Identification and functional significance of N-glycosylation of the 5-ht5A receptor.
The presence and roles of N-glycosylation of the human (h) 5-ht(5A) receptor were investigated using a heterologous expression system. Following transient transfection of COS-7 cells with h5-ht(5A) receptor cDNA, SDS-PAGE/Western blot analysis of immunoreactivity demonstrated two protein species; a predominant species with a molecular weight of approximately 35-45 kDa and a minor species of approximately 45-55 kDa. Transfected cells grown in the presence of the N-glycosylation inhibitor tunicamycin, failed to express the minor immunoreactive species indicating this represented the N-glycosylated form of the h5-ht(5A) receptor. Comparison of the molecular weights of immunoreactive bands arising from the wild-type and each of the mutant 5-ht(5A) receptors with disruption of the predicted N-glycosylation sites (N6S and N21S) demonstrated that both identified asparagines were N-glycosylated. Immunocytochemical and ELISA studies demonstrated that the [N6S]h5-ht(5A) receptor mutation, but not the [N21S]h5-ht(5A) receptor mutation, reduced protein expression in the cell membrane, indicating that N-glycosylation of the N6 residue is important for the membrane expression of this neurotransmitter receptor; a requirement for receptor function.
Dutton
Alice C
AC
Cellular and Molecular Neuropharmacology Research Group, Division of Neuroscience, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Massoura
Andrew N
AN
Dover
Terri J
TJ
Andrews
Nick A
NA
Barnes
Nicholas M
NM
D000818:Animals; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*; D009435:Synaptic Transmission / Q000235:genetics; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D020033:Protein Isoforms; D011985:Receptors, Serotonin; C078730:serotonin 5 receptor; D014415:Tunicamycin; D012701:Serotonin; D009584:Nitrogen