Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1^–/– and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.

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.

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.

Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca²⁺ and cGMP. Ca²⁺ regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC‐activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a‐wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild‐type flash responses in the absence of GCAP2.

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.