Retinal cGMP Phosphodiesterase Research Articles

Clinical and functional analyses of AIPL1 variants reveal mechanisms of pathogenicity linked to different forms of retinal degeneration

Disease-causing sequence variants in the highly polymorphic AIPL1 gene are associated with a broad spectrum of inherited retinal diseases ranging from severe autosomal recessive Leber congenital amaurosis to later onset retinitis pigmentosa. AIPL1 is a photoreceptor-specific co-chaperone that interacts with HSP90 to facilitate the stable assembly of retinal cGMP phosphodiesterase, PDE6. In this report, we establish unequivocal correlations between patient clinical phenotypes and in vitro functional assays of uncharacterized AIPL1 variants. We confirm that missense and nonsense variants in the FKBP-like and tetratricopeptide repeat domains of AIPL1 lead to the loss of both HSP90 interaction and PDE6 activity, confirming these variants cause LCA. In contrast, we report the association of p.G122R with milder forms of retinal degeneration, and show that while p.G122R had no effect on HSP90 binding, the modulation of PDE6 cGMP levels was impaired. The clinical history of these patients together with our functional assays suggest that the p.G122R variant is a rare hypomorphic allele with a later disease onset, amenable to therapeutic intervention. Finally, we report the primate-specific proline-rich domain to be dispensable for both HSP90 interaction and PDE6 activity. We conclude that variants investigated in this domain do not cause disease, with the exception of p.A352_P355del associated with autosomal dominant cone-rod dystrophy.

The Leber Congenital Amaurosis-Linked Protein AIPL1 and Its Critical Role in Photoreceptors.

Mutations in the photoreceptor/pineal-expressed gene, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), are mainly associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy that occurs in early childhood. AIPL1 functions as a photoreceptor-specific molecular co-chaperone that interacts specifically with the molecular chaperones HSP90 and HSP70 to facilitate the correct folding and assembly of the retinal cGMP phosphodiesterase (PDE6) holoenzyme. The absence of AIPL1 leads to a dramatic degeneration of rod and cone cells and a complete loss of any light-dependent electrical response. Here we review the important role of AIPL1 in photoreceptor functionality.

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The Inhibitory γ Subunit of the Type 6 Retinal cGMP Phosphodiesterase Functions to Link c-Src and G-protein-coupled Receptor Kinase 2 in a Signaling Unit That Regulates p42/p44 Mitogen-activated Protein Kinase by Epidermal Growth Factor

The inhibitory gamma subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEgamma) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEgamma is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEgamma produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEgamma lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEgamma, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEgamma.

Binding of cGMP to GAF Domains in Amphibian Rod Photoreceptor cGMP Phosphodiesterase (PDE): IDENTIFICATION OF GAF DOMAINS IN PDE αβ SUBUNITS AND DISTINCT DOMAINS IN THE PDE γ SUBUNIT INVOLVED IN STIMULATION OF cGMP BINDING TO GAF DOMAINS

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.

The positive role of the carboxyl terminus of the γ subunit of retinal cGMP-phosphodiesterase in maintaining phosphodiesterase activity in vivo

The inhibitory rod cyclic GMP-phosphodiesterase γ subunit, PDEγ, is a key component of the photoresponse and is required to support rod integrity. Pdeg tm1 / Pdeg tm1 mice that lack PDEγ due to a targeted disruption of the gene encoding PDEγ, ( Pdeg) suffer from a very rapid and severe photoreceptor degeneration. Previously, deletions in the carboxyl-terminal domain of PDEγ blocked its ability to inhibit trypsin-activated PDE activity, in vitro. In other words, these mutations eliminated PDEγ's control on the catalytic activity of PDEα and PDEβ. To study the in vivo effects resulting from the deletion of the last seven amino acids of the PDEγ carboxyl terminal, this PDEγ allele ( Del7C) was introduced as a transgene Pdeg tm1 / Pdeg tm1 mice. These animals could only synthesize transgenic mutant PDEγ. The mutant retinas were expected to display a higher basal level of PDE activity and lower cGMP levels in light and darkness than the PDEγ 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 PDEα and PDEβ content.

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit (Pgamma) of retinal cgmp phosphodiesterase (PDE6): its implications in phototransduction.

Cyclic GMP phosphodiesterase (PDE6) is a key enzyme in vertebrate retinal phototransduction. After GTP/GDP exchange on the a subunit of transducin (Talpha) by illuminated rhodopsin, the GTP-bound form Talpha (GTP/Talpha) interacts with the regulatory subunit (Pgamma) of PDE6 to activate cGMP hydrolytic activity. The regulatory mechanism of PDE6 has been believed to be a typical G protein-mediated signal transduction process. We found that cyclin-dependent protein kinase 5 (Cdk5) phosphorylates Pgamma complexed with GTP/Talpha in vitro and in vivo. Phosphorylated Py dissociates from GTP/Talpha without GTP hydrolysis and interacts effectively with catalytic subunits of PDE6 to inhibit the enzyme activity. These observations provide new twists to the current model of retinal phototransduction. In this article, in addition to the details of Py phosphorylation by Cdk5, we review previous studies implying the Pgamma phosphorylation and the turnoff of PDE6 without GTP hydrolysis and indicate the direction for future studies of Py phosphorylation, including the possible involvement of Ca2+/Ca2+-binding proteins.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase: II. ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VIVO

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950-32957), we have shown that all known Pgammas contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent Pgamma phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.

29] Structural analysis of protein prenyl groups and associated C-terminal modifications

The prenylation of proteins, including several in the visual transduction pathway, is one of the most recently discovered modifications of eukaryotic cell proteins.1–4 Some prenylated proteins, including rhodopsin kinase, the γ subunit of transducin, the α subunit of retinal cGMP phosphodiesterase, Ras, and lamin B, are initially produced with a C-terminal sequence motif CaaX (C is cysteine, a is usually but not necessarily an aliphatic residue, and X is typically M, S, or Q). Prenylation occurs by enzyme-catalyzed attachment of a 15-carbon farnesyl group to cysteine via a thioether linkage. After prenylation, aaX is released by a membrane-bound endoprotease,5–8 and the newly exposed S-farnesylcysteine is methylated on its α-carboxyl group by a membrane-bound methyltransferase.9,10 Other prenylated proteins, including the γ subunits of many heterotrimeric G proteins, the β subunit of retinal cGMP phosphodiesterase, and a subset of small GTP-binding proteins, contain a CaaX motif (X = L, F) and are modified by the attachment of the 20-carbon geranylgeranyl group. Removal of aaX and C-terminal methylation occurs as for farnesylated proteins. Finally, the subset of GTP-binding proteins termed Rab contains two cysteines near or at the C terminus (CCXX, XXCC, CXC), and both cysteine sulfhydryls contain a thioether-linked geranylgeranyl group.11 The attachment of prenyl groups to these three classes of proteins is catalyzed by three distinct protein prenyltranferases.12,13 Structures of protein prenyl groups have been rigorously established by releasing the protein-bound lipids with Raney nickel (to cleave reductively the bond between cysteine S and C-1 of the prenyl group) and analyzing the released hydrocarbons by combined gas chromatography–mass spectrometry versus authentic standards.14–17 Protein prenyl groups can also be released by treatment with methyl iodide to produce farnesol and geranylgeraniol along with isomerized prenols.16,18 This method has been used when relatively small amounts of prenylated protein are available, typically from tissue culture cells that have been grown in the presence of radiolabeled mevalonic acid (the precursor of prenyl groups) or its lactone in the presence of statins, which block the production of endogenous mevalonic acid. The radiolabeled prenol is analyzed versus standards by reversed phase high-performance liquid chromatography (HPLC).16 While this approach does not establish the chemical structure of the prenyl group, it provides strong circumstantial evidence for the presence of protein-bound farnesyl and geranylgeranyl groups. However, in our hands, we have experienced difficulties with methyl iodide cleavage, in that yields are highly variable and can be quite low in some cases (<10% cleavage). Work in our laboratory has shown that the Raney nickel cleavage method is superior to methyl iodide treatment, and the details of this procedure, combined with HPLC analysis of the released radiolabeled hydrocarbons, are described in this chapter. This method has been used to analyze protein prenyl groups from protein extracted from sodium dodecyl sulfate (SDS)–polyacrylamide gel slices. Also described here is the use of Raney nickel cleavage followed by combined gas chromatography–mass spectrometry to determine the structure of prenyl groups released from delipidated total cell protein. The rigorous structural analysis of aaX removal and C-terminal methylation has been carried out by fast atom bombardment mass spectrometry of C-terminal peptides from prenylated proteins.11,19 A less rigorous approach has been used to explore C-terminal methylation: HPLC analysis of the product of oxidative scission of the prenyl–cysteine linkage combined with amide bond hydrolysis to yield cysteic acid methyl ester.20 In the present chapter, a method for electrospray mass spectrometric analysis of protein C-terminal peptides is presented, which has been applied to the analysis of prenylated peptides in a complex mixture (a tryptic digest of partially purified bovine rhodopsin kinase). This method is facilitated by the availability of prenylated peptide standards, and a convenient synthesis of peptides and peptide methyl esters radiolabeled with high specific activity farnesyl and geranylgeranyl groups is described.

Delineation of two functionally distinct gammaPDE binding sites on the bovine retinal cGMP phosphodiesterase by a mutant gammaPDE subunit.

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In this work, we describe a full length, doubly point-mutated gamma subunit, C68S, Y84C gammaPDE, which binds to PDE with increased affinity but has a decreased ability to inhibit the enzyme. Fluorescence studies monitoring the competition between wild-type gammaPDE and the C68S, Y84C gammaPDE mutant suggest that the mutant gammaPDE binds with high affinity to only half of the total sites occupied by wild-type gammaPDE. Competition studies between wild-type gammaPDE and the mutant further suggest that the wild-type protein is able to fully inhibit PDE activity even when the mutant gammaPDE occupies its high-affinity binding site on PDE. Taken together, our findings are consistent with a model in which there are two distinguishable binding sites for gammaPDE on the PDE enzyme but that only one of the two sites mediates PDE inhibition.

Lead-induced alterations in retinal cGMP phosphodiesterase trigger calcium overload, mitochondrial dysfunction and rod photoreceptor apoptosis

Lead exposure results in the selective apoptotic loss of rods and bipolar cells. During and following developmental lead exposure rod/retinal cGMP phosphodiesterase expression and activity are delayed in onset and decreased, [Ca2+] is elevated, and mitochondrial ATP synthesis is decreased. In vitro studies, using retinas incubated in Ca2+ and/or Pb2+, demonstrate that rods selectively die by apoptosis, retinal mitochondrial ATP synthesis is decreased, mitochondrial cytochrome c is released and caspase activity is increased. These results suggest that lead-induced rod and bipolar cell apoptosis is triggered by Ca2+ and Pb2+ overload due to altered cGMP phosphodiesterase activity and that mitochondrial alterations play a central role in this process.

Cloning of a cyclic GMP phosphodiesterase γ subunit from the ground squirrel retina

Recent evidence suggests that the two main classes of cones are not only equipped with different photopigments, but also exhibit differences in their downstream phototransduction cascade. An antibody against the γ subunit of retinal cGMP phosphodiesterase (PDE) had previously been found to label all photoreceptors in the ground squirrel ( Spermophilus tridecemlineatus). This property was utilized for the cloning of a cDNA fragment encoding the corresponding polypeptide. A λgt11 cDNA library was constructed and screened with the antibody. Positive clones were isolated, subcloned, and sequenced. Clones were used as probes in RNA blot hybridization, and the obtained sequence information was compared to other available γ-PDE sequences. Four virtually identical cDNA clones were isolated. Both nucleic acid and amino acid sequence alignment placed this gene in the same group as bovine and human cone γ-phosphodiesterase subunits. The predicted length of the translated protein was 84 amino acids, and its molecular weight 11 kD. The cDNA hybridized with an 0.6-kb transcript in retinal RNA from ground squirrel, Syrian golden hamster, and mouse, and also with a 1.4-kb transcript in the ground squirrel. We conclude that the isolated cDNA fragment encodes a γ subunit of cone cGMP-PDE. This subunit is expressed in middle-wavelength sensitive cones (the predominant photoreceptor type in this species). It remains to be determined if this subunit is expressed in short-wavelength sensitive cones as well, or if these photoreceptors are equipped with another, unique γ-PDE subunit.

Transcriptional activation of the human rod cGMP-phosphodiesterase beta-subunit gene is mediated by an upstream AP-1 element.

During photoactivation retinal cGMP-phosphodiesterase (PDE) mediates signal transduction in the photoreceptor outer segments. Mutations in the beta-subunit gene of rod-specific PDE (beta-PDE) have been associated with inherited retinal degeneration in a number of species, including human. Here we have investigated the proximal upstream sequences that participate in transcriptional activation of this gene. Transient transfections demonstrated that the sequence from -72 to +53 bp contained sufficient information to direct high levels of gene expression in cells of retinal origin. Deletion or mutagenesis of an AP-1 motif present in this region caused 90-95% reduction in reporter gene expression. By gel mobility shift assay we demonstrated specific interactions between putative nuclear transcription factors and this AP-1 element. These findings indicate that the proximal AP-1 site in the human beta-PDE promoter is functionally relevant and necessary for transcriptional activation of this gene.

Real Time Conformational Changes in the Retinal Phosphodiesterase γ Subunit Monitored by Resonance Energy Transfer

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of the holoenzyme, gammaPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluorescein specifically labeled a cysteine residue at position 68 and served as a resonance energy transfer donor. The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, was determined to be within the first seven residues of the amino terminus of gammaPDE. Energy transfer between the labeled sites on free, unbound gammaPDE indicated that they were separated by a distance of 63 A, consistent with a random conformation. Upon binding the catalytic alphabeta subunits of the PDE, the distance between the two probes on gammaPDE increased to 77 A. Binding of the labeled gammaPDE by alphaT.guanosine 5'-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated. These data are consistent with the view that the binding of activated alphaT to gammaPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gammaPDE molecule. They also support a model for PDE activation that places active alphaT in a complex with the holoenzyme.

Visualizing signal transduction: receptors, G-proteins, and adenylate cyclases.

1. The first glimpses of heterotrimeric G-proteins came with the discoveries of the ubiquitous adenylate cyclase activator, Gs, and the specialized retinal cGMP phosphodiesterase activator, Gi or transducin. The model that evolved for regulation of adenylate cyclase activity by G-proteins soon proved to be a general paradigm for a large number of signalling pathways. Although many different G-proteins interact with a diverse array of receptors and effectors, each is composed of a guaninenucleotide-binding alpha-subunit and a tightly associated complex of a beta- and a gamma-subunit. 2. Receptors catalyse the activation of G-proteins by promoting exchange of GDP for GTP, while G-proteins catalyse their own deactivation as a result of their intrinsic GTPase activity. Crystallographic analysis has described several of the various conformational states that G-proteins undergo as they are activated and deactivated and has provided great insight into the kinetic models of G-protein-mediated signal transduction. 3. The regulation of adenylate cyclase has proven to be intriguing and complex. Gsx activates all forms of mammalian adenylate cyclase; other G-proteins (Gi, Go and Gz) inhibit certain isoforms of the enzyme. The discovery of new isoforms of adenylate cyclase has revealed synergistic and conditional mechanisms of regulation. These include activation or inhibition by the G-protein beta gamma-subunit complex, activation by Ca(2+)-calmodulin, and phosphorylation by protein kinases. The large number of receptors, G-proteins and adenylate cyclases provides a complex signalling network that integrates and interprets a multitude of convergent inputs.

Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems.

We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.

1] Purification of bovine retinal cGMP phosphodiesterase

1] Purification of bovine retinal cGMP phosphodiesterase

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.

Genetic mapping demonstrates that the α-subunit of retinal cGMP-phosphodiesterase is not the site of the rd mutation

In the inherited degenerative retinal disease of the rd mouse, rod cGMP levels rise above normal due to depressed cGMP-phosphodiesterase (cGMP-PDE) function a few days before degeneration begins. The subnormal activity of the cGMP-PDE may be due to a lesion in the enzyme itself, or in any of several proteins that regulate it. We have used a bovine cDNA for the α-subunit of cGMP-PDE to map its gene Pdea to mouse chromosome 18 at a distance of 21 centimorgans (cM) from the Mbp locus. Since the locus of the rd mutation is on mouse chromosome 5, a defect in the Pdea gene is ruled out as the cause of this inherited retinal degeneration.