Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Nov 15;90(22):10449–10453. doi: 10.1073/pnas.90.22.10449

Functional expression and photoaffinity labeling of a cloned P2U purinergic receptor.

L Erb 1, K D Lustig 1, D M Sullivan 1, J T Turner 1, G A Weisman 1
PMCID: PMC47794  PMID: 8248130

Abstract

ATP and UTP can function as extracellular signaling molecules by activating plasma membrane receptors termed P2 purinergic receptors. In the present study a P2U receptor cDNA has been expressed in K-562 human leukemia cells, one of the few available mammalian cell lines that lacks an endogenous P2U receptor. In stably transfected cells, low micromolar concentrations of ATP or UTP activated the receptor, resulting in the mobilization of intracellular calcium but not the influx of extracellular calcium. A photoaffinity agonist of the P2U receptor, 3'-O-(4-benzoylbenzoyl)adenosine 5'-[alpha-32P]triphosphate ([alpha-32P]BzATP), photolabeled several proteins in plasma membranes from the stable transfectant or from untransfected K-562 cells. The photolabeling of a 53-kDa protein was significantly greater in plasma membranes from the stable transfectant than from untransfected cells. A mutant receptor containing six consecutive histidine residues at its carboxyl terminus was constructed and used to verify that this 53-kDa protein was the P2U receptor. In plasma membranes from cells expressing the histidine-tagged P2U receptor, but not from cells expressing the wild-type receptor, a single [alpha-32P]BzATP-labeled protein with a molecular mass of 53 kDa was retained on a Ni(2+)-charged Sepharose column, which binds many proteins containing a polyhistidine tag. Photolabeling of the 53-kDa protein by [alpha-32P]BzATP was inhibited by ATP but not by UTP, raising the possibility that the P2U receptor may have distinct binding sites for each nucleotide.

Full text

PDF
10449

Images in this article

10450Fig. 1
on p.10450
10452Fig. 4
on p.10452
10452Fig. 6
on p.10452

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnbaumer L. Receptor-to-effector signaling through G proteins: roles for beta gamma dimers as well as alpha subunits. Cell. 1992 Dec 24;71(7):1069–1072. doi: 10.1016/s0092-8674(05)80056-x. [DOI] [PubMed] [Google Scholar]
  2. Boeynaems J. M., Pearson J. D. P2 purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. Trends Pharmacol Sci. 1990 Jan;11(1):34–37. doi: 10.1016/0165-6147(90)90039-b. [DOI] [PubMed] [Google Scholar]
  3. Boyer J. L., Cooper C. L., Harden T. K. [32P]3'-O-(4-benzoyl)benzoyl ATP as a photoaffinity label for a phospholipase C-coupled P2Y-purinergic receptor. J Biol Chem. 1990 Aug 15;265(23):13515–13520. [PubMed] [Google Scholar]
  4. Brown H. A., Lazarowski E. R., Boucher R. C., Harden T. K. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. Mol Pharmacol. 1991 Nov;40(5):648–655. [PubMed] [Google Scholar]
  5. Burnstock G. Overview. Purinergic mechanisms. Ann N Y Acad Sci. 1990;603:1–18. doi: 10.1111/j.1749-6632.1990.tb37657.x. [DOI] [PubMed] [Google Scholar]
  6. Conklin B. R., Chabre O., Wong Y. H., Federman A. D., Bourne H. R. Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C. J Biol Chem. 1992 Jan 5;267(1):31–34. [PubMed] [Google Scholar]
  7. Dubyak G. R. Signal transduction by P2-purinergic receptors for extracellular ATP. Am J Respir Cell Mol Biol. 1991 Apr;4(4):295–300. doi: 10.1165/ajrcmb/4.4.295. [DOI] [PubMed] [Google Scholar]
  8. Erb L., Lustig K. D., Ahmed A. H., Gonzalez F. A., Weisman G. A. Covalent incorporation of 3'-O-(4-benzoyl)benzoyl-ATP into a P2 purinoceptor in transformed mouse fibroblasts. J Biol Chem. 1990 May 5;265(13):7424–7431. [PubMed] [Google Scholar]
  9. Fine J., Cole P., Davidson J. S. Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. Biochem J. 1989 Oct 15;263(2):371–376. doi: 10.1042/bj2630371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Giannattasio B., Powers K., Scarpa A. Photoaffinity labeling and expression cloning of extracellular ATP receptors of cardiac myocytes. Ann N Y Acad Sci. 1992 Nov 30;671:471–477. doi: 10.1111/j.1749-6632.1992.tb43833.x. [DOI] [PubMed] [Google Scholar]
  11. Gonzalez F. A., Ahmed A. H., Lustig K. D., Erb L., Weisman G. A. Permeabilization of transformed mouse fibroblasts by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate and the desensitization of the process. J Cell Physiol. 1989 Apr;139(1):109–115. doi: 10.1002/jcp.1041390116. [DOI] [PubMed] [Google Scholar]
  12. Gordon J. L. Extracellular ATP: effects, sources and fate. Biochem J. 1986 Jan 15;233(2):309–319. doi: 10.1042/bj2330309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  14. Knowles M. R., Clarke L. L., Boucher R. C. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med. 1991 Aug 22;325(8):533–538. doi: 10.1056/NEJM199108223250802. [DOI] [PubMed] [Google Scholar]
  15. Le Grice S. F., Grüninger-Leitch F. Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J Biochem. 1990 Jan 26;187(2):307–314. doi: 10.1111/j.1432-1033.1990.tb15306.x. [DOI] [PubMed] [Google Scholar]
  16. Lin T. A., Lustig K. D., Sportiello M. G., Weisman G. A., Sun G. Y. Signal transduction pathways coupled to a P2U receptor in neuroblastoma x glioma (NG108-15) cells. J Neurochem. 1993 Mar;60(3):1115–1125. doi: 10.1111/j.1471-4159.1993.tb03262.x. [DOI] [PubMed] [Google Scholar]
  17. Ljungquist C., Breitholtz A., Brink-Nilsson H., Moks T., Uhlén M., Nilsson B. Immobilization and affinity purification of recombinant proteins using histidine peptide fusions. Eur J Biochem. 1989 Dec 22;186(3):563–569. doi: 10.1111/j.1432-1033.1989.tb15245.x. [DOI] [PubMed] [Google Scholar]
  18. Lustig K. D., Erb L., Landis D. M., Hicks-Taylor C. S., Zhang X., Sportiello M. G., Weisman G. A. Mechanisms by which extracellular ATP and UTP stimulate the release of prostacyclin from bovine pulmonary artery endothelial cells. Biochim Biophys Acta. 1992 Feb 19;1134(1):61–72. doi: 10.1016/0167-4889(92)90028-a. [DOI] [PubMed] [Google Scholar]
  19. Lustig K. D., Shiau A. K., Brake A. J., Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5113–5117. doi: 10.1073/pnas.90.11.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lustig K. D., Sportiello M. G., Erb L., Weisman G. A. A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Biochem J. 1992 Jun 15;284(Pt 3):733–739. doi: 10.1042/bj2840733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Majid M. A., Okajima F., Kondo Y. Characterization of ATP receptor which mediates norepinephrine release in PC12 cells. Biochim Biophys Acta. 1992 Sep 9;1136(3):283–289. doi: 10.1016/0167-4889(92)90118-u. [DOI] [PubMed] [Google Scholar]
  22. Mason S. J., Paradiso A. M., Boucher R. C. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol. 1991 Jul;103(3):1649–1656. doi: 10.1111/j.1476-5381.1991.tb09842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murgo A. J., Sistare F. D. K562 leukemia cells express P2T (adenosine diphosphate) purinergic receptors. J Pharmacol Exp Ther. 1992 May;261(2):580–585. [PubMed] [Google Scholar]
  24. O'Connor S. E., Dainty I. A., Leff P. Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci. 1991 Apr;12(4):137–141. doi: 10.1016/0165-6147(91)90530-6. [DOI] [PubMed] [Google Scholar]
  25. Pfeilschifter J. Comparison of extracellular ATP and UTP signalling in rat renal mesangial cells. No indications for the involvement of separate purino- and pyrimidino-ceptors. Biochem J. 1990 Dec 1;272(2):469–472. doi: 10.1042/bj2720469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pirotton S., Raspe E., Demolle D., Erneux C., Boeynaems J. M. Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells. J Biol Chem. 1987 Dec 25;262(36):17461–17466. [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seifert R., Schultz G. Involvement of pyrimidinoceptors in the regulation of cell functions by uridine and by uracil nucleotides. Trends Pharmacol Sci. 1989 Sep;10(9):365–369. doi: 10.1016/0165-6147(89)90009-6. [DOI] [PubMed] [Google Scholar]
  29. Simon M. I., Strathmann M. P., Gautam N. Diversity of G proteins in signal transduction. Science. 1991 May 10;252(5007):802–808. doi: 10.1126/science.1902986. [DOI] [PubMed] [Google Scholar]
  30. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Toneguzzo F., Hayday A. C., Keating A. Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells. Mol Cell Biol. 1986 Feb;6(2):703–706. doi: 10.1128/mcb.6.2.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Turner J. T., James-Kracke M. R., Camden J. M. Regulation of the neurotensin receptor and intracellular calcium mobilization in HT29 cells. J Pharmacol Exp Ther. 1990 Jun;253(3):1049–1056. [PubMed] [Google Scholar]
  33. Williams N., Ackerman S. H., Coleman P. S. Benzophenone-ATP: a photoaffinity label for the active site of ATPases. Methods Enzymol. 1986;126:667–682. doi: 10.1016/s0076-6879(86)26070-x. [DOI] [PubMed] [Google Scholar]
  34. Wu H., Franklin C. C., Kim H. D., Turner J. T. Regulation of calcium-activated potassium efflux by neurotensin and other agents in HT-29 cells. Am J Physiol. 1991 Jan;260(1 Pt 1):C35–C42. doi: 10.1152/ajpcell.1991.260.1.C35. [DOI] [PubMed] [Google Scholar]
  35. Yu H. X., Turner J. T. Functional studies in the human submandibular duct cell line, HSG-PA, suggest a second salivary gland receptor subtype for nucleotides. J Pharmacol Exp Ther. 1991 Dec;259(3):1344–1350. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES