A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin.
In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007930:Leucine; D008239:Lysine; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012168:Retinal Pigments / Q000032:analysis*; D012243:Rhodopsin / Q000032:analysis* / Q000235:genetics; D013329:Structure-Activity Relationship; D015293:Transducin
D008565:Membrane Proteins; D012168:Retinal Pigments; D012243:Rhodopsin; D020558:GTP Phosphohydrolases; D015293:Transducin; D007930:Leucine; D008239:Lysine