Inhibition of N-linked glycosylation of the human type 1α metabotropic glutamate receptor by tunicamycin: effects on cell-surface receptor expression and function

Abstract

The potential role of N-linked glycosylation of the human type 1α metabotropic glutamate (mGlu1α) receptor was studied in a recombinant, inducible expression system, where receptor expression was induced in the absence and presence of tunicamycin. In the absence of tunicamycin the mGlu1α receptor appeared to be expressed, at least in part, as a dimer consisting of monomers of approx. 145 and 160 KDa relative molecular mass (M_r). In the presence of tunicamycin only a single monomeric protein could be detected approximating the M_r predicted for the human mGlu1α receptor based on its primary amino acid sequence (130 KDa). Exposure to tunicamycin during receptor induction did not appear to affect the cell surface expression of the mGlu1α receptor as determined immunocytochemically or using a cell-surface biotinylation strategy, but reduced agonist-stimulated phosphoinositide hydrolysis by approximately 50% compared to control cell populations. Our data suggest that non-N-glycosylated human mGlu1α receptors can traffic to the cell surface and activate phospholipase C.

Introduction

Metabotropic glutamate (mGlu) receptors, together with GABA_B, Ca²⁺-sensing, and a subset of pheromone receptors, are G protein-coupled receptors (GPCRs) which possess unique regulatory and structural features, making them only distant relatives of the other members of the GPCR superfamily (Conn and Pin, 1997, Bockaert and Pin, 1999). These so-called family 3 GPCRs share some key structural features. Thus, in addition to a 7-transmembrane domain topology, each receptor possesses a large N-terminal extracellular domain, which often comprises as much as 50% of the entire amino acid sequence, and has either been shown, or is hypothesised, to contain the ligand-binding site (Brown et al., 1993, O’Hara et al., 1993).

Family 3 receptors also exhibit other commonalities; for example, either homo-, or hetero-dimerization has been shown to be important for both receptor expression and function (Romano et al., 1996, Bai et al., 1998, White et al., 1998). In common with the vast majority of GPCRs, family 3 receptors possess consensus sites for N-linked glycosylation within the extracellular domain(s) (Masu et al., 1991, Brown et al., 1993, White et al., 1998), although few studies have addressed which of the potential sites are glycosylated and the importance of such post-translational modification for receptor expression and coupling to cell signalling pathways. Very recently, Spiegel and colleagues have performed an extensive investigation exploring the potential role(s) of N-linked glycosylation of the human Ca²⁺-sensing receptor (Ray et al., 1998). These workers found evidence for extensive N-linked glycosylation of the receptor and a correlation between glycosylation and receptor cell-surface expression (Ray et al., 1998).

The human mGlu1α receptor possesses four putative N-linked glycosylation sites in the N-terminal domain (Desai et al., 1995). Although Western blot analyses have provided evidence for post-translational modification of this receptor (Alaluf et al., 1995a, Alaluf et al., 1995b, Carruthers et al., 1997, Hermans et al., 1998), surprisingly little is presently known about the significance of such modification. In the present study we have investigated the potential role of N-linked glycosylation of the human mGlu1α receptor in an inducible expression system (Hermans et al., 1998, Hermans et al., 1999), where the induction of mGlu1α receptor expression was performed in the absence or presence of tunicamycin, an inhibitor of the N-linked glycosylation of proteins.