title: Biochemical and pharmacological study of N-linked glycosylation of the human serotonin 5-HT₇a receptor.
abstract: The 5-hydroxytryptamine (5-HT)(7(a))  receptor is a G-protein-coupled receptor critically involved in human psychiatric and neurological disorders. In the present study, we evaluate the presence and the functional role of N-glycosylation of the human 5-HT(7)  receptor. Western blot analysis of HEK293T cells transiently expressing the 5-HT(7(a))  receptor in the presence of tunicamycin gave rise to a band shift, indicating the existence of an N-glycosylated form of the 5-HT(7(a))  receptor. To further investigate this, we mutated the two predicted N-glycosylation sites (N5Q and N66Q) and compared the molecular mass of the immunoreactive bands with those of the wild-type receptor, indicating that both asparagines were N-glycosylated. The mutant receptors had the same binding affinity for [(3) H]5-CT and the same potency and efficacy with regard to 5-HT-induced activation of adenylyl cyclase. However, there was a reduction in maximal ligand binding for the single and double mutants compared to the wild-type receptor. Next, membrane labelling and immunocytochemical studies demonstrated that the N-glycosylation mutants were expressed at the cell surface. We conclude that N-glycosylation is not important for cell surface expression of the 5-HT(7)  receptor.
authors.affiliation: Laboratory for Eukaryotic Gene Expression and Signal Transduction (LEGEST), Ghent University, Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Gellynck
authors.forename: Kjetil W
authors.initials: KW
authors.lastname: Andressen
authors.forename: Béatrice
authors.initials: B
authors.lastname: Lintermans
authors.forename: Guy
authors.initials: G
authors.lastname: Haegeman
authors.forename: Finn O
authors.initials: FO
authors.lastname: Levy
authors.forename: Peter
authors.initials: P
authors.lastname: Vanhoenacker
authors.forename: Kathleen
authors.initials: K
authors.lastname: Van Craenenbroeck
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011869:Radioligand Assay; D011985:Receptors, Serotonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000031:analogs & derivatives / Q000378:metabolism; D014162:Transfection
chemical_list: D011985:Receptors, Serotonin; C104425:serotonin 7 receptor; D005973:Glutamine; D012701:Serotonin; D001216:Asparagine; C047087:5-carboxamidotryptamine; D000262:Adenylyl Cyclases