Mutant rhodopsin transgene expression on a null background.

PURPOSE: To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation. METHODS: Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG). RESULTS: Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS: GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.

Characterization of human GRK7 as a potential cone opsin kinase.

PURPOSE: Homozygous inactivation of the mouse gene for GRK1 (G protein-coupled receptor kinase 1, or rhodopsin kinase) causes severe defects in the recovery of cone phototransduction. However, electroretinographic (ERG) analyses of human oguchi patients with defective GRK1 alleles showed normal or slightly abnormal photopic responses. It remains unclear why the loss of GRK1 yields such different phenotypes in the recovery of mouse and human cones. We examined the localization and enzyme activity of GRK7, the human ortholog of the seventh member of the GRK family, in an attempt to understand its potential role in photopic vision. METHODS: Bioinformatic approaches were used to identify the human GRK7 gene. Human and bovine GRK7 cDNAs were isolated by RT-PCR. Recombinant GRK7, expressed in insect cells, was used to phosphorylate activated rhodopsin. Antibodies raised against GRK7 peptides were used to examine the retina specific expression of GRK7 by immunoblotting and its subcellular localization by immunocytochemistry. RESULTS: The human GRK7 gene is located on chromosome 3q21, spans at least 10 Kb and consists of 4 exons. In human, GRK7 is expressed exclusively in the retina and is found in all retinal neurons, and specifically, in cone outer segments. Recombinant human GRK7 catalyzes rhodopsin phosphorylation in a light dependent manner. We provide evidence that GRK1 and GRK7 are co-expressed in human cones. In contrast, mouse GRK7 is expressed in many tissues including retina where photoreceptors apparently do not express GRK7. CONCLUSIONS: The presence of GRK7 in human, but not in mouse, cone outer segments suggests that GRK7 may function to provide the normal photopic vision reported by oguchi patients with a defective GRK1 gene. The absence of GRK7 expression in cone outer segments of mice is consistent with the notion that mouse cones rely solely on GRK1 to shutoff cone visual pigments.