The identification of N-glycosylated residues of the human 5-HT3B receptor subunit: importance for cell membrane expression.
The 5-hydroxytryptamine 3 (5-HT(3)) receptor is a pentameric ligand-gated ion channel with potential molecular isoforms arising from different subunit combinations and/or different post-translational modifications of the individual subunits. Since N-glycosylation of the 5-HT3A subunit impacts cell surface trafficking, the presence of N-glycosylation of the human (h) 5-HT3B subunit and the influence upon cell membrane expression was investigated. Following transient expression of the h5-HT3B subunit by human embryonic kidney cells (HEK293 cells) stably expressing the h5-HT3A subunit, the N-glycosylation inhibitor tunicamycin reduced the size of the predominant h5-HT3B-immunoreactive protein (∼ 55 kDa reduced to ∼ 40 kDa). Disruption of each consensus N-glycosylation sequences in the h5-HT3B subunit (N31S, N75S, N117S, N147S and N182S) resulted in a reduced molecular weight (by ∼ 2-4 kDa) of each mutant when expressed by HEK293 cells stably expressing the h5-HT3A subunit. Immunocytochemical studies demonstrated that disruption of each of the N-glycosylation sequences (individually or combined) reduced the expression of the mutant h5-HT3B subunit protein in the cell membrane when co-expressed with the h5-HT3A subunit. The present study has identified utilised N-glycosylation sites of the h5-HT3B subunit and demonstrated that they promote subunit expression in the cell membrane; a prerequisite for 5-HT(3) receptor function.
Neuropharmacology and Neurobiology Section, Clinical and Experimental Medicine, The Medical School, University of Birmingham, Birmingham, UK.
Andrew N
AN
Massoura
Terri J
TJ
Dover
Amy S
AS
Newman
Nicholas M
NM
Barnes
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006031:Glycosylation / Q000187:drug effects; D057809:HEK293 Cells / Q000166:cytology; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein / Q000379:methods; D014162:Transfection / Q000379:methods
D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase