Characterization of Human and Mouse Rod cGMP Phosphodiesterase δ Subunit (PDE6D) and Chromosomal Localization of the Human Gene (original) (raw)
Related papers
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
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.
Cellular Signalling, 2011
The inhibitory subunit of rod cyclic guanosine monophosphate (cGMP) phosphodiesterase, PDE6γ, is a major component of rod transduction and is required to support photoreceptor integrity. The N74A allele of PDE6γ has previously been shown in experiments carried out in vitro to reduce the regulatory inhibition on the PDE6 catalytic core subunits, PDE6αβ. This should, in intact rods, lead to an increase in basal (dark) PDE6 activity producing a state equivalent to light adaptation in the rods and we have examined this possibility using ERG and suction-electrode measurements. The murine opsin promoter was used to drive the expression of a mutant N74A and a wild-type PDE6γ control transgene in the photoreceptors of +/Pde6g tm1 mice. This transgenic line was crossed with Pde6g tm1 /Pde6g tm1 mice to generate animals able to synthesize only the transgenic mutant PDE6γ. We find that the N74A mutation did not produce a significant decrease in circulating current, a decrease in sensitivity or affect the kinetics of the light response, all hallmarks of the light-adapted state. In an in vitro assay of the PDE purified from the N74A transgenic mice and control mice we could find no increase in basal activity of the mutant PDE6. Both the results from the physiology and the biochemistry experiments are consistent with the interpretation that the mutation causes a much milder phenotype in vivo than was predicted from observations made using a cell-free assay system. The in vivo regulation of PDE6γ on PDE6αβ may be more dynamic and context-dependent than was replicated in vitro.
Rod phosphodiesterase-6 PDE6A and PDE6B Subunits Are Enzymatically Equivalent
Journal of Biological Chemistry, 2010
Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K m (20-23 M) and k cat (4200-5100 s ؊1) values. Likewise, the K i values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone-and rod-specific PDE6 ␥-subunits (P␥) were comparable. Recombinant cone transducin-␣ (G␣ t2) and native rod G␣ t1 fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of G␣ t2 or G␣ t1. Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with P␥ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.
Cellular Signalling, 2012
The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g tm1 /Pde6g tm1 mice that does not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile 86 and Ile 87 residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100 µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile 86 and Ile 87 residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.
Regulatory Sequences in the 3' Untranslated Region of the Human cGMP-Phosphodiesterase -Subunit Gene
Investigative Ophthalmology & Visual Science, 2009
PURPOSE-Rod cGMP-phosphodiesterase, a key enzyme in visual transduction, is important for retinal integrity and function. Mutations in the gene encoding its β-subunit (PDEβ) cause retinal degeneration in animals and humans. Here we tested the hypothesis that elements in the 3'UTR of the PDEβ gene are involved in the regulation of PDEβ expression. METHODS-Involvement of the 3' untranslated region (3'UTR) of PDEβ mRNA in the regulation of PDEβ expression was assessed by transfecting Y-79 retinoblastoma cells or the heads of Xenopus laevis tadpoles with constructs containing the SV40 or PDEβ promoter, the luciferase cDNA and either the SV40 or PDEβ 3'UTR (or fragments of its sequence). RESULTS-Compared to the SV40 3'UTR (used as control), the entire PDEβ 3'UTR decreased reporter gene expression in Y-79 retinoblastoma cells as well as in SY5Y neuroblastoma and 293 human embryonic kidney cell lines. However, we observed that two 100 nt fragments from the PDEβ 3'UTR increased while its non-canonical polyadenylation signal abolished reporter gene expression both in Y-79 retinoblastoma cells and in ex vivo experiments using Xenopus tadpole heads. In particular, an 11 nt element (EURE) in one of the 100 nt fragments was responsible for upregulation of luciferase expression. CONCLUSION-Our studies indicate that the 3'UTR of the PDEβ mRNA is involved in the very complex regulation of this gene's expression in the retina. Moreover, they show that the PDEβ poly-A signal has a dominant inhibitory effect over two other regions in the 3'UTR that stimulate gene expression.
Journal of Biological Chemistry, 2003
The catalytic core of photoreceptor-specific cGMPphosphodiesterase (PDE) consists of two subunits, PDE␣ and PDE, that are homologous and have similar domain organization but are encoded by different genes. We have examined the PDE␣ and PDE mRNA steady-state and protein levels as well as the biosynthesis rate of these proteins in developing and fully differentiated retinas. We have also determined the translational efficiency of PDE subunits and the role of their mRNA structures in regulating protein synthesis. In mature retinas, PDE␣ and PDE are represented by ϳ1.5 ؋ 10 8 and 7.5 ؋ 10 8 copies/g retinal mRNA, respectively. The levels of these transcripts in developing photoreceptors (P10) are approximately 75% of those at P30. Quantification of protein concentration indicated that PDE␣ and PDE are equally expressed in developing and fully differentiated photoreceptors. Furthermore, the PDE␣/PDE ratios obtained throughout a 2-h pulsechase period revealed a similar turnover rate for both subunits. The observed discordance between the mRNA and protein levels of PDE␣ and PDE suggested posttranscriptional regulation of their expression. We found that PDE␣ mRNA is translated more efficiently than either of the two PDE transcripts expressed in retina. Therefore, the lower level of PDE␣ mRNA is compensated by its more efficient translation to achieve equimolar expression with PDE. We also analyzed the effect of PDE␣ and PDE mRNA 5-and 3-untranslated regions as well as that of their coding regions on protein synthesis. We determined that the PDE-coding regions play a critical role in the differential translation of these subunits. Signal transduction in the vertebrate rod and cone photoreceptors is mediated by a cascade of protein interactions/activations that lead to hydrolysis of cGMP by a photoreceptor-specific cGMP-phosphodiesterase (PDE) 1 followed by closure of the
Journal of Biological Chemistry, 2010
The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P␥ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of P␥ to effectively relieve its inhibitory constraint. . 2 The abbreviations used are: PDE6, photoreceptor cyclic nucleotide phosphodiesterase; P␣, catalytic dimer of PDE6 ␣and -subunits; P␥, inhibitory ␥ subunit of PDE6; T␣*, activated transducin ␣-subunit; GTP␥S, guanosine 5Ј-O-(thiotriphosphate).
Investigative Ophthalmology Visual Science, 2009
PURPOSE-Rod cGMP-phosphodiesterase, a key enzyme in visual transduction, is important for retinal integrity and function. Mutations in the gene encoding its β-subunit (PDEβ) cause retinal degeneration in animals and humans. Here we tested the hypothesis that elements in the 3'UTR of the PDEβ gene are involved in the regulation of PDEβ expression. METHODS-Involvement of the 3' untranslated region (3'UTR) of PDEβ mRNA in the regulation of PDEβ expression was assessed by transfecting Y-79 retinoblastoma cells or the heads of Xenopus laevis tadpoles with constructs containing the SV40 or PDEβ promoter, the luciferase cDNA and either the SV40 or PDEβ 3'UTR (or fragments of its sequence). RESULTS-Compared to the SV40 3'UTR (used as control), the entire PDEβ 3'UTR decreased reporter gene expression in Y-79 retinoblastoma cells as well as in SY5Y neuroblastoma and 293 human embryonic kidney cell lines. However, we observed that two 100 nt fragments from the PDEβ 3'UTR increased while its non-canonical polyadenylation signal abolished reporter gene expression both in Y-79 retinoblastoma cells and in ex vivo experiments using Xenopus tadpole heads. In particular, an 11 nt element (EURE) in one of the 100 nt fragments was responsible for upregulation of luciferase expression. CONCLUSION-Our studies indicate that the 3'UTR of the PDEβ mRNA is involved in the very complex regulation of this gene's expression in the retina. Moreover, they show that the PDEβ poly-A signal has a dominant inhibitory effect over two other regions in the 3'UTR that stimulate gene expression.