N-Glycosylation Is Not a Prerequisite for Glutamate Receptor Function but Is Essential for Lectin Modulation

Abstract

SUMMARYAll ionotropic glutamate receptor (iGluR) subunits analyzed so far are heavily N-glycosylated at multiple sites on their aminoterminal extracellular domains. Although the exact functional significance of this glycosylation remains to be determined, it has been suggested that N-glycosylation may be a precondition for the formation of functional ion channels. In particular, it has been argued that N-glycosylation is required for the formation of functional ligand binding sites. We analyzed heterologously expressed recombinant glutamate receptors (GluRs) of all three pharmacological subclasses of glutamate receptors, N-methyl-D-aspartate (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and kainate receptors. By expressing the GluR subunits in tunicamycin-treated, nonglycosylating Xenopus laevis oocytes, we determined that in neither case is N-glycosylation required for ion channel function, although for NMDA receptors, functional expression in the absence of Nglycosylation is very low. Furthermore, we analyzed and compared the interaction of the desensitization-inhibiting lectin concanavalin A (ConA) with all functional GluR subunits. We show that although ConA has its most pronounced effects on kainate receptors, it potentiates currents at most other receptor subtypes as well, including certain NMDA receptor subunits, although to a much lesser extent. One notable exception is the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor GluR2, which is not affected by ConA. Furthermore, we show that ConA acts directly via binding to the carbohydrate side chains of the receptor protein.iGluRs are the prevalent excitatory neurotransmitter receptors in the central nervous system of vertebrates (1). They can be classified into three major subfamilies on the basis of pharmacological and electrophysiological profiles: NMDA, AMPA, and KA receptors. Being the main mediators of cellto-cell signaling, GluRs are regulated and functionally modulated by a multitude of post-transcriptional and post-translational mechanisms such as alternative splicing, RNA editing, protein phosphorylation, palmitoylation, and N-glycosylation (for a review, see Ref.2). iGluRs are thought to consist of five subunits and may form heteromeric receptors containing different subunits, in many cases resulting in a receptor complex with different properties than their constituent subunits. For some receptor subunits, function can be demonstrated only on coexpression with another subunit of the same subfamily (for a review, see Ref. 2).The amino acid sequences of iGluR subunits contain 4 -12 potential extracellular sites for N-glycosylation, which conform to the universal consensus sequence N-X-S/T, with X P. These sites are marked in Fig. 1 and occur in the two large domains of iGluRs that according to the recently proposed three-transmembrane domain model, are located in the two putatively extracellular domains, the amino terminus and the loop between transmembrane domains B and C (3, 4). Many but not all of the sites are con...

Subunit Composition and Alternative Splicing Regulate Membrane Delivery of Kainate Receptors

“…2C), suggesting that incompletely glycosylated GluR5 subunits might have trafficked to the plasma membrane. In keeping with this suggestion, glycosylation is not necessary for the proper function and expression of KARs in oocytes (Everts et al, 1997). Alternatively, GluR5 isoforms might exist under other different post-translational states.…”

The glutamate receptor subunit delta2 is capable of gating its intrinsic ion channel as revealed by ligand binding domain transplantation

“…AMPA does not activate GluR6 receptors (20), and in contrast to GluR1, GluR6 is most sensitive to DA (21). In addition, KA receptor desensitization is potently inhibited by treatment with the lectin Concanavalin A (ConA), while the effect on GluR1 is only moderate (22). We find that after ConA treatment of oocytes Author contributions: S.M.S.…”

The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration

“…The abundance of NMDA-R NR1 subunit in Schwann cell extracts was somewhat lower than in extracts of DRGs and PC12 cells. The mobility of the NR1 subunit band in Schwann cell extracts was slightly increased compared with the controls, probably reflecting differential glycosylation of this highly glycosylated gene product (Everts et al, 1997;Lichnerova et al, 2015).…”