Identification and importance of N-glycosylation of the human 5-hydroxytryptamine3A receptor subunit

Abstract We investigated the presence and potential role of N -glycosylation of the human (h) 5-hydroxytryptamine 3 (5-HT 3A ) 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-HT 3A subunit species (from ≈59 to 45 kDa) indicating that the h5-HT 3A 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 ≈3 kDa) suggesting that each asparagine residue was subject to N -glycosylation. In addition, 5-HT 3 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-HT 3 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-HT 3A receptor subunit.