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Identification and importance of N-glycosylation of the human 5-hydroxytryptamine3A receptor subunit.
Monk, Sarah A; Williams, Julie M; Hope, Anthony G; Barnes, Nicholas M.
Affiliation
  • Monk SA; Cellular and Molecular Neuropharmacology Research Group, Department of Pharmacology, Division of Neuroscience, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Biochem Pharmacol ; 68(9): 1787-96, 2004 Nov 01.
Article in En | MEDLINE | ID: mdl-15450944
ABSTRACT
We investigated the presence and potential role of N-glycosylation of the human (h) 5-hydroxytryptamine3 (5-HT3A) receptor subunit expressed in COS-7 cells. Incubation of cells with the N-glycosylation inhibitor, tunicamycin, reduced the molecular weight of the predominant immunoreactive h5-HT3A subunit species (from approximately 59 to 45 kDa) indicating that the h5-HT3A subunit is normally N-glycosylated. Site-directed mutagenesis studies individually substituting four identified N-terminal asparagines (N5, N81, N147, N163) demonstrated that each expressed mutant displayed a reduced molecular weight (by approximately 3 kDa) suggesting that each asparagine residue was subject to N-glycosylation. In addition, 5-HT3 receptor binding studies indicated that prevention of N-glycosylation, by individual amino acid substitution at each of the four asparagine residues, either prevented (N81, N147, N163) or greatly reduced (N5) the production of a 5-HT3 receptor binding site. Corresponding with the radioligand binding studies, immunocytochemical studies demonstrated that substitution of each asparagine either prevented (N81, N147, N163) or reduced considerably (N5) mutant protein expression within the cell membrane. The present study demonstrates an important role for N-glycosylation at multiple identified asparagine residues in the N-terminus of the h5-HT3A receptor subunit.
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Collection: 01-internacional Database: MEDLINE Main subject: Protein Subunits / Receptors, Serotonin, 5-HT3 Type of study: Diagnostic_studies Limits: Animals / Humans Language: En Journal: Biochem Pharmacol Year: 2004 Document type: Article Affiliation country: United kingdom Publication country: ENGLAND / ESCOCIA / GB / GREAT BRITAIN / INGLATERRA / REINO UNIDO / SCOTLAND / UK / UNITED KINGDOM
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Collection: 01-internacional Database: MEDLINE Main subject: Protein Subunits / Receptors, Serotonin, 5-HT3 Type of study: Diagnostic_studies Limits: Animals / Humans Language: En Journal: Biochem Pharmacol Year: 2004 Document type: Article Affiliation country: United kingdom Publication country: ENGLAND / ESCOCIA / GB / GREAT BRITAIN / INGLATERRA / REINO UNIDO / SCOTLAND / UK / UNITED KINGDOM