CHARACTERIZATION OF MUTANT RHODOPSINS RESPONSIBLE FOR AUTOSOMAL-DOMINANT RETINITIS-PIGMENTOSA - MUTATIONS ON THE CYTOPLASMIC SURFACE AFFECTTRANSDUCIN ACTIVATION

Rhodopsin mutants responsible for autosomal dominant retinitis pigment osa (ADRP) were prepared by site-directed mutagenesis and characterize d. The aim was to evaluate ADRP mutations that occur at three location s 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 cyt oplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It wa s 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 deleti on (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 co mplementary structure-function information. The four-amino acid deleti on mutant failed to bind 11-cis-retinal. However, each of the Thr-58 a nd Arg- 135 mutants bound 11-cis-retinal to form a pigment with a visi ble absorbance maximum (lambda(max)) of 500 nm. Upon illumination, eac h pigment was converted to a metarhodopsin II-like spectral form (lamb da(max) = 380 nm). However, each of these spectrally normal ADRP mutan ts was defective in activating guanine nucleotide exchange by transduc in. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.