Molecular organization of bovine rod cGMP-phosphodiesterase 6 (original) (raw)
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Structural Characterization of the Rod cGMP Phosphodiesterase 6
Journal of Molecular Biology, 2010
Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6α, PDE6β, and two regulatory PDE6γ subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6α and PDE6β and can form a complex with prenyl-binding protein δ (PrBP/δ), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/δ binding and the mechanism by which the PDE6-PrBP/δ complex assembles have not been fully characterized, and the location of regulatory PDE6γ subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6-PrBP/δ from bovine rod outer segments utilizing recombinant PrBP/δ. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/δ and, thus, where the carboxyl-termini of PDE6α and PDE6β must be located. The threedimensional structure of the PDE6αβγ complex was determined up to 18 Å resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6γ subunits, and catalytic sites.
Journal of Structural Biology, 2002
Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Pa and Pb) and two identical regulatory subunits (Pcs). Pc regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Pabcc, and compare its average image with those of Pc-released PDE. The structure of Pabcc appears to be a flattened bell-shape, with dimensions of 150 Â 108 Â 60 A A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Pabcc is determined by hetero-dimerization of Pa and Pb, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Pabcc with those of Pc-released PDE suggests that Pc release does not affect the overall structure of Pab, and that the Pab C-terminus, but not Pc, is a determinant for the Pab orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Pab is regulated by its regional interaction with Pc. Ó 2002 Elsevier Science (USA). All rights reserved.
cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits
Proceedings of the National Academy of Sciences, 1988
The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits ar...
The human rod photoreceptor cGMP phosphodiesterase β-subunit
FEBS Letters, 1993
cDNA clones encoding the @bunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed a-, p-and a'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant 1 phage clones containing 26 kb of the human PDE/%ubunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4-22 of the PDE /?-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the p-subunit gene was observed between exons 20 and 21 which led to the formation of the /3'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE /3'-subunit in human retina.
Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits
FEBS Letters, 1993
Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-I 12641, and the rod PDE 11 kDa inhibitory subunit in E. coli, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar K, values (-80 PM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has K, values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDEy.
European Journal of Human Genetics, 1998
Rod-specific cGMP phosphodiesterase (PDE) is a key enzyme of the phototransduction cascade, and mutations in its catalytic subunits have been associated with retinal degenerative diseases. The bovine δ-subunit solubilises the normally membrane-bound PDE and is the only subunit expressed in extraocular tissues. We isolated the human and mouse orthologs, and found 78% identity at the DNA level and 98% identity at the protein level. The Caenorhabditis elegans homolog shows 69% identity at the protein level. The human PDED gene consisted of 5 exons spanning at least 30 kb of genomic DNA. Northern blot analysis showed a 1.3 kb transcript in human retina, heart, brain, placenta, liver, and skeletal muscle. Fluorescence in situ hybridisation (FISH) and radiation hybrid mapping localised the human PDED gene to chromosome 2q37. A preliminary screen of all 5 exons in 20 unrelated patients with autosomal recessive retinitis pigmentosa revealed no PDED mutations. * The genomic position of the forward (-) and reverse (+) primer in relation to exon boundaries. PDED gene structure B Lorenz et al 285
Vision Research, 2002
The inhibitory rod cyclic GMP-phosphodiesterase c subunit, PDEc, is a key component of the photoresponse and is required to support rod integrity. Pdeg tm1 =Pdeg tm1 mice that lack PDEc due to a targeted disruption of the gene encoding PDEc, (Pdeg) suffer from a very rapid and severe photoreceptor degeneration. Previously, deletions in the carboxyl-terminal domain of PDEc blocked its ability to inhibit trypsin-activated PDE activity, in vitro. In other words, these mutations eliminated PDEc's control on the catalytic activity of PDEa and PDEb. To study the in vivo effects resulting from the deletion of the last seven amino acids of the PDEc carboxyl terminal, this PDEc allele (Del7C) was introduced as a transgene Pdeg tm1 =Pdeg tm1 mice. These animals could only synthesize transgenic mutant PDEc. The mutant retinas were expected to display a higher basal level of PDE activity and lower cGMP levels in light and darkness than the PDEc knockout mice, which would allow the rescue of their photoreceptors. Instead, our results showed that the Del7C transgene could not complement the Pdeg tm1 =Pdeg tm1 mutant for photoreceptor survival. In fact, animals carrying the Del7C transgene have low PDE activity as well as reduced PDEa and PDEb content.
Genomics, 1998
rods and two a in cones) and two copies of inhibitory The mammalian multisubunit photoreceptor cGMP small subunits (rod g and cone g) (Baehr et al., 1979; phosphodiesterase PDEabg 2 (PDE6 family) is a pe- Hurwitz et al., 1985; Deterre et al., 1988; Gillespie and ripherally membrane-associated enzyme. A novel sub- . Under physiological unit, termed PDEd (HGMW-approved symbol, PDE6D; conditions, most of PDE is peripherally bound to rod MW 17 kDa), is able to detach PDE partially from boor cone outer segment membranes via methylated and vine rod outer segment membranes under physiologiisoprenylated C-termini of the large subunits (Swanson cal conditions. Cloning of human and mouse PDEd and Applebury, 1984; cDNAs revealed that PDEd is a nearly perfectly con-1992; . A 17-kDa served polypeptide of 150 amino acids that shows parpolypeptide, referred to as PDEd subunit, copurified tial sequence homology to photoreceptor RG4 of unwith a soluble fraction of bovine rod and cone PDE and known function. Multiple-species Southern blot analywas cloned recently Florio et al., sis demonstrates that the PDEd gene has been well 1996). PDEd does not affect the PDE catalytic activity conserved during evolution and is detectable at high in vitro, but is thought to directly interact with isoprenstringency in invertebrates. The human and mouse ylated C-termini, preventing membrane anchoring. genes are contained in less than 8 kb of genomic DNA The physiological significance of partial PDE solubiliand consist of four exons and three introns (0.7-4 kb zation is unclear, but PDE detachment may serve as a in human, 0.7-2.2 kb in mouse). The PDEd gene strucmechanism to desensitize the phototransduction casture is identical to that of the C27H5.1 gene identified cade by removing active PDE from the membrane surin the eyeless nematode Caenorhabditis elegans. The face ( Florio et al., 1996).