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Structural characterization of the rod cGMP phosphodiesterase 6.

ABSTRACT: Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6alpha, PDE6beta, and two regulatory PDE6gamma subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6alpha and PDE6beta and can form a complex with prenyl-binding protein delta (PrBP/delta), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/delta binding and the mechanism by which the PDE6-PrBP/delta complex assembles have not been fully characterized, and the location of regulatory PDE6gamma subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6-PrBP/delta from bovine rod outer segments utilizing recombinant PrBP/delta. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/delta and, thus, where the carboxyl-termini of PDE6alpha and PDE6beta must be located. The three-dimensional structure of the PDE6alphabetagamma complex was determined up to 18 A resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6gamma subunits, and catalytic sites.

SUBMITTER: Goc A

PROVIDER: S-EPMC2917047 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Structural characterization of the rod cGMP phosphodiesterase 6.

Goc Anna A Chami Mohamed M Lodowski David T DT Bosshart Patrick P Moiseenkova-Bell Vera V Baehr Wolfgang W Engel Andreas A Palczewski Krzysztof K

Journal of molecular biology 20100701 3

Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6alpha, PDE6beta, and two regulatory PDE6gamma subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6alpha and PDE6beta and can form a complex with prenyl-binding protein delta (PrBP/delta), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/delta binding and the mechanism by which the PDE6- ...[more]

PMID: 20600113

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Project description:We report the chromosomal localization, mutant gene identification, ophthalmic appearance, histology, and functional analysis of two new hereditary mouse models of retinal degeneration not having the Pde6brd1("r", "rd", or "rodless") mutation. One strain harbors an autosomal recessive mutation that maps to mouse chromosome 5. Sequence analysis showed that the retinal degeneration is caused by a missense point mutation in exon 13 of the beta-subunit of the rod cGMP phosphodiesterase (beta-PDE) gene (Pde6b). The gene symbol for this strain was set as Pde6brd10, abbreviated rd10 hereafter. Mice homozygous for the rd10 mutation showed histological changes at postnatal day 16 (P16) of age and sclerotic retinal vessels at four weeks of age, consistent with retinal degeneration. Retinal sections were highly positive for TUNEL and activated caspase-3 immunoreactivity, specifically in the outer nuclear layer (ONL). ERGs were never normal, but rod and cone ERG a- and b-waves were easily measured at P18 and steadily declined over 90% by two months of age. Protein extracts from rd10 retinas were positive for beta-PDE immunoreactivity starting at about the same time as wild-type (P10), though signal averaged less than 40% of wild-type. Interestingly, rearing rd10 mice in total darkness delayed degeneration for at least a week, after which morphological and functional loss progressed irregularly. With the second strain, a complementation test with rd1 mice revealed that the retinal degeneration phenotype observed represents a possible new allele of Pde6b. Sequencing demonstrated a missense point mutation in exon 16 of the beta-subunit of rod phosphodiesterase gene, different from the point mutations in rd1 and rd10. The gene symbol for this strain was set as Pde6bnmf137, abbreviated nmf137 hereafter. Mice homozygous for this mutation showed retinal degeneration with a mottled retina and white retinal vessels at three weeks of age. The exon 13 missense mutation (rd10) is the first known occurrence of a second mutant allele spontaneously arising in the Pde6b gene in mice and may provide a model for studying the pathogenesis of autosomal recessive retinitis pigmentosa (arRP) in humans. It may also provide a better model for experimental pharmaceutical-based therapy for RP because of its later onset and milder retinal degeneration than rd1 and nmf137.

Dataset Information

Structural characterization of the rod cGMP phosphodiesterase 6.

Publications

Structural characterization of the rod cGMP phosphodiesterase 6.

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