RHODOPSIN FROM THE FISH, ASTYANAX - ROLE OF TYROSINE-261 IN THE RED SHIFT

Purpose. To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on it s spectral tuning. Methods. The rhodopsin cDNA was cloned using revers e transcription-polymerase chain reaction amplification and then seque nced. A mutant, Y261F, was generated by site-directed mutagenesis. Bot h wild type and mutant were transiently expressed in COS-1 cells, rege nerated with 11-cis retinal, and purified by immunoaffinity chromatogr aphy. Ultraviolet-visible spectrophotometry was used to determine wave length of maximum absorption. Results. A. fasciatus rhodopsin cDNA exh ibits 80% amino acid identity with bovine rhodopsin. In contrast to al l known rhodopsins, this rhodopsin contains a tyrosine instead of a ph enylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of t he Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. Conclusions. A naturall y occurring fish rhodopsin is red shifted about 8 nm due to one critic al amino acid substitution.