ACS Publications. Most Trusted. Most Cited. Most Read
N-Linked Glycosylation Is Essential for the Functional Expression of the Recombinant P2X2 Receptor

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Biochemistry 1998, 37, 42, 14845-14851
    Article

    N-Linked Glycosylation Is Essential for the Functional Expression of the Recombinant P2X2 Receptor†
    Click to copy article linkArticle link copied!

    View Author Information
    Department of Pharmacological and Physiological Sciences, St. Louis University Health Sciences Center, St. Louis, Missouri 63104
    Other Access Options

    Biochemistry

    Cite this: Biochemistry 1998, 37, 42, 14845–14851
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi981209g
    Published September 29, 1998
    Copyright © 1998 American Chemical Society
    Request reuse permissions

    Abstract

    Click to copy section linkSection link copied!

    P2X receptors are integral membrane proteins that belong to the growing family of transmitter-gated ion channels. The extracellular domain of these receptors contains several consensus sequences for N-linked glycosylation that may contribute to the functional expression of the channel. We have previously reported the extracellular orientation of asparagine residues 182, 239, and 298 of the P2X2 receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEBS Lett.425, 19−23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X2 receptor by using two different approaches, tunicamycin treatment or site-directed mutagenesis. HEK-293 cells stably transfected with the P2X2 receptor subunit showed little or no response to ATP after tunicamycin treatment. In addition, loss of function was observed with the elimination of all three N-linked glycosylation sites from P2X2. Cell surface labeling with biotin or indirect immunofluorescence revealed that the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the cell surface, indicating that the nonglycosylated P2X2 receptors are retained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expression of a P2X receptor subunit.

    Copyright © 1998 American Chemical Society

    Subjects

    what are subjects

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.

    Recommended

    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. Add or change your institution or let them know you’d like them to include access.

    †

     This work was supported by NIH Grants NS35534 (M.M.V.) and HL56236 (T.M.E.).

    *

     To whom correspondence should be addressed at 1402 S. Grand Blvd., St. Louis, MO 63104. Phone:  (314) 577-8545. Fax:  (314) 577-8233. E-mail:  [email protected].

    Cited By

    Click to copy section linkSection link copied!
    Citation Statements
    Explore this article's citation statements on scite.ai
    powered by  Scite

    This article is cited by 51 publications.

    1. Jerry M. Troutman and Barbara Imperiali. Campylobacter jejuni PglH Is a Single Active Site Processive Polymerase that Utilizes Product Inhibition to Limit Sequential Glycosyl Transfer Reactions. Biochemistry 2009, 48 (12) , 2807-2816. https://doi.org/10.1021/bi802284d
    2. Kenneth A. Jacobson,, Michael F. Jarvis, and, Michael Williams. Purine and Pyrimidine (P2) Receptors as Drug Targets. Journal of Medicinal Chemistry 2002, 45 (19) , 4057-4093. https://doi.org/10.1021/jm020046y
    3. Chiara Guidi, Xevi Biarnés, Antoni Planas, Marjan De Mey. Controlled processivity in glycosyltransferases: A way to expand the enzymatic toolbox. Biotechnology Advances 2023, 63 , 108081. https://doi.org/10.1016/j.biotechadv.2022.108081
    4. Jasmeet Kaur, Sanchit Dora. Purinergic signaling: Diverse effects and therapeutic potential in cancer. Frontiers in Oncology 2023, 13 https://doi.org/10.3389/fonc.2023.1058371
    5. Felix M. Bennetts, Jesse I. Mobbs, Sabatino Ventura, David M. Thal. The P2X1 receptor as a therapeutic target. Purinergic Signalling 2022, 18 (4) , 421-433. https://doi.org/10.1007/s11302-022-09880-4
    6. Mark T. Young. Studying Purinoceptor Cell-Surface Expression by Protein Biotinylation. 2020, 137-146. https://doi.org/10.1007/978-1-4939-9717-6_9
    7. Benjamin Caballero, Yipeng Wang, Antonio Diaz, Inmaculada Tasset, Yves Robert Juste, Barbara Stiller, Eva‐Maria Mandelkow, Eckhard Mandelkow, Ana Maria Cuervo. Interplay of pathogenic forms of human tau with different autophagic pathways. Aging Cell 2018, 17 (1) https://doi.org/10.1111/acel.12692
    8. Jin Wang, Ye Yu. Insights into the channel gating of P2X receptors from structures, dynamics and small molecules. Acta Pharmacologica Sinica 2016, 37 (1) , 44-55. https://doi.org/10.1038/aps.2015.127
    9. Jane A. Cox, Angela LaMora, Stephen L. Johnson, Mark M. Voigt. Novel role for carbamoyl phosphate synthetase 2 in cranial sensory circuit formation. International Journal of Developmental Neuroscience 2014, 33 (1) , 41-48. https://doi.org/10.1016/j.ijdevneu.2013.11.003
    10. Karina Kaczmarek-Hájek, Éva Lörinczi, Ralf Hausmann, Annette Nicke. Molecular and functional properties of P2X receptors—recent progress and persisting challenges. Purinergic Signalling 2012, 8 (3) , 375-417. https://doi.org/10.1007/s11302-012-9314-7
    11. Marie Jindrichova, Pavlo Kuzyk, Shuo Li, Stanko S. Stojilkovic, Hana Zemkova. Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking. Purinergic Signalling 2012, 8 (2) , 317-325. https://doi.org/10.1007/s11302-012-9291-x
    12. Liam E. Browne. Structure of P2X receptors. Wiley Interdisciplinary Reviews: Membrane Transport and Signaling 2012, 1 (1) , 56-69. https://doi.org/10.1002/wmts.24
    13. Betsy Navarro, Kiyoshi Miki, David E. Clapham. ATP-activated P2X2 current in mouse spermatozoa. Proceedings of the National Academy of Sciences 2011, 108 (34) , 14342-14347. https://doi.org/10.1073/pnas.1111695108
    14. F. Vacca, N. D'Ambrosi, V. Nestola, S. Amadio, M. Giustizieri, M. L. Cucchiaroni, A. Tozzi, M. C. Velluz, N. B. Mercuri, C. Volonte. N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP. Glycobiology 2011, 21 (5) , 634-643. https://doi.org/10.1093/glycob/cwq211
    15. Richard J Evans. Structural interpretation of P2X receptor mutagenesis studies on drug action. British Journal of Pharmacology 2010, 161 (5) , 961-971. https://doi.org/10.1111/j.1476-5381.2010.00728.x
    16. Sebastian Kracun, Vincent Chaptal, Jeff Abramson, Baljit S. Khakh. Gated Access to the Pore of a P2X Receptor. Journal of Biological Chemistry 2010, 285 (13) , 10110-10121. https://doi.org/10.1074/jbc.M109.089185
    17. Kerstin Wirkner, Doychin Stanchev, Doreen Milius, Lars Hartmann, Erzsebet Kato, Zoltan S. Zadori, Peter P. Mager, Patrizia Rubini, Wolfgang Nörenberg, Peter Illes. Regulation of the pH sensitivity of human P2X 3 receptors by N‐linked glycosylation. Journal of Neurochemistry 2008, 107 (5) , 1216-1224. https://doi.org/10.1111/j.1471-4159.2008.05655.x
    18. Mark T. Young, Yi-Hong Zhang, Lishuang Cao, Helen Broomhead, Lin-Hua Jiang. Role of the domain encompassing Arg304–Ile328 in rat P2X2 receptor conformation revealed by alterations in complex glycosylation at Asn298. Biochemical Journal 2008, 416 (1) , 137-143. https://doi.org/10.1042/BJ20081182
    19. Guillaume Guerlet, Antoine Taly, Lia Prado de Carvalho, Adeline Martz, Ruotian Jiang, Alexandre Specht, Nicolas Le Novère, Thomas Grutter. Comparative models of P2X2 receptor support inter-subunit ATP-binding sites. Biochemical and Biophysical Research Communications 2008, 375 (3) , 405-409. https://doi.org/10.1016/j.bbrc.2008.08.030
    20. Daniel I. Levy, Steve A.N. Goldstein. The Effects of Electrolyte Disorders on Excitable Membranes. 2008, 1407-1427. https://doi.org/10.1016/B978-012088488-9.50053-X
    21. Doreen Milius, Helke Gröger-Arndt, Doychin Stanchev, Christine Lange-Dohna, Steffen Rossner, Beata Sperlagh, Kerstin Wirkner, Peter Illes. Oxygen/glucose deprivation increases the integration of recombinant P2X7 receptors into the plasma membrane of HEK293 cells. Toxicology 2007, 238 (1) , 60-69. https://doi.org/10.1016/j.tox.2007.05.028
    22. K. Barth, K. Weinhold, A. Guenther, M. T. Young, H. Schnittler, M. Kasper. Caveolin‐1 influences P2X 7 receptor expression and localization in mouse lung alveolar epithelial cells. The FEBS Journal 2007, 274 (12) , 3021-3033. https://doi.org/10.1111/j.1742-4658.2007.05830.x
    23. V.H.J. Roberts, R.P. Webster, D.E. Brockman, B.A. Pitzer, L. Myatt. Post-Translational Modifications of the P2X4 Purinergic Receptor Subtype in the Human Placenta are Altered in Preeclampsia. Placenta 2007, 28 (4) , 270-277. https://doi.org/10.1016/j.placenta.2006.04.008
    24. Mark T. Young, Pablo Pelegrin, Annmarie Surprenant. Amino Acid Residues in the P2X7 Receptor that Mediate Differential Sensitivity to ATP and BzATP. Molecular Pharmacology 2007, 71 (1) , 92-100. https://doi.org/10.1124/mol.106.030163
    25. Peter P Mager, Peter Illes. The h-P2X3 glycoprotein receptor as an example of integrating bioinformatics and structural research. Expert Opinion on Drug Discovery 2006, 1 (4) , 303-309. https://doi.org/10.1517/17460441.1.4.303
    26. Theresa Puthussery, Erica L. Fletcher. P2X 2 receptors on ganglion and amacrine cells in cone pathways of the rat retina. Journal of Comparative Neurology 2006, 496 (5) , 595-609. https://doi.org/10.1002/cne.20889
    27. Kazuhiro Mio, Yoshihiro Kubo, Toshihiko Ogura, Tomomi Yamamoto, Chikara Sato. Visualization of the trimeric P2X2 receptor with a crown-capped extracellular domain. Biochemical and Biophysical Research Communications 2005, 337 (3) , 998-1005. https://doi.org/10.1016/j.bbrc.2005.09.141
    28. Joanna Napp, Francisco Monje, Walter Stühmer, Luis A. Pardo. Glycosylation of Eag1 (Kv10.1) Potassium Channels. Journal of Biological Chemistry 2005, 280 (33) , 29506-29512. https://doi.org/10.1074/jbc.M504228200
    29. Liqin Wang, Ying-Hong Feng, George I. Gorodeski. Epidermal Growth Factor Facilitates Epinephrine Inhibition of P2X7-Receptor-Mediated Pore Formation and Apoptosis: A Novel Signaling Network. Endocrinology 2005, 146 (1) , 164-174. https://doi.org/10.1210/en.2004-1026
    30. Séverine Chaumont, Lin-Hua Jiang, Aubin Penna, R. Alan North, Francois Rassendren. Identification of a Trafficking Motif Involved in the Stabilization and Polarization of P2X Receptors. Journal of Biological Chemistry 2004, 279 (28) , 29628-29638. https://doi.org/10.1074/jbc.M403940200
    31. Clare A Jones, Catherine Vial, Lynda A Sellers, Pat P. A Humphrey, Richard J Evans, Iain P Chessell. Functional Regulation of P2X6 Receptors by N-Linked Glycosylation: Identification of a Novel αβ-Methylene ATP-Sensitive Phenotype. Molecular Pharmacology 2004, 65 (4) , 979-985. https://doi.org/10.1124/mol.65.4.979
    32. Xiaotian Zhong, Ron Kriz, Jasbir Seehra, Ravindra Kumar. N‐linked glycosylation of platelet P 2 Y 12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression. FEBS Letters 2004, 562 (1-3) , 111-117. https://doi.org/10.1016/S0014-5793(04)00191-7
    33. Peter Illes, J. Alexandre Ribeiro. Molecular physiology of P2 receptors in the central nervous system. European Journal of Pharmacology 2004, 483 (1) , 5-17. https://doi.org/10.1016/j.ejphar.2003.10.030
    34. Sylvia O. Suadicani, Mara Helena De Pina‐Benabou, Marcia Urban‐Maldonado, David C. Spray, Eliana Scemes. Acute downregulation of Cx43 alters P2Y receptor expression levels in mouse spinal cord astrocytes. Glia 2003, 42 (2) , 160-171. https://doi.org/10.1002/glia.10197
    35. Amanda Taylor Boyce, Erik M Schwiebert. Extracellular ATP-Gated P2X Purinergic Receptor Channels. 2003, 97-150. https://doi.org/10.1016/S1063-5823(03)01004-4
    36. Lisa R. Conti, Carolyn M. Radeke, Carol A. Vandenberg. Membrane Targeting of ATP-sensitive Potassium Channel. Journal of Biological Chemistry 2002, 277 (28) , 25416-25422. https://doi.org/10.1074/jbc.M203109200
    37. Qiuming Gong, Corey L. Anderson, Craig T. January, Zhengfeng Zhou. Role of glycosylation in cell surface expression and stability of HERG potassium channels. American Journal of Physiology-Heart and Circulatory Physiology 2002, 283 (1) , H77-H84. https://doi.org/10.1152/ajpheart.00008.2002
    38. Bing Hu, Carol Senkler, Alexander Yang, Florentina Soto, Bruce T. Liang. P2X4 Receptor Is a Glycosylated Cardiac Receptor Mediating a Positive Inotropic Response to ATP. Journal of Biological Chemistry 2002, 277 (18) , 15752-15757. https://doi.org/10.1074/jbc.M112097200
    39. R. Alan North. Molecular Physiology of P2X Receptors. Physiological Reviews 2002, 82 (4) , 1013-1067. https://doi.org/10.1152/physrev.00015.2002
    40. Nicholas J. Greco, Giovanni Tonon, Weidong Chen, Xunyi Luo, Rakhi Dalal, G. A. Jamieson. Novel structurally altered P2X1 receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells. Blood 2001, 98 (1) , 100-107. https://doi.org/10.1182/blood.V98.1.100
    41. Miran Kim, Valeria Spelta, Joan Sim, R. Alan North, Annmarie Surprenant. Differential Assembly of Rat Purinergic P2X7 Receptor in Immune Cells of the Brain and Periphery. Journal of Biological Chemistry 2001, 276 (26) , 23262-23267. https://doi.org/10.1074/jbc.M102253200
    42. Guy Vassort. Adenosine 5′-Triphosphate: a P2-Purinergic Agonist in the Myocardium. Physiological Reviews 2001, 81 (2) , 767-806. https://doi.org/10.1152/physrev.2001.81.2.767
    43. Baljit S. Khakh. Molecular physiology of p2x receptors and atp signalling at synapses. Nature Reviews Neuroscience 2001, 2 (3) , 165-174. https://doi.org/10.1038/35058521
    44. Baljit S. Khakh, Geoffrey Burnstock, Charles Kennedy, Brian F. King, R. Alan North, Philippe Séguéla, Mark Voigt, Patrick P.A. Humphrey. International Union of Pharmacology. XXIV. Current Status of the Nomenclature and Properties of P2X Receptors and Their Subunits. Pharmacological Reviews 2001, 53 (1) , 107-118. https://doi.org/10.1016/S0031-6997(24)01482-0
    45. Maria E. Rubio, Florentina Soto. Distinct Localization of P2X Receptors at Excitatory Postsynaptic Specializations. The Journal of Neuroscience 2001, 21 (2) , 641-653. https://doi.org/10.1523/JNEUROSCI.21-02-00641.2001
    46. Mary C. Ryan, Lucia Notterpek, Andreas R. Tobler, Ning Liu, Eric M. Shooter. Role of the Peripheral Myelin Protein 22 N‐Linked Glycan in Oligomer Stability. Journal of Neurochemistry 2000, 75 (4) , 1465-1474. https://doi.org/10.1046/j.1471-4159.2000.0751465.x
    47. Jürgen Rettinger, Armaz Aschrafi, Günther Schmalzing. Roles of Individual N-Glycans for ATP Potency and Expression of the Rat P2X1 Receptor. Journal of Biological Chemistry 2000, 275 (43) , 33542-33547. https://doi.org/10.1074/jbc.M002918200
    48. Liping Chen, James P. Hardwick, Peter McPhie, Michail V. Sitkovsky, Kenneth A. Jacobson. Purification and recognition of recombinant mouse P2X1 receptors expressed in a baculovirus system. Drug Development Research 2000, 51 (1) , 7-19. https://doi.org/10.1002/1098-2299(20000901)51:1<7::AID-DDR2>3.0.CO;2-W
    49. C. E. Clarke, C. D. Benham, A. Bridges, A. R. George, H. J. Meadows. Mutation of histidine 286 of the human P2X 4 purinoceptor removes extracellular pH sensitivity. The Journal of Physiology 2000, 523 (3) , 697-703. https://doi.org/10.1111/j.1469-7793.2000.00697.x
    50. GATTADAHALLI S. SEETHARAMAIAH, JUN ZHUANG, JUAN HUANG, SAI A. PATIBANDLA, SHASHI KAITHAMANA, KAZUO TAHARA, LEONARD D. KOHN, BELLUR S. PRABHAKAR. Selective Binding of Thyrotropin Receptor Autoantibodies to Recombinant Extracellular Domain of Thyrotropin/Lutropin-Chorionic Gonadotropin Receptor Chimeric Proteins. Thyroid 1999, 9 (9) , 879-886. https://doi.org/10.1089/thy.1999.9.879
    51. Baljit S Khakh, Henry A Lester. Dynamic Selectivity Filters in Ion Channels. Neuron 1999, 23 (4) , 653-658. https://doi.org/10.1016/S0896-6273(01)80025-8
    Download PDF
    Go to Biochemistry
    Get e-Alerts
    Get e-Alerts

    Biochemistry

    Cite this: Biochemistry 1998, 37, 42, 14845–14851
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi981209g
    Published September 29, 1998
    Copyright © 1998 American Chemical Society
    Request reuse permissions

    Article Views

    436

    Altmetric

    -

    Citations

    51
    Learn about these metrics

    Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

    Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

    The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.