Structure and function in rhodopsin: The role of asparagine-linked glycosylation (membrane protein/signal transduction/carbohydrate/structure-function relationship)

Rhodopsin, the dim light photoreceptor of the 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. Nongly- cosylated opsin was prepared by expression of a wild-type bovine opsin gene in COS-1 cells in the presence of tunicamy- cin, an inhibitor of asparagine-linked glycosylation. The non- glycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (Amax, 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 significant 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 GT very poorly. We conclude that Asn-15 glycosylation is important in signal transduction.