The missense mutation Lys-296 --Glu (K296E) in the rhodopsin gene produces an opsin with no chromophore binding site and therefore is not activated by light. Nevertheless, the mutant opsin constitutively activates transducin in vitro and causes photoreceptor degeneration in vivo, possibly by continuously activating the phototransduction cascade, analogous to constant exposure to environmental light. We studied the K296E mutation in eight lines of transgenic mice. Each line developed photoreceptor degeneration with the rate of degeneration increasing monotonically as the ratio of mutant:wild-type opsin mRNA increased. At no time in the course of degeneration was there endogenous light adaptation in the retina as measured by the electroretinogram. The mutant opsin was found to be invariably phosphorylated and stably bound to arrestin. Light-independent activation of transducin was demonstrated only after the removal of arrestin and dephosphorylation of K296E opsin. Thus, K296E opsin in vivo does not activate the phototransduction cascade because it is shut off by photoreceptor inactivation mechanisms. Our data show that the K296E mutation does not cause photoreceptor degeneration by continuous activation of phototransduction.Over 60 different mutations in the rhodopsin gene have been found to cause autosomal dominant retinitis pigmentosa (RP), a progressive degeneration of the neural retina that typically leads to blindness in middle age. The mechanisms by which any of these mutant alleles leads to degeneration of the rod and cone photoreceptors remain obscure. One testable hypothesis (1) is that the mutant opsins continuously activate transducin, resulting in a pathogenic overstimulation of photoreceptors. Support for this hypothesis comes from several observations. Constant exposure to light damages photoreceptor cells (2, 3). Many rhodopsin mutants regenerate poorly with the chromophore, 11-cis-retinal. Opsin, the apoprotein without the chromophore, was reported to activate phototransduction without light [albeit at six orders of magnitude lower efficiency compared to photo-activated metarhodopsin II (4)]. Thus defective regeneration might lead to a pool of weakly active opsin molecules in the photoreceptors. In addition, a rhodopsin mutant, K296E, that causes RP (5, 24) has been found to activate transducin constitutively in vitro (6). To gain insight into the consequences of a constitutively active mutant in vivo, we generated transgenic mice bearing the K296E (Lys-296 -> Glu) mutation. We examined the characteristics of retinal degeneration in these mice and analyzed the mutant opsin from the transgenic mouse retinas. MATERIALS AND METHODSConstruction of Transgene and Generation of Transgenic Mice. The K296E transgene was generated by site-directedThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. mutagenesis on a genomic DNA fragment encompass...
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