Structure and function in rhodopsin: the role of asparagine-linked glycosylation.

Abstract

Rhodopsin, the dim light photoreceptor ofthe rod cell, is an integral membrane protein that is glycosylated at Asn-2 and Asn-15. Here we report experiments on the role of the glycosylation in rhodopsin folding and function. Nonglycosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamycin, an inhibitor of asparagine-linked glycosylation. The nonglycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (Aa, 500 nm) with 11-cis-retinal. However, the nonglycosylated rhodopsin showed strikingly low light-dependent activation of GT at concentration levels comparable with those of glycosylated rhodopsin. Amino acid replacements at positions 2 and 15 and the cognate tripeptide consensus sequence [Asn-2 -* Gln, Gly-3 -+ Cys (Pro), Thr-4 -+ Lys, Asn-15 Ala (Cys, Glu, Lys, Gln, Arg), Lys-16 -* Cys (Arg), Thr-17 Met (Val)] showed that the substitutions at Asn-2, Gly-3, and Thr-4 had no sgnificant effect on the folding, cellular transport, and/or function of rhodopsin, whereas those at Asn-15 and Lys-16 caused poor folding and were defective in transport to the cell surface.Further, mutant pigments with amino acid replacements at Asn-15 and Thr-17 activated Gr very poorly. We conclude that Asn-15 glycosylation is important in signal tanduction.Asparagine-linked (N-linked) glycosylation of membrane and secreted proteins is observed frequently, although the role that glycosylation may serve is not always evident (1-3). Bovine rhodopsin is glycosylated at Asn residues 2 and 15 (Fig. 1) by the hexasaccharide sequence Man3GlcNac3 (4, 5). We have now examined the role of N-linked glycosylation in rhodopsin folding and function. We expressed the wild-type bovine opsin gene (6) in the presence of the glycosylation inhibitor tunicamycin (TM). The resulting nonglycosylated opsin was normally palmitoylated, was transported to the cell surface, and formed the characteristic rhodopsin chromophore with 11-cisretinal. However, the nonglycosylated rhodopsin showed strikingly diminished light-dependent activation of transducin (GT) when compared with glycosylated rhodopsin. We next studied opsin mutants that contained amino acid replacements in the regions ofthe two glycosylation sites (Fig. 1). Mutations at or near Asn-2 had little effect on cell-surface expression, chromophore formation, and/or GT activation. In contrast, mutations at Asn-15 and Lys-16 resulted in opsins that were defective in cellular transport and formed little or no chromophore with il-cis-retinal. The mutations at Asn-15 and Thr-17 resulted in pigments that were defective in signal transduction. These results show that while glycosylation of rhodopsin is not required for its folding to an apparently correct ground-state structure, it is necessary for full activity in signal transduction.

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

“…Early studies by Khorana and others (50,(55)(56)(57)(58)(59) led to the hypothesis that the rhodopsin intradiscal domain plays a crucial role in maintaining proper protein folding, correct posttranslational modifications, trafficking, and 11-cis-retinal binding. Consistent with this theory, a number of point mutations that naturally occur in this region result in ADRP, an inherited human disease causing retina degradation (23, 24, 26, 60 -62).…”

“…3B), (50). Mutants capable of regenerating with 11-cisretinal formed characteristic rhodopsin-like pigments and could be purified to obtain spectral ratios (A 280 /A 500 ) between 1.6 and 1.8.…”

N-Linked Glycosylations at Asn26 and Asn114 of Human MD-2 Are Required for Toll-Like Receptor 4-Mediated Activation of NF-κB by Lipopolysaccharide

“…The other consists of biological functions. Recent reports have provided evidence that N-linked glycosylation may be required for ligand recognition or signaling (27)(28)(29)(30). However, what role N-linked glycosylation plays in ligand binding and signaling remains uncertain.…”

G Protein-Coupled Receptor Rhodopsin: A Prospectus

“…Mutant rhodopsins lacking Asn 15 -glycosylation exhibited poor folding and were defective in transport to the cell surface. They were also poor transducin activators, perhaps owing to their intrinsic instability (71). These studies have not yet been extended to mouse animal models.…”