Characterization of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Mutations on the cytoplasmic surface affect transducin activation.
Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It was hypothesized that amino acid changes at these sites would result in mutant rhodopsins with normal spectral properties but defects in their ability to interact with the rod outer segment G protein, transducin. A set of 12 mutant opsin genes was prepared. Four of the mutants were known to cause ADRP: Thr-58 replaced by Arg, a four-amino acid deletion (Leu-68/Arg-69/Thr-70/Pro-71), Arg-135 replaced by Leu, and Arg-135 replaced by Trp. Eight additional mutants were prepared to provide complementary structure-function information. The four-amino acid deletion mutant failed to bind 11-cis-retinal. However, each of the Thr-58 and Arg-135 mutants bound 11-cis-retinal to form a pigment with a visible absorbance maximum (lambda max) of 500 nm. Upon illumination, each pigment was converted to a metarhodopsin II-like spectral form (lambda max = 380 nm). However, each of these spectrally normal ADRP mutants was defective in activating guanine nucleotide exchange by transducin. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.
Min
K C
KC
Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
Zvyaga
T A
TA
Cypess
A M
AM
Sakmar
T P
TP
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D005799:Genes, Dominant; D005813:Genes, Synthetic; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012174:Retinitis Pigmentosa / Q000235:genetics*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D012243:Rhodopsin; D015293:Transducin