title: N-glycan-dependent cell-surface expression of the P2Y2receptor and N-glycan-independent distribution to lipid rafts.
abstract: P2Y2receptor (P2Y2R) is a G-protein-coupled receptor (GPCR) that couples with Gαq/11 and is stimulated by ATP and UTP. P2Y2R is involved in pain, proinflammatory changes, and blood pressure control. Some GPCRs are localized in lipid rafts for interaction with other signaling molecules. In this study, we prepared N-glycan-deficient mutants by mutating the two consensus Asn residues for N-glycosylation to Gln to examine intracellular localization and association with lipid rafts. Western blotting of the wild type (WT) protein and mutants (N9Q, N13Q, N9Q/N13Q) in COS-7 cells showed that both Asn residues were glycosylated in the WT. Fluorescent microscopy analysis showed that WT, N9Q and N13Q were expressed in the endoplasmic reticulum (ER), Golgi body, and cell membrane, but N9Q/N13Q was only found in the ER. WT, N9Q and N13Q moved from the cell surface to endosomes within 15 min after UTP stimulation. WT and the N9Q/N13Q glycosylation-deficient mutant appeared in the detergent insoluble membrane fraction, lipid raft. These findings suggest that P2Y2R is localized in lipid rafts in the ER during biosynthesis, and that N-glycosylation is required for subsequent expression in the cell membrane. In the presence of epoxomicin, a proteasome inhibitor, there was a significant increase in the level of N9Q/N13Q, which suggests that N-glycan-deficient P2Y2R undergoes proteasomal degradation.
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Tetsuto
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Takahashi
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Matsuzaki
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Shohei
authors.initials: S
authors.lastname: Takeyama
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Shinpei
authors.initials: S
authors.lastname: Sato
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Sato
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kuroda
authors.affiliation: Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981-8558, Japan. Electronic address: hhigashi@tohoku-mpu.ac.jp.
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Higashi
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D058473:Receptors, Purinergic P2Y2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D011134:Polysaccharides; D061988:Proteasome Inhibitors; D058473:Receptors, Purinergic P2Y2; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin
keywords: Cell surface expression; ER-associated protein degradation; Lipid raft; N-linked glycosylation; P2Y(2) receptor