Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions

• Published in: International journal of molecular sciences Vol. 21; no. 14; p. 5101
• Main Authors: Morise, Jyoji, Yamamoto, Saki, Midorikawa, Ryosuke, Takamiya, Kogo, Nonaka, Motohiro, Takematsu, Hiromu, Oka, Shogo
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.07.2020; MDPI
• Subjects: Amino Acid Substitution; AMPA-type glutamate receptor; Binding Sites - genetics; Cell Membrane - metabolism; Cell membranes; Cell surface; Gene Expression; GluA1; GluA2; Glutamic acid receptors; Glycan; Glycosylation; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Mutants; Mutation; N-glycan; N-glycans; Polysaccharides; Protein Structure, Quaternary; Receptors, Glutamate - chemistry; Receptors, Glutamate - genetics; Receptors, Glutamate - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors; GluA2; N-glycan; GluA1; AMPA-type glutamate receptor
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21145101

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.