N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor.
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.
Department of Pharmacological and Physiological Sciences, St. Louis University Health Sciences Center, Missouri 63104, USA.
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D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D016384:Consensus Sequence / Q000235:genetics; D004594:Electrophysiology; D019085:Fluorescent Antibody Technique, Direct; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011233:Precipitin Tests; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D009842:Oligopeptides; D010455:Peptides; D018048:Receptors, Purinergic P2; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D001216:Asparagine; C085706:FLAG peptide