Cysteine residues 110 and 187 are essentialforthe formation of correctstructureinbovine rhodopsin (heterologousexpression/glycosylation/transmembranereceptor/site -specificmutagenesis/synthetic gene)

To investigatethe role of differentcysteine residuesin bovine rhodopsin, a seriesofmutants were prepared in which the cysteine residues were systematicallyreplaced by serines. The mutant genes were expressed in monkey kidney cells(COS -1) and the mutant opsins were evaluated for their levelsof expression, glycosylationpatterns,and abilityto form the chromophore characteristicof rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane- embedded cysteines(Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutionsof Cys-185 gave risetoa wild-type phenotype . In contrast, substitutionof the three intradiscalcysteines(Cys- 110, Cys-185, and Cys-187) or singlesubstitutionofCys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal . Thus, of the 10 cysteinesin bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essentialfor the correct tertiary structure of the protein.