Structure and function in rhodopsin: the role of asparagine-linked glycosylation.
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. 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 non-glycosylated opsin folded correctly as shown by its normal palmitoylation, transport to the cell surface, and the formation of the characteristic rhodopsin chromophore (lambda max, 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.
Kaushal
S
S
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Ridge
K D
KD
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D002417:Cattle; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D012243:Rhodopsin / Q000737:chemistry* / Q000502:physiology*; D017299:Rod Opsins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism; D014162:Transfection
D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D017299:Rod Opsins; D001216:Asparagine; D012243:Rhodopsin; D015293:Transducin