Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors

PLoS One. 2015 Aug 13;10(8):e0135644. doi: 10.1371/journal.pone.0135644. eCollection 2015.

Abstract

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD57 Antigens / metabolism
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation*
  • Humans
  • Immunoprecipitation
  • Polysaccharides / genetics*
  • Polysaccharides / metabolism
  • Receptors, AMPA / genetics*
  • Receptors, AMPA / metabolism
  • Receptors, Glutamate / genetics*
  • Receptors, Glutamate / metabolism

Substances

  • CD57 Antigens
  • Polysaccharides
  • Receptors, AMPA
  • Receptors, Glutamate

Grant support

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 23110006 to S.O.) from MEXT of Japan and Grants-in-Aid for Scientific Research (B) (No. 26291021 to S.O.) and Scientific Research (C) (No. 24590078 to H.T.) from the Japan Society for the Promotion of Science.