The mouse Pde6d gene encodes a ubiquitous prenyl binding protein, termed PrBP/delta, of largely unknown physiological function. PrBP/delta was originally identified as a putative rod cGMP phosphodiesterase (PDE6) subunit in the retina, where it is relatively abundant. To investigate the consequences of Pde6d deletion in retina, we generated a Pde6d(-/-) mouse by targeted recombination. Although manifesting reduced body weight, the Pde6d(-/-) mouse was viable and fertile and its retina developed normally. Immunocytochemistry showed that farnesylated rhodopsin kinase (GRK1) and prenylated rod PDE6 catalytic subunits partially mislocalized in Pde6d(-/-) rods, whereas rhodopsin was unaffected. In Pde6d(-/-) rod single-cell recordings, sensitivity to single photons was increased and saturating flash responses were prolonged. Pde6d(-/-) scotopic paired-flash electroretinograms indicated a delay in recovery of the dark state, likely due to reduced levels of GRK1 in rod outer segments. In Pde6d(-/-) cone outer segments, GRK1 and cone PDE6alpha' were present at very low levels and the photopic b-wave amplitudes were reduced by 70%. Thus the absence of PrBP/delta in retina impairs transport of prenylated proteins, particularly GRK1 and cone PDE, to rod and cone outer segments, resulting in altered photoreceptor physiology and a phenotype of a slowly progressing rod/cone dystrophy.

The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH–/–) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH–/– mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.

Highly abundant short-chain alcohol dehydrogenases (RDHs) in the retina were assumed to be involved in the recycling of 11- cis -retinal chromophore in the visual cycle. Mutations in human RDH genes are associated with Fundus albipunctatus , a mild form of night blindness ( RDH5 ) and an autosomal recessive, childhood-onset severe retinal dystrophy ( RDH12 ). Rdh12 knockout mice were found to be susceptible to light-induced photoreceptor apoptosis, whereas Rdh5 and Rdh8 knockout mice displayed only delayed dark adaptation. However, each knockout mouse eventually regenerated normal levels of visual pigments, suggesting that RDHs compensate for each other in the visual cycle. Here, we established RDH double knockout ( Rdh8 ^−/− Rdh12 ^−/− ) and triple knockout ( Rdh5 ^−/− Rdh8 ^−/− Rdh12 ^−/− ) mice generated on various genetic backgrounds including a rod α-transducin knockout to test cone function. RDH activity was severely reduced in Rdh8 ^−/− Rdh12 ^−/− retina extracts, whereas Rdh8 ^−/− RDH activity was intermediate and Rdh12 ^−/− RDH activity was reduced only slightly. Surprisingly, all multiple knockout mice produced sufficient amounts of the chromophore to regenerate rhodopsin and cone pigments in vivo . Three-month-old Rdh8 ^−/− Rdh12 ^−/− mice characteristically displayed a slowly progressing rod–cone dystrophy accompanied by accumulation of N -retinylidene- N -retinylethanolamine (A2E), a toxic substance known to contribute to retinal degeneration. A2E accumulation and retinal degeneration were prevented by application of retinylamine, a potent retinoid cycle inhibitor. The results suggest that RDH8 and RDH12 are dispensable in support of the visual cycle but appear to be key components in clearance of free all- trans -retinal, thereby preventing A2E accumulation and photoreceptor cell death.

Bovine PDEδ was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEδ can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEδ-specific antibody that PDEδ is present in rods and cones. We find by yeast two-hybrid screening with a PDEδ bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEδ fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEδ and dansylated prenyl cysteines as fluorescent ligands, we show that PDEδ specifically binds geranylgeranyl and farnesyl moieties with a K_d of 19.06 and 0.70 μm, respectively. Our experiments establish that PDEδ functions as a prenyl-binding protein interacting with multiple prenylated proteins.

Arf-like protein 3 (ARL3) is a ubiquitous small GTPase expressed in ciliated cells of plants and animals. Germline deletion of Arl3 in mice causes multiorgan ciliopathy reminiscent of Bardet-Biedl or Joubert syndromes. As photoreceptors are elegantly compartmentalized and have cilia, we probed the function of ARL3 (ADP-ribosylation factor (Arf)-like 3 protein) by generating rod photoreceptor-specific (prefix ^rod) and retina-specific (prefix ^ret) Arl3 deletions. In predegenerate ^rodArl3^−/− mice, lipidated phototransduction proteins showed trafficking deficiencies, consistent with the role of ARL3 as a cargo displacement factor for lipid-binding proteins. By contrast, ^retArl3^−/− rods and cones expressing Cre recombinase during embryonic development formed neither connecting cilia nor outer segments and degenerated rapidly. Absence of cilia infers participation of ARL3 in ciliogenesis and axoneme formation. Ciliogenesis was rescued, and degeneration was reversed in part by subretinal injection of adeno-associated virus particles expressing ARL3-EGFP. The conditional knock-out phenotypes permitted identification of two ARL3 functions, both in the GTP-bound form as follows: one as a regulator of intraflagellar transport participating in photoreceptor ciliogenesis and the other as a cargo displacement factor transporting lipidated protein to the outer segment. Surprisingly, a farnesylated inositol polyphosphate phosphatase only trafficked from the endoplasmic reticulum to the Golgi, thereby excluding it from a role in photoreceptor cilia physiology.