihydrotestosterone and dihydroprogesterone. Moreover, both cell lines are also able to convert these metabolites further into their corresponding 3 alpha-OH derivatives, 5 alpha-androstan-3 alpha, 7 beta-diol (3 alpha-diol) and tetrahydroprogesterone. On the basis of these observations, the possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned as well as in fetal rat neurons and in neonatal rat type 1 astrocytes has been considered. To this purpose, cultures of the different cellular types have been exposed to the conditioned medium in which the other cells were grown. The results obtained indicate that: 1. Neurons are able to stimulate, in a statistically significant fashion, the formation of dihydrotestosterone (DHT), 3 alpha-diol, and tetrahydraprogesterone (THP) in C6 cells. 2. Type 1 astrocytes, on the contrary, are unable to modify steroid metabolism in C6 cells. 3. C6 cell product(s) decrease(s) the formation of DHP in type 1 astrocytes, without modifying that of DHT. 4. C6 cells do not influence the metabolism of testosterone (T) and progesterone (P) in neurons. In conclusion, the present observations show that the conditioned medium of normal neurons is able to increase the metabolism of testosterone and progesterone occurring in a tumoral glial cell line, and that the conditioned media of the two tumoral cell lines analyzed are able to decrease the conversion of P into DHP occurring in normal type 1 astrocytes. The surprising result that these conditioned media do not alter the formation of DHT is discussed. Work is presently in progress to identify the principle(s) responsible respectively for the activating and inhibiting actions here described.

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.

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.

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.

The G12 coupled thrombin receptor stimulates mitogenesis through the Shc SH2 domain.

Our previous studies in 1321N1 astrocytoma cells demonstrate that thrombin stimulates Ras-dependent mitogenesis through the pertussis toxin insensitive G protein G12. While the direct effectors of G12 are unknown, G12 can transform fibroblasts, utilize Ras and Rac dependent signaling pathways and stimulate GTP loading of Ras. Here we have examined the role of the Shc adaptor protein in mitogenic signaling by the thrombin receptor in 1321N1 cells. As has been reported in other systems, thrombin stimulation results in tyrosine phosphorylation of Shc in 1321N1 cells. We also show that transient expression of G alpha12 results in tyrosine phosphorylation of Shc, thereby identifying Shc as the most proximal G12 effector to date. In addition, we demonstrate by microinjection that thrombin stimulated mitogenesis requires Shc and occurs specifically through the Shc SH2 domain. Expression of the SH2 domain of Shc also inhibits G alpha12 mediated induction of an AP-1 dependent reporter gene demonstrating that G12 utilizes Shc to propagate downstream signals. Our data indicate that Shc is essential for stimulation of Ras dependent mitogenesis and gene expression by the G12 coupled thrombin receptor.

The 6H1 Orphan Receptor, Claimed to Be the p2y5 Receptor, Does Not Mediate Nucleotide-Promoted Second Messenger Responses

An orphan G protein-coupled receptor, termed 6H1, with ∼30% sequence identity to P2Y receptors has been proposed to be a P2Y receptor (p2y5) based solely on a radioligand binding assay with [35S]dATPαS [Webb et al. (1997) Biochem. Biophys. Res. Commun. 219:105-110]. Previous work in our laboratory has shown that [35S]-dATPαS is not a general ligand for P2Y receptors, and thus inclusion of the p2y5 receptor in the family of P2Y receptors is questionable. To define unambiguously whether the p2y5 receptor is a P2Y receptor, we have cloned the turkey homologue of the chick p2y5 receptor. Sequence analysis indicated that the turkey receptor contains an additional 32 amino acids at its carboxy terminus compared to the published chick sequence. HA epitope-tagged turkey p2y5 receptors were stably expressed in 1321N1 human astrocytoma cells, and cells shown to express the HA-tagged p2y5 receptor by an intact cell-based ELISA were used to determine whether changes in second messenger levels occurred in response to a series of nucleotides. ATP, ADP, UTP, UDP, dATPαS, and A2P4had no effect on either inositol phosphate or cyclic AMP concentrations in cells expressing the p2y5 receptor. Robust inositol phosphate and cyclic AMP responses occurred to other G protein-coupled receptors expressed in 1321N1 cells, which indicate that these cells contain all of the necessary signaling components to generate these second messenger responses. These data indicate that the 6H1/p2y5 receptor is not a member of the P2Y receptor family of signaling proteins.

Cloning of a Human Heptahelical Receptor Closely Related to the P2Y5Receptor

The 6H1 receptor cloned from activated chicken T cells was initially considered an orphan G-coupled receptor, but was later included in the P2Y family of receptors for purine and pyrimidine nucleotides on the basis of a significant amino acid identity and was renamed P2Y5. Analysis of the expressed sequence tag database revealed the presence of a related sequence exhibiting 63% amino acid identity with this receptor. Starting from this partial sequence, we have isolated a complete clone and identified a 1113 base pair open reading frame encoding a new G-coupled receptor that we have called P2Y5-like. This sequence exhibits 61% identity with the chicken P2Y5sequence and 30-33% with other P2Y subtypes. A construct encoding this P2Y5-like receptor was transfected into COS-7, 1321N1, and CHO-K1 cells, and expression was documented by Northern blotting. None of the 40 nucleotides and nucleosides tested was able to elicit a response in any of four functional assays: inositol phosphate formation, stimulation or inhibition of cAMP formation, and extracellular acidification measured with a microphysiometer. These results suggest either that the natural ligand of the P2Y5-like receptor is an uncommon nucleotide or alternatively that despite its structural similarity to the P2Y family it is not a nucleotide receptor.

Lack of Nucleotide-Promoted Second Messenger Signaling Responses in 1321N1 Cells Expressing the Proposed P2Y Receptor, p2y7

A recently cloned G protein-coupled receptor (named the p2y7 receptor) with relatively low sequence identity to previously cloned P2Y receptors was proposed to be a member of this family of receptors on the basis of both a radioligand binding assay with [35S]dATPαS and an inositol phosphate response to ATP in COS-7 cells transiently transfected with receptor cDNA. Previous work in our laboratory has shown that [35S]dATPαS is not a general radioligand for the identification of P2Y receptors and that COS-7 cells express an endogenous P2Y receptor (P2Y2) that complicates the analysis of nucleotide-promoted inositol phosphate responses. Thus, data supporting inclusion of the p2y7 receptor in the P2Y family of receptors are equivocal. To determine unambiguously whether the p2y7 receptor is a P2Y receptor subtype, a p2y7 receptor bearing an epitope-tag at its NH2-terminus was expressed in 1321N1 cells and cell surface expression of the receptor was demonstrated by an intact cell-based ELISA. Cells shown to express epitope-tagged p2y7 receptors by ELISA were examined for their second messenger signaling properties in response to a range of nucleotides. ATP, UTP, ADP, UDP, and dATPαS had no effect on phospholipase C or adenylyl cyclase activities in cells expressing the p2y7 receptor. Experimental controls utilizing expression of other G protein-coupled receptors showed that 1321N1 cells displayed robust responses for each of these signaling pathways. These data, together with the low sequence identity of the p2y7 receptor to other P2Y receptors, indicate that the p2y7 is not a member of the P2Y family of signaling molecules.

An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor.

1. The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]-inositol phosphate accumulation. Half-maximal responses were obtained at approximately 30 nM for 2-methylthioadenosine-5'-triphosphate (2MeSATP), 300 nM for dATP alpha S, and 1000 nM for adenosine 5'-triphosphate (ATP). dATP alpha S produced a maximal response that was only 37 +/- 4% of that produced by ATP or 2MeSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644 +/- 14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y1 receptors. 3. The selectivity of dATP alpha S for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATP alpha S was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors. 4. Although [35S]-dATP alpha S bound to a relatively high density (ca 10 pmol mg-1 protein) of binding sites in membranes from 1321N1 or Cos-7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild-type, empty vector-transfected, or P2Y1 receptor-expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]-dATP alpha S binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor-expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]-dATP alpha S binding in wild-type Cos-7 cell membranes. These data from 1321N1 and Cos-7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]-dATP alpha S that are not related to P2Y receptor subtypes.