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. 2007 May 22;104(21):8857-62.
doi: 10.1073/pnas.0701681104. Epub 2007 May 11.

Deletion of PrBP/delta impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments

H Zhang  1 , S Li, T Doan, F Rieke, P B Detwiler, J M Frederick, W Baehr
Affiliations

Deletion of PrBP/delta impedes transport of GRK1 and PDE6 catalytic subunits to photoreceptor outer segments

H Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Knockout strategy and phenotype of Pde6d−/− mice. (A) Generation of Pde6d−/− mice. Structure of the WT Pde6d gene (a), the targeting vector (b), and the disrupted Pde6d gene (c) are shown schematically. Blue triangles denote loxP, and black rectangles denote exons; F1, F2, and R are primers used for genotyping. TK, thymidine kinase. (B) Genotyping of Pde6d−/− mice by PCR. Amplification with primers PDE6D-F2 and PDE6D-R verified the deletion of sequence between loxP sites. Amplification with primers PDE6D-F1 and PDE6D-R confirmed the WT Pde6d allele. (C) Immunoblots of 1- to 2-month-old WT and Pde6d−/− retina lysates. PDE, rod PDE6 catalytic subunits; cPDE, cone PDE6 catalytic subunit; RK, GRK1; Tγ, rod Tγ subunit; Arr, rod arrestin; Rho, rhodopsin; Tα, rod Tα subunit; β-T, β-tubulin as an internal control. Prenylated PDE6 subunits, farnesylated GRK1, and Tγ appear down-regulated, whereas rod arrestin is up-regulated. Acylated Tα and Rho protein levels appear unaffected by deletion of the Pde6d gene. (D) Labeling of WT and Pde6d−/− frozen sections with polyclonal anti-PrBP/δ antibody. Sections were subjected to antigen retrieval (see Methods) before blocking and incubating with primary antibody. PrBP/δ (red) is most prominent in the inner segments of rods and cones. (Scale bar: 10 μm.) (E) Pde6d−/− mice (2 months old) were consistently smaller. (Upper) Female (−/−) and (+/+) littermates. (Lower) Male (+/+) and (−/−) littermates.
Fig. 2.
Fig. 2.
Confocal immunolocalizations of GRK1, rod PDE6, and Tγ in WT (A, C, and E) and Pde6d−/− (B, D, and F) retina cryosections. (A and B) Immunoreactivity for GRK1 is prominent over rod and cone outer segments (OS) in WT retina although nearly undetectable in Pde6d−/− rod and cone OS. (C and D) Localizations of rod PDE6 in WT and Pde6d−/− retinas, respectively (MOE antibody; Cytosignal). Rod PDE6 labeling is typically restricted to rod OS in WT retina. Aberrant distribution of rod PDE6 was observed in inner segments and synaptic terminals of Pde6d−/− rods. (E and F) Distributions of rod Tγ in WT and Pde6d−/− retinas, respectively. Tγ mislocalizes, in part, to the inner segment, perinuclear, and synaptic regions (white arrows) of Pde6d−/− photoreceptors. In each image, propidium iodide (red) demonstrates the extent of the ONL by binding nucleic acids (both DNA and RNA). (Scale bars: 10 μm.) IS, inner segments; OPL, outer plexiform layer.
Fig. 3.
Fig. 3.
Localization of cone phototransduction components in WT (A, C, E, and G) and Pde6d−/− (B, D, F, and H) retina sections. In A and B, cones are labeled from their pedicles to outer segments using anti-cone arrestin antibody. The cone numbers in WT and knockout retinas are comparable at 1–2 months. (C and D) Labeling with anti-cone PDE6α′ antibody. Cone PDE is nearly absent in Pde6d−/− COS but is present at low levels in inner segments. (E–H) WT and Pde6d−/− sections were probed with anti-cone Tα (E and F) or anti-cone Tγ (G and H) antibodies. In contrast to geranylgeranylated cone PDEα′, the farnesylated Tα and acylated Tγ subunits show normal distributions in Pde6d−/− cones. OS, outer segments; IS, inner segments; OPL, outer plexiform layer.
Fig. 4.
Fig. 4.
Scotopic and photopic ERGs of WT and Pde6d−/− mice. (A and B) Scotopic a- and b-wave amplitudes, respectively, of 2-month-old WT and Pde6d−/− mice as a function of light intensity. Error bars represent mean ± SD (n = 3–7). (C and D) Paired-flash ERGs. The intensity of the first and second flash is 1.4 log cd·s·m−2. The interval between two flashes increases from 600 ms to 5 sec. (E) Photopic ERG traces of 2-month-old WT and Pde6d−/− mice at 0.87 and 2.9 log cd·s·m2. (F) Photopic b-wave amplitudes as a function of light intensity. Error bars represent mean ± SD (n = 3–7).
Fig. 5.
Fig. 5.
Flash responses in WT and Pde6d−/− single-rod photoreceptors. (A) Average dim flash responses scaled to 1 photoisomerization for WT (n = 17) and Pde6d−/− (n = 11). Pde6d−/− average single-photon response was prolonged relative to WT average single-photon response (time to peak 300 ± 20 vs. 213 ± 12 ms, respectively, mean ± SEM; integration time 736 ± 42 vs. 300 ± 38 ms). (B) Normalized saturating flash responses of WT (n = 11) and of Pde6d−/− (n = 9). Pde6d−/− saturating flash response was prolonged in comparison with WT saturating flash response. (C) Pepperberg plot (delay period Tsat for recovery of the dark current vs. log flash intensity) for WT (n = 16) and Pde6d−/− (n = 9). Tsat for Pde6d−/− rods was higher than for WT rods at every flash strength measured. Linear regression over the flash strengths measured yielded slopes of 492 ms for WT and 655 ms for Pde6d−/−. Error bars represent ± SEM. (D) Mean stimulus response curves of WT (n = 17) and Pde6d−/− (n = 10) with error bars representing ± SEM. Fits are saturating exponential functions, used to estimate the half-saturating flash intensity (WT 14 ± 1 photons per μm2, mean ± SEM; Pde6d−/− 13 ± 1 photons per μm2).
Fig. 6.
Fig. 6.
Pde6d−/− photoreceptor degeneration over time. Localization of rod PDE6 in retina cryosections of 5-month-old WT (A), 5-month-old Pde6d−/− (B), and 20-month-old Pde6d−/− (C) mice is shown. Colocalization of GRK1 (green) and cone arrestin (red) in 2-month-old WT (D), 2-month-old Pde6d−/− (E), and 20-month-old Pde6d−/− (F) retina is also shown. The ONL, consisting of 5–6 rows of photoreceptor nuclei in the knockout, is half that of WT retina at 20 months. RIS, rod inner segments; OPL, outer plexiform layer.
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