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Lack of Nucleotide-Promoted Second Messenger Signaling Responses in 1321N1 Cells Expressing the Proposed P2Y Receptor, p2y7
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
235, 717–721 (1997)
RC976884
Lack of Nucleotide-Promoted Second Messenger Signaling Responses in 1321N1 Cells Expressing the Proposed P2Y Receptor, p2y7 Christopher L. Herold, Qing Li, Joel B. Schachter, T. Kendall Harden, and Robert A. Nicholas Department of Pharmacology, CB #7365, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365
Received May 27, 1997
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]dATPaS 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]dATPaS 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 dATPaS 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. q 1997 Academic Press
receptors are G protein-coupled signaling proteins that respond to both adenine and uridine nucleotides and either activate phospholipase C (PLC) (1-5) or inhibit adenylyl cyclase (6-10). Although receptors claiming the monographs P2Y1-P2Y7 have been reported, only four of these, the P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors, have been cloned from mammalian species and shown unequivocally to possess nucleotide-promoted functional activity. All four of these receptors activate PLC and promote mobilization of intracellular Ca2/. The P2Y1 receptor is activated specifically by adenine nucleotides, with a preference for diphosphates over triphosphates (11), the P2Y2 receptor is a triphosphatespecific receptor activated equipotently by ATP and UTP (12,13), the P2Y4 receptor is activated primarily by UTP (13), and the P2Y6 receptor is activated selectively by UDP (13). These four receptors share 35-52% sequence identity. A receptor with high sequence identity (65%) to the rat P2Y6 receptor has been cloned from chick brain and designated as the p2y3 receptor1 (14). Like the mammalian P2Y6 receptor, the p2y3 receptor couples to PLC and is activated most potently by UDP. Two other receptors, tentatively designated the chick p2y5 receptor and the human p2y7 receptor1, have been purported to be P2Y receptors (15,16). These two receptors, which exhibit low sequence identity (28-33%) to the known mammalian P2Y receptors, were proposed to be members of the P2Y receptor family of signaling proteins largely on the basis of a radioligand binding assay with [35S]dATPaS. We recently have shown (17) that [35S]dATPaS is not a general radioligand for P2Y receptors, and thus the use of [35S]dATPaS to identify 1
Extracellular nucleotides exert their diverse biological effects through two classes of P2 receptors. P2X receptors are cation-selective ion channels that respond primarily to adenine nucleotides, whereas P2Y
According to the recommendations of the IUPHAR nomenclature committee (30), we have used the designation p2y3 for this P2Y receptor since it is not from a mammalian species and no mammalian homolog has been identified. Likewise, the same designation has been used for the p2y5 and p2y7 receptors, which have not been shown unequivocally to possess nucleotide-promoted functional activity.
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new P2Y receptors in the absence of nucleotide-promoted functional activity is suspect. Although the p2y7 receptor also was claimed to be a P2Y receptor on the basis of an inositol phosphate response to ATP in transfected COS-7 cells, these results also are difficult to interpret, since we have shown that COS-7 cells express an endogenous P2Y2 receptor that activates PLC in response to ATP and UTP (17). Thus, the data supporting the claim that the p2y7 receptor is a functional P2Y receptor are ambiguous. Since the identity of the p2y7 receptor as a bona fide P2Y receptor is in question, we have stably expressed an epitope-tagged p2y7 receptor in 1321N1 human astrocytoma cells and assessed both receptor expression and nucleotide-promoted second messenger responses in these cells. Neither adenine nor uridine nucleotides promoted any effect on inositol phosphate or cyclic AMP accumulation in cells shown by an intact cell-based ELISA to express the p2y7 receptor. We conclude from these data that the so-called p2y7 receptor is not a member of the P2Y family of G protein-coupled receptors. MATERIALS AND METHODS Materials. ATP, ADP, UTP, and UDP were from Boehringer Mannheim Biochemicals (Indianapolis, IN). dATPaS was from Research Biochemicals International (Natick, MA). Carbachol, goat anti-mouse IgG peroxidase conjugate, IBMX, isoproterenol, and OPD peroxidase substrate tablet set were from Sigma Chemical (St. Louis, MO). Mouse 12CA5 monoclonal antibody was from BAbCO (Richmond, CA). [8-3H]-Adenine (18 Ci/mmol) and myo-[2-3H]-inositol (20 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). All tissue culture reagents were from the Lineberger Comprehensive Cancer Center tissue culture facilities (University of North Carolina at Chapel Hill, NC). Construction of expression vectors harboring the native and HA epitope-tagged p2y7 receptor. The human p2y7 receptor was amplified by PCR from 200 ng of human genomic DNA with primers based on the published sequence of the p2y7 receptor (16,18,19). The upstream primer contained an EcoRI site at its 5*-end and was designed to include 7 bp of upstream noncoding sequence, whereas the downstream primer contained a XhoI restriction site at its 5*-end and was designed to include 24 bp of 3*-untranslated sequence in the amplified fragment. The amplified fragment was purified, digested with EcoRI and XhoI, and subcloned into the similarly digested retroviral expression vector pLXSN. A p2y7 receptor harboring a tandem repeat of the 12CA5 epitope (hereafter referred to as the HA epitope) (20) was constructed by PCR amplification of 1 ng of pLXSNp2y7 with the same downstream primer as above and an upstream primer designed to include an MluI restriction site at its 5*-end and the coding sequence for an HA epitope tag immediately prior to the second codon of the p2y7 receptor. The modified fragment was used to replace the coding sequence of the P2Y1 receptor in the plasmid pLXSN-HA-P2Y1, which contained an initiating Met codon and an HA-epitope tag sequence followed by an MluI site at the start of the P2Y1 receptor. This resulted in the incorporation of a tandem repeat of the HA-epitope tag at the NH2-terminus of the expressed p2y7 receptor (hereafter referred to as the HA-p2y7 receptor). Inclusion of an eptitope tag at the NH2-terminus of a G protein-coupled receptor has been shown to have no effect on either its pharmacological selectivity or second messenger coupling specificity (21,22).
Expression of p2y7 and HA-p2y7 receptors in 1321N1 human astrocytoma cells. 1321N1 human astrocytoma cells, a cell line that does not demonstrate functional responses to P2Y receptor agonists, were grown in monolayer culture at 377 C in 5% CO2 in high-glucose DMEM supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. Recombinant retroviruses were produced by calcium phosphate-mediated transfection of PA317 cells with pLXSN vector containing the appropriate p2y7 receptor sequence as described by Comstock et al. (23). 1321N1 cells were infected with retroviruses harboring recombinant p2y7 receptor sequences or with control retrovirus, and geneticin-resistant cells were selected after approximately two weeks in culture with 1 mg/ml G-418. Populations of resistant cells were maintained in medium supplemented with 0.4 mg/ml G-418. The generation of 1321N1 cells expressing HA epitope-tagged P2Y1 and M2muscarinic receptors were accomplished in a similar manner. ELISA of HA epitope-tagged receptors. Cells seeded in 24-well plates at 1 1 105 cells/well were tested for the expression of tagged receptors essentially as described (24). Following the washing and blocking steps, cells were incubated for 1 hr at 377 C with a 1:5000 dilution of mouse anti-HA monoclonal antibody. Cells were washed and then incubated with goat anti-mouse IgG peroxidase conjugate at a 1:5000 dilution for 1 hr at 377 C. Following three washes, cells were incubated with peroxidase substrate for 30 min at room temperature in the dark. Aliquots were transferred to microtiter plates and product formation was detected at 450 nm on an EL 312 microplate reader. Determination of inositol phosphate accumulation. Cells were seeded in 24-well plates at 1 1 105 cells/well and assayed after 3 days in culture. Inositol lipids were radiolabeled by incubation of the cells for 18 h in 0.2 ml of inositol-free DMEM containing 1.0 g/liter glucose and 0.4 mCi of myo-[2-3H]inositol. No changes of medium were made subsequent to the addition of [3H]inositol. Drug challenges were initiated by addition of 50 ml of a 51 concentrated solution of the appropriate agonist in 50 mM LiCl, 250 mM HEPES, pH 7.25. Following a 10 min incubation at 377 C, the medium was aspirated and the assay terminated with 0.75 ml boiling 10 mM EDTA, pH 8.0. [3H]Inositol phosphates were resolved by Dowex AG1-X8 columns as described previously (25,26). Determination of cyclic AMP accumulation. Cells were seeded in 24-well plates at 1 1 105 cells/well in 0.5 ml growth medium 2 days prior to assay. Cells were labeled with 1 mCi [8-3H]-adenine in 225 ml for 2 h, and then 25 ml of 350 mM HEPES, pH 7.25, and 50 ml of 1.2 mM IBMX were added for 10 min at 377 C prior to a 10 min drug challenge with 50 ml of 71 concentrated agonist. Incubations were terminated by aspiration of the medium and addition of 1 ml icecold 5% TCA. The extract was applied directly to Dowex and alumina columns and [3H]-cAMP was resolved as described previously (27).
RESULTS Expression of the p2y7 receptor in 1321N1 cells. The coding sequence of the p2y7 receptor was amplified from human genomic DNA by PCR with primers based on the published DNA sequence and cloned into the retroviral vector pLXSN. Two other constructs in the same vector were made to allow for the verification of expression by ELISA. These constructs contained either a single copy or a tandem repeat of the HA epitope (YPYDVPDYA) immediately following the initiating methionine residue. Retroviruses from these constructs were produced as described in Materials and Methods and used to infect 1321N1 human astrocytoma cells. Following selection with geneticin, cell populations
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FIG. 1. Expression of HA-epitope tagged receptors in 1321N1 cells as measured by ELISA. 1321N1 cells were infected with control retrovirus (pLXSN) or retroviruses harboring either the human HAtagged p2y7 receptor (HA-p2y7) or the human HA-tagged P2Y1 receptor (HA-P2Y1). The data are presented as the mean { s.d. from one experiment performed with quadruplicate samples and are representative of three experiments.
were examined for receptor expression and nucleotidepromoted signaling responses. Verification of expression. Expression of the epitope-tagged p2y7 receptor was determined with an intact cell-based ELISA with the 12CA5 monoclonal antibody. Unexpectedly, significant ELISA activity was not detected with p2y7 receptors containing a single epitope tag at the NH2-terminus (data not shown). Lack of detection potentially could result from either low expression of the tagged receptor, an inability of the antibody to recognize the epitope in context of the short (õ 20 amino acids) NH2-terminus of the p2y7 receptor, or the close proximity of a putative N-linked glycosylation site. We also have observed a lack of ELISA activity in 1321N1 cells expressing other epitope-tagged receptors containing a short NH2-terminus. In contrast, cells expressing the p2y7 receptor with a tandem repeat of the epitope showed levels of ELISA activity that were 1.5 to 2-fold higher than those observed for 1321N1 cells expressing the P2Y1 receptor bearing a single epitope tag (Fig. 1). These data indicate that the p2y7 receptor was expressed on the surface of 1321N1 cells at levels similar to that of the functionally active P2Y1 receptor. Assessment of nucleotide-promoted activity of the p2y7 receptor. Akbar et al. (16) reported that ATP promoted an increase in [3H]inositol phosphate accumulation in COS-7 cells transiently transfected with p2y7 receptor cDNA. Thus, we tested the capacity of four natural nucleotides (ATP, ADP, UTP, and UDP, each at 100 mM) and the nucleotide analog dATPaS to promote increases in [3H]inositol phosphate accumulation in 1321N1 cells expressing either the HA-p2y7 receptor
or the HA-P2Y1 receptor (Fig. 2). In contrast to the results reported by Akbar et al. (16), these nucleotides were completely inactive in promoting increases in [3H]inositol phosphate accumulation in 1321N1-HAp2y7 cells. Nucleotides were also inactive in promoting [3H]inositol phosphate accumulation in 1321N1 cells expressing the untagged p2y7 receptor (data not shown). Incubation of 1321N1-HA-p2y7 cells with 1 mM carbachol, which activates an endogenous M3-muscarinic receptor, promoted a 5-fold increase in [3H]inositol phosphate accumulation over vehicle (Fig. 2). For comparison to a bona fide P2Y receptor, we also tested the ability of these nucleotides to activate the HA-P2Y1 receptor expressed in 1321N1 cells. ATP and ADP promoted a 5-8 fold increase in [3H]inositol phosphate accumulation, whereas dATPaS promoted a 2fold increase in [3H]inositol phosphate accumulation, consistent with its partial agonist activity at the P2Y1 receptor (11). P2Y receptors also have been reported to couple either to stimulation (28,29) or inhibition (6-10) of adenylyl cyclase. Thus, we also assessed the potential effects of nucleotides on cyclic AMP accumulation in 1321N1 cells expressing the p2y7 receptor. 1321N1HA-p2y7 cells were labeled with [3H]adenine, and cyclic AMP levels were quantitated following a 10 min incubation with 100 mM concentrations of ATP, UTP, ADP, UDP, or dATPaS. None of the nucleotides produced any effect on cyclic AMP levels in these cells (Fig. 3). Isoproterenol promoted an 20-fold increase in cyclic AMP levels through activation of an endogenous b2adrenergic receptor. To examine the potential capacity of nucleotides to inhibit cyclic AMP accumulation
FIG. 2. Nucleotide-promoted inositol phosphate accumulation in 1321N1 cells expressing the human p2y7 receptor. 1321N1-pLXSN, 1321N1-HA-p2y7, and 1321N1-HA-P2Y1 cell populations were assayed for nucleotide-promoted inositol phosphate accumulation in the presence of vehicle (10 mM LiCl), 100 mM nucleotide, or 1 mM carbachol. The data are the mean { s.d. from one experiment performed with triplicate samples and are representative of three experiments.
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FIG. 3. Nucleotide-promoted cyclic AMP responses in 1321N1 cells expressing the human p2y7 receptor. (A) 1321N1-HA-p2y7 cell populations were assayed for cyclic AMP accumulation in the presence of either vehicle, 100 mM nucleotides, or 10 mM isoproterenol (Iso). (B) 1321N1-HA-p2y7 cell populations were assayed for inhibition of isoproterenol-stimulated cyclic AMP accumulation in the presence of either vehicle or 100 mM nucleotides. The panel on the right shows the capacity of 1 mM carbachol to inhibit isoproterenol-promoted cyclic AMP accumulation in 1321N1 cells expressing the HAM2-muscarinic receptor. The data are the mean { s.d. from one experiment performed with triplicate samples and are representative of three experiments.
through the p2y7 receptor, 1321N1-HA-p2y7 cells were labeled with [3H]adenine and incubated with nucleotides in the presence of isoproterenol. Again, ATP, UTP, ADP, UDP, or dATPaS had no effect on cyclic AMP levels in these cells. As a positive control, we expressed the m2-muscarinic receptor in 1321N1 cells and assessed the capacity of 1 mM carbachol to inhibit isoproterenol-stimulated cyclic AMP accumulation. Activation of the m2-muscarinic receptor resulted in a 50% decrease in isoproterenol-promoted cyclic AMP accumulation (Fig. 3). DISCUSSION The p2y7 receptor was purported to be a P2Y receptor based on two criteria: the ‘‘specific’’ binding of [35S]dATPaS and a small (£ 2-fold) increase in inositol
phosphate accumulation in response to a single concentration of ATP in COS-7 cells transiently expressing the p2y7 receptor (16). Both of these criteria are ambiguous. [35S]dATPaS displays an unusually high level of binding to non-P2Y receptor proteins and the unlabeled compound has very low potency at established P2Y receptor subtypes (17). Thus, [35S]dATPaS is a questionable ligand for radiolabeling P2Y receptors. Furthermore, COS-7 cells endogenously express a P2Y2 receptor that activates PLC, thus complicating the use of these cells to express recombinant P2Y receptors (17). It is likely that the increase in [3H]inositol phosphates in response to ATP reported by Akbar et al. (16) was due to activation of the endogenous P2Y2 receptor. Thus, there are no unequivocal data supporting inclusion of the p2y7 receptor as a member of the P2Y family of nucleotide receptors. Construction of a recombinant p2y7 receptor bearing an HA epitope tag at the amino terminus permitted verification of receptor expression in 1321N1 cells. However, no nucleotide-promoted increases in inositol phosphate accumulation were detected under conditions in which activation of an endogenous M3-muscarinic receptor or a heterologously expressed P2Y1 receptor produced 5-15-fold increases in inositol phosphates over basal levels. Similarly, p2y7 receptor-expressing cells did not respond to nucleotides with changes in cyclic AMP accumulation under conditions in which activation of an endogenous b2-adrenergic receptor increased, and activation of a heterologously expressed M2-muscarinic receptor decreased cyclic AMP accumulation. Since 1321N1 cells display robust responses to ligands that activate receptors coupled to Gq, Gs, or Gi, the lack of response of 1321N1-HA-p2y7 cells to nucleotides cannot be ascribed to expression in a cell line that lacks the appropriate cellular signaling mechanisms. Although it might be argued that the presence of an epitope tag may have altered the functional activity of the receptor, previous studies in our laboratory with HA-tagged P2Y1 and P2Y2 receptors have shown that the addition of an epitope tag at the NH2-terminus has no effect on either pharmacological selectivity or signaling activity. The lack of effect of an HA epitope on selectivity and functional activity of other receptors also has been reported (21,22). Thus, it is highly unlikely that addition of one or two copies of the HA epitope has any contribution to the lack of functional activity of the p2y7 receptor. An identical lack of function activity was observed in 1321N1 cells expressing the non-tagged p2y7 receptor. The overall sequence identities of the four functionally confirmed mammalian P2Y receptors range between 35 and 52%, while the homology of the p2y7 receptor to these four P2Y receptors is in the range of 28-30%. Indeed, many peptide hormone receptors have much higher homology to the p2y7 receptor than do P2Y receptors. In fact, two other groups have reported
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the cloning of a sequence identical to the p2y7 receptor that was identified in each case as a putative chemokine receptor (18,19). Given the relatively low homology of the p2y7 receptor to other P2Y receptors, it is perhaps predictable that it does not display nucleotidepromoted activity. The p2y7 receptor is likely a member of another family of G protein-coupled receptors, possibly one of those activated by peptide hormones. The rather low sequence identity between P2Y receptor subtypes makes it difficult to determine a priori whether a newly cloned or orphan receptor displaying a sequence identity in the range of 30-40% with P2Y receptors are in fact members of this family of signaling proteins. This property, along with the lack of generally applicable radioligand binding assays, makes it imperative that newly identified receptors be shown to mediate nucleotide-promoted second messenger signaling responses. Another receptor, the 6H1 orphan receptor, falls into this category. This receptor shows 30-33% sequence identity to the four mammalian subtypes of P2Y receptors, and has been reported to be a P2Y receptor on the basis of [35S]dATPaS binding (15). However, no functional activity in response to nucleotides has been reported for this receptor and its entry as a member of the P2Y family of receptors by Webb et al. (15) would appear to be premature. Application of the approach used here for the p2y7 receptor will be needed to determine whether the 6H1 receptor also is a member of a non-P2Y receptor class of G protein-coupled receptors. ACKNOWLEDGMENTS We thank Jose´ Boyer for critically reading the manuscript and Sue Sromek for help in optimizing the ELISA. This work was supported by USPHS Grant GM38213 and Grant-In-Aid 9301067 from the American Heart Association. R.A.N. is an Established Investigator of the American Heart Association.
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7. Pianet, I., Merle, M., and Labouesse, J. (1989) Biochem. Biophys. Res. Commun. 163, 1150–1157. 8. Valeins, H., Merle, M., and Labouesse, J. (1992) Mol. Pharmacol. 42, 1033–1041. 9. Yamada, M., Hamamori, Y., Akita, H., and Yokoyama, M. (1992) Circ. Res. 70, 477–485. 10. Boyer, J. L., Lazarowski, E. R., Chen, X. H., and Harden, T. K. (1993) J. Pharmacol. Exp. Ther. 267, 1140–1146. 11. Schachter, J. B., Li, Q., Boyer, J. L., Nicholas, R. A., and Harden, T. K. (1996) Br. J. Pharmacol. 118, 167–173. 12. Parr, C. E., Sullivan, D. M., Paradiso, A. M., Lazarowski, E. R., Burch, L. H., Olsen, J. C., Erb, L., Weisman, G. A., Boucher, R. C., and Turner, J. T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 13067. 13. Nicholas, R. A., Watt, W. C., Lazarowski, E. R., Li, Q., and Harden, T. K. (1996) Mol. Pharmacol. 50, 224–229. 14. Webb, T. E., Henderson, D., King, B. F., Wang, S., Simon, J., Bateson, A. N., Burnstock, G., and Barnard, E. A. (1996) Mol. Pharmacol. 50, 258–265. 15. Webb, T. E., Kaplan, M. G., and Barnard, E. A. (1996) Biochem. Biophys. Res. Commun. 219, 105–110. 16. Akbar, G. K. M., Dasari, V. R., Webb, T. E., Ayyanathan, K., Pillarisetti, K., Sandhu, A. K., Athwal, R. S., Daniel, J. L., Ashby, B., Barnard, E. A., and Kunapuli, S. P. (1996) J. Biol. Chem. 271, 18363–18367. 17. Schachter, J. B., and Harden, T. K. (1997) Br. J. Pharmacol. [in press] 18. Raport, C. J., Schweickart, V. L., Chantry, D., Eddy, R. L., Jr., Shows, T. B., Godiska, R., and Gray, P. W. (1996) J. Leukocyte Biol. 59, 18–23. 19. Owman, C., Nillson, C., and Lolait, S. J. (1996) Genomics 37, 187–194. 20. Kolodziej, P. A., and Young, R. A. (1991) Methods Enzymol. 194, 508–519. 21. Ali, H., Richardson, R. M., Tomhave, E. D., Didsbury, J. R., and Snyderman, R. (1993) J. Biol. Chem. 268, 24247–24254. 22. Ali, H., Richardson, R. M., Tomhave, E. D., DuBose, R. A., Haribabu, B., and Snyderman, R. (1994) J. Biol. Chem. 269, 24557– 24563. 23. Comstock, K. E., Watson, N. F., and Olsen, J. C. (1997) in Methods in Molecular Biology: Recombinant Gene Expression Protocols (Tuan, R., Ed.), Vol. 62, pp. 207–222, Humana Press, Totowa. 24. Liu, J., Schoneberg, T., van Rhee, M., and Wess, J. (1995) J. Biol. Chem. 270, 19532–19539. 25. Lazarowski, E. R., Watt, W. C., Stutts, M. J., Boucher, R. C., and Harden, T. K. (1995) Br. J. Pharmacol. 116, 1619–1627. 26. Filtz, T. A., Li, Q., Boyer, J. L., Nicholas, R. A., and Harden, T. K. (1994) Mol. Pharmacol. 46, 8–14. 27. Harden, T. K., Scheer, A. G., and Smith, M. M. (1982) Mol. Pharmacol. 21, 570–580. 28. Matsuoka, I., Zhou, Q., Ishimoto, H., and Nakanishi, H. (1995) Mol. Pharmacol. 47, 855–862. 29. Jiang, L., Foster, F. M., Ward, P., Tasevski, V., Luttrell, B. M., and Conigrave, A. D. (1997) Biochem. Biophys. Res. Commun. 232, 626–630. 30. Vanhoutte, P. M., Humphrey, P. A. A., and Spedding, M. (1996) Pharmacol. Rev. 48, 1–2.
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RC976884
Lack of Nucleotide-Promoted Second Messenger Signaling Responses in 1321N1 Cells Expressing the Proposed P2Y Receptor, p2y7 Christopher L. Herold, Qing Li, Joel B. Schachter, T. Kendall Harden, and Robert A. Nicholas Department of Pharmacology, CB #7365, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365
Received May 27, 1997
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]dATPaS 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]dATPaS 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 dATPaS 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. q 1997 Academic Press
receptors are G protein-coupled signaling proteins that respond to both adenine and uridine nucleotides and either activate phospholipase C (PLC) (1-5) or inhibit adenylyl cyclase (6-10). Although receptors claiming the monographs P2Y1-P2Y7 have been reported, only four of these, the P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors, have been cloned from mammalian species and shown unequivocally to possess nucleotide-promoted functional activity. All four of these receptors activate PLC and promote mobilization of intracellular Ca2/. The P2Y1 receptor is activated specifically by adenine nucleotides, with a preference for diphosphates over triphosphates (11), the P2Y2 receptor is a triphosphatespecific receptor activated equipotently by ATP and UTP (12,13), the P2Y4 receptor is activated primarily by UTP (13), and the P2Y6 receptor is activated selectively by UDP (13). These four receptors share 35-52% sequence identity. A receptor with high sequence identity (65%) to the rat P2Y6 receptor has been cloned from chick brain and designated as the p2y3 receptor1 (14). Like the mammalian P2Y6 receptor, the p2y3 receptor couples to PLC and is activated most potently by UDP. Two other receptors, tentatively designated the chick p2y5 receptor and the human p2y7 receptor1, have been purported to be P2Y receptors (15,16). These two receptors, which exhibit low sequence identity (28-33%) to the known mammalian P2Y receptors, were proposed to be members of the P2Y receptor family of signaling proteins largely on the basis of a radioligand binding assay with [35S]dATPaS. We recently have shown (17) that [35S]dATPaS is not a general radioligand for P2Y receptors, and thus the use of [35S]dATPaS to identify 1
Extracellular nucleotides exert their diverse biological effects through two classes of P2 receptors. P2X receptors are cation-selective ion channels that respond primarily to adenine nucleotides, whereas P2Y
According to the recommendations of the IUPHAR nomenclature committee (30), we have used the designation p2y3 for this P2Y receptor since it is not from a mammalian species and no mammalian homolog has been identified. Likewise, the same designation has been used for the p2y5 and p2y7 receptors, which have not been shown unequivocally to possess nucleotide-promoted functional activity.
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new P2Y receptors in the absence of nucleotide-promoted functional activity is suspect. Although the p2y7 receptor also was claimed to be a P2Y receptor on the basis of an inositol phosphate response to ATP in transfected COS-7 cells, these results also are difficult to interpret, since we have shown that COS-7 cells express an endogenous P2Y2 receptor that activates PLC in response to ATP and UTP (17). Thus, the data supporting the claim that the p2y7 receptor is a functional P2Y receptor are ambiguous. Since the identity of the p2y7 receptor as a bona fide P2Y receptor is in question, we have stably expressed an epitope-tagged p2y7 receptor in 1321N1 human astrocytoma cells and assessed both receptor expression and nucleotide-promoted second messenger responses in these cells. Neither adenine nor uridine nucleotides promoted any effect on inositol phosphate or cyclic AMP accumulation in cells shown by an intact cell-based ELISA to express the p2y7 receptor. We conclude from these data that the so-called p2y7 receptor is not a member of the P2Y family of G protein-coupled receptors. MATERIALS AND METHODS Materials. ATP, ADP, UTP, and UDP were from Boehringer Mannheim Biochemicals (Indianapolis, IN). dATPaS was from Research Biochemicals International (Natick, MA). Carbachol, goat anti-mouse IgG peroxidase conjugate, IBMX, isoproterenol, and OPD peroxidase substrate tablet set were from Sigma Chemical (St. Louis, MO). Mouse 12CA5 monoclonal antibody was from BAbCO (Richmond, CA). [8-3H]-Adenine (18 Ci/mmol) and myo-[2-3H]-inositol (20 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). All tissue culture reagents were from the Lineberger Comprehensive Cancer Center tissue culture facilities (University of North Carolina at Chapel Hill, NC). Construction of expression vectors harboring the native and HA epitope-tagged p2y7 receptor. The human p2y7 receptor was amplified by PCR from 200 ng of human genomic DNA with primers based on the published sequence of the p2y7 receptor (16,18,19). The upstream primer contained an EcoRI site at its 5*-end and was designed to include 7 bp of upstream noncoding sequence, whereas the downstream primer contained a XhoI restriction site at its 5*-end and was designed to include 24 bp of 3*-untranslated sequence in the amplified fragment. The amplified fragment was purified, digested with EcoRI and XhoI, and subcloned into the similarly digested retroviral expression vector pLXSN. A p2y7 receptor harboring a tandem repeat of the 12CA5 epitope (hereafter referred to as the HA epitope) (20) was constructed by PCR amplification of 1 ng of pLXSNp2y7 with the same downstream primer as above and an upstream primer designed to include an MluI restriction site at its 5*-end and the coding sequence for an HA epitope tag immediately prior to the second codon of the p2y7 receptor. The modified fragment was used to replace the coding sequence of the P2Y1 receptor in the plasmid pLXSN-HA-P2Y1, which contained an initiating Met codon and an HA-epitope tag sequence followed by an MluI site at the start of the P2Y1 receptor. This resulted in the incorporation of a tandem repeat of the HA-epitope tag at the NH2-terminus of the expressed p2y7 receptor (hereafter referred to as the HA-p2y7 receptor). Inclusion of an eptitope tag at the NH2-terminus of a G protein-coupled receptor has been shown to have no effect on either its pharmacological selectivity or second messenger coupling specificity (21,22).
Expression of p2y7 and HA-p2y7 receptors in 1321N1 human astrocytoma cells. 1321N1 human astrocytoma cells, a cell line that does not demonstrate functional responses to P2Y receptor agonists, were grown in monolayer culture at 377 C in 5% CO2 in high-glucose DMEM supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. Recombinant retroviruses were produced by calcium phosphate-mediated transfection of PA317 cells with pLXSN vector containing the appropriate p2y7 receptor sequence as described by Comstock et al. (23). 1321N1 cells were infected with retroviruses harboring recombinant p2y7 receptor sequences or with control retrovirus, and geneticin-resistant cells were selected after approximately two weeks in culture with 1 mg/ml G-418. Populations of resistant cells were maintained in medium supplemented with 0.4 mg/ml G-418. The generation of 1321N1 cells expressing HA epitope-tagged P2Y1 and M2muscarinic receptors were accomplished in a similar manner. ELISA of HA epitope-tagged receptors. Cells seeded in 24-well plates at 1 1 105 cells/well were tested for the expression of tagged receptors essentially as described (24). Following the washing and blocking steps, cells were incubated for 1 hr at 377 C with a 1:5000 dilution of mouse anti-HA monoclonal antibody. Cells were washed and then incubated with goat anti-mouse IgG peroxidase conjugate at a 1:5000 dilution for 1 hr at 377 C. Following three washes, cells were incubated with peroxidase substrate for 30 min at room temperature in the dark. Aliquots were transferred to microtiter plates and product formation was detected at 450 nm on an EL 312 microplate reader. Determination of inositol phosphate accumulation. Cells were seeded in 24-well plates at 1 1 105 cells/well and assayed after 3 days in culture. Inositol lipids were radiolabeled by incubation of the cells for 18 h in 0.2 ml of inositol-free DMEM containing 1.0 g/liter glucose and 0.4 mCi of myo-[2-3H]inositol. No changes of medium were made subsequent to the addition of [3H]inositol. Drug challenges were initiated by addition of 50 ml of a 51 concentrated solution of the appropriate agonist in 50 mM LiCl, 250 mM HEPES, pH 7.25. Following a 10 min incubation at 377 C, the medium was aspirated and the assay terminated with 0.75 ml boiling 10 mM EDTA, pH 8.0. [3H]Inositol phosphates were resolved by Dowex AG1-X8 columns as described previously (25,26). Determination of cyclic AMP accumulation. Cells were seeded in 24-well plates at 1 1 105 cells/well in 0.5 ml growth medium 2 days prior to assay. Cells were labeled with 1 mCi [8-3H]-adenine in 225 ml for 2 h, and then 25 ml of 350 mM HEPES, pH 7.25, and 50 ml of 1.2 mM IBMX were added for 10 min at 377 C prior to a 10 min drug challenge with 50 ml of 71 concentrated agonist. Incubations were terminated by aspiration of the medium and addition of 1 ml icecold 5% TCA. The extract was applied directly to Dowex and alumina columns and [3H]-cAMP was resolved as described previously (27).
RESULTS Expression of the p2y7 receptor in 1321N1 cells. The coding sequence of the p2y7 receptor was amplified from human genomic DNA by PCR with primers based on the published DNA sequence and cloned into the retroviral vector pLXSN. Two other constructs in the same vector were made to allow for the verification of expression by ELISA. These constructs contained either a single copy or a tandem repeat of the HA epitope (YPYDVPDYA) immediately following the initiating methionine residue. Retroviruses from these constructs were produced as described in Materials and Methods and used to infect 1321N1 human astrocytoma cells. Following selection with geneticin, cell populations
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FIG. 1. Expression of HA-epitope tagged receptors in 1321N1 cells as measured by ELISA. 1321N1 cells were infected with control retrovirus (pLXSN) or retroviruses harboring either the human HAtagged p2y7 receptor (HA-p2y7) or the human HA-tagged P2Y1 receptor (HA-P2Y1). The data are presented as the mean { s.d. from one experiment performed with quadruplicate samples and are representative of three experiments.
were examined for receptor expression and nucleotidepromoted signaling responses. Verification of expression. Expression of the epitope-tagged p2y7 receptor was determined with an intact cell-based ELISA with the 12CA5 monoclonal antibody. Unexpectedly, significant ELISA activity was not detected with p2y7 receptors containing a single epitope tag at the NH2-terminus (data not shown). Lack of detection potentially could result from either low expression of the tagged receptor, an inability of the antibody to recognize the epitope in context of the short (õ 20 amino acids) NH2-terminus of the p2y7 receptor, or the close proximity of a putative N-linked glycosylation site. We also have observed a lack of ELISA activity in 1321N1 cells expressing other epitope-tagged receptors containing a short NH2-terminus. In contrast, cells expressing the p2y7 receptor with a tandem repeat of the epitope showed levels of ELISA activity that were 1.5 to 2-fold higher than those observed for 1321N1 cells expressing the P2Y1 receptor bearing a single epitope tag (Fig. 1). These data indicate that the p2y7 receptor was expressed on the surface of 1321N1 cells at levels similar to that of the functionally active P2Y1 receptor. Assessment of nucleotide-promoted activity of the p2y7 receptor. Akbar et al. (16) reported that ATP promoted an increase in [3H]inositol phosphate accumulation in COS-7 cells transiently transfected with p2y7 receptor cDNA. Thus, we tested the capacity of four natural nucleotides (ATP, ADP, UTP, and UDP, each at 100 mM) and the nucleotide analog dATPaS to promote increases in [3H]inositol phosphate accumulation in 1321N1 cells expressing either the HA-p2y7 receptor
or the HA-P2Y1 receptor (Fig. 2). In contrast to the results reported by Akbar et al. (16), these nucleotides were completely inactive in promoting increases in [3H]inositol phosphate accumulation in 1321N1-HAp2y7 cells. Nucleotides were also inactive in promoting [3H]inositol phosphate accumulation in 1321N1 cells expressing the untagged p2y7 receptor (data not shown). Incubation of 1321N1-HA-p2y7 cells with 1 mM carbachol, which activates an endogenous M3-muscarinic receptor, promoted a 5-fold increase in [3H]inositol phosphate accumulation over vehicle (Fig. 2). For comparison to a bona fide P2Y receptor, we also tested the ability of these nucleotides to activate the HA-P2Y1 receptor expressed in 1321N1 cells. ATP and ADP promoted a 5-8 fold increase in [3H]inositol phosphate accumulation, whereas dATPaS promoted a 2fold increase in [3H]inositol phosphate accumulation, consistent with its partial agonist activity at the P2Y1 receptor (11). P2Y receptors also have been reported to couple either to stimulation (28,29) or inhibition (6-10) of adenylyl cyclase. Thus, we also assessed the potential effects of nucleotides on cyclic AMP accumulation in 1321N1 cells expressing the p2y7 receptor. 1321N1HA-p2y7 cells were labeled with [3H]adenine, and cyclic AMP levels were quantitated following a 10 min incubation with 100 mM concentrations of ATP, UTP, ADP, UDP, or dATPaS. None of the nucleotides produced any effect on cyclic AMP levels in these cells (Fig. 3). Isoproterenol promoted an 20-fold increase in cyclic AMP levels through activation of an endogenous b2adrenergic receptor. To examine the potential capacity of nucleotides to inhibit cyclic AMP accumulation
FIG. 2. Nucleotide-promoted inositol phosphate accumulation in 1321N1 cells expressing the human p2y7 receptor. 1321N1-pLXSN, 1321N1-HA-p2y7, and 1321N1-HA-P2Y1 cell populations were assayed for nucleotide-promoted inositol phosphate accumulation in the presence of vehicle (10 mM LiCl), 100 mM nucleotide, or 1 mM carbachol. The data are the mean { s.d. from one experiment performed with triplicate samples and are representative of three experiments.
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FIG. 3. Nucleotide-promoted cyclic AMP responses in 1321N1 cells expressing the human p2y7 receptor. (A) 1321N1-HA-p2y7 cell populations were assayed for cyclic AMP accumulation in the presence of either vehicle, 100 mM nucleotides, or 10 mM isoproterenol (Iso). (B) 1321N1-HA-p2y7 cell populations were assayed for inhibition of isoproterenol-stimulated cyclic AMP accumulation in the presence of either vehicle or 100 mM nucleotides. The panel on the right shows the capacity of 1 mM carbachol to inhibit isoproterenol-promoted cyclic AMP accumulation in 1321N1 cells expressing the HAM2-muscarinic receptor. The data are the mean { s.d. from one experiment performed with triplicate samples and are representative of three experiments.
through the p2y7 receptor, 1321N1-HA-p2y7 cells were labeled with [3H]adenine and incubated with nucleotides in the presence of isoproterenol. Again, ATP, UTP, ADP, UDP, or dATPaS had no effect on cyclic AMP levels in these cells. As a positive control, we expressed the m2-muscarinic receptor in 1321N1 cells and assessed the capacity of 1 mM carbachol to inhibit isoproterenol-stimulated cyclic AMP accumulation. Activation of the m2-muscarinic receptor resulted in a 50% decrease in isoproterenol-promoted cyclic AMP accumulation (Fig. 3). DISCUSSION The p2y7 receptor was purported to be a P2Y receptor based on two criteria: the ‘‘specific’’ binding of [35S]dATPaS and a small (£ 2-fold) increase in inositol
phosphate accumulation in response to a single concentration of ATP in COS-7 cells transiently expressing the p2y7 receptor (16). Both of these criteria are ambiguous. [35S]dATPaS displays an unusually high level of binding to non-P2Y receptor proteins and the unlabeled compound has very low potency at established P2Y receptor subtypes (17). Thus, [35S]dATPaS is a questionable ligand for radiolabeling P2Y receptors. Furthermore, COS-7 cells endogenously express a P2Y2 receptor that activates PLC, thus complicating the use of these cells to express recombinant P2Y receptors (17). It is likely that the increase in [3H]inositol phosphates in response to ATP reported by Akbar et al. (16) was due to activation of the endogenous P2Y2 receptor. Thus, there are no unequivocal data supporting inclusion of the p2y7 receptor as a member of the P2Y family of nucleotide receptors. Construction of a recombinant p2y7 receptor bearing an HA epitope tag at the amino terminus permitted verification of receptor expression in 1321N1 cells. However, no nucleotide-promoted increases in inositol phosphate accumulation were detected under conditions in which activation of an endogenous M3-muscarinic receptor or a heterologously expressed P2Y1 receptor produced 5-15-fold increases in inositol phosphates over basal levels. Similarly, p2y7 receptor-expressing cells did not respond to nucleotides with changes in cyclic AMP accumulation under conditions in which activation of an endogenous b2-adrenergic receptor increased, and activation of a heterologously expressed M2-muscarinic receptor decreased cyclic AMP accumulation. Since 1321N1 cells display robust responses to ligands that activate receptors coupled to Gq, Gs, or Gi, the lack of response of 1321N1-HA-p2y7 cells to nucleotides cannot be ascribed to expression in a cell line that lacks the appropriate cellular signaling mechanisms. Although it might be argued that the presence of an epitope tag may have altered the functional activity of the receptor, previous studies in our laboratory with HA-tagged P2Y1 and P2Y2 receptors have shown that the addition of an epitope tag at the NH2-terminus has no effect on either pharmacological selectivity or signaling activity. The lack of effect of an HA epitope on selectivity and functional activity of other receptors also has been reported (21,22). Thus, it is highly unlikely that addition of one or two copies of the HA epitope has any contribution to the lack of functional activity of the p2y7 receptor. An identical lack of function activity was observed in 1321N1 cells expressing the non-tagged p2y7 receptor. The overall sequence identities of the four functionally confirmed mammalian P2Y receptors range between 35 and 52%, while the homology of the p2y7 receptor to these four P2Y receptors is in the range of 28-30%. Indeed, many peptide hormone receptors have much higher homology to the p2y7 receptor than do P2Y receptors. In fact, two other groups have reported
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the cloning of a sequence identical to the p2y7 receptor that was identified in each case as a putative chemokine receptor (18,19). Given the relatively low homology of the p2y7 receptor to other P2Y receptors, it is perhaps predictable that it does not display nucleotidepromoted activity. The p2y7 receptor is likely a member of another family of G protein-coupled receptors, possibly one of those activated by peptide hormones. The rather low sequence identity between P2Y receptor subtypes makes it difficult to determine a priori whether a newly cloned or orphan receptor displaying a sequence identity in the range of 30-40% with P2Y receptors are in fact members of this family of signaling proteins. This property, along with the lack of generally applicable radioligand binding assays, makes it imperative that newly identified receptors be shown to mediate nucleotide-promoted second messenger signaling responses. Another receptor, the 6H1 orphan receptor, falls into this category. This receptor shows 30-33% sequence identity to the four mammalian subtypes of P2Y receptors, and has been reported to be a P2Y receptor on the basis of [35S]dATPaS binding (15). However, no functional activity in response to nucleotides has been reported for this receptor and its entry as a member of the P2Y family of receptors by Webb et al. (15) would appear to be premature. Application of the approach used here for the p2y7 receptor will be needed to determine whether the 6H1 receptor also is a member of a non-P2Y receptor class of G protein-coupled receptors. ACKNOWLEDGMENTS We thank Jose´ Boyer for critically reading the manuscript and Sue Sromek for help in optimizing the ELISA. This work was supported by USPHS Grant GM38213 and Grant-In-Aid 9301067 from the American Heart Association. R.A.N. is an Established Investigator of the American Heart Association.
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