Abstract
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 ∼35–45 kDa and a minor species of ∼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.
Published Version
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