Carriers of the mouse rd gene have reduced levels of the beta subunit of the retinal cyclic GMP phosphodiesterase.

Polyclonal antipeptide antisera have been utilized to quantitate the amount of retinal rod outer segment cGMP phosphodiesterase alpha and beta catalytic subunits present in retinas from C57BL/6J mice which are normal or carriers for the rd gene defect. Results suggest that the quantity of PDE-beta subunit is reduced in carrier mice while PDE-alpha and PDE-gamma are not affected. In 21-day-old mice, the PDE-beta was reduced by about one-half while adult carrier mice had even more reduced levels of PDE-beta. Since PDE alpha was not reduced, this suggests that synthesis of PDE alpha and PDE beta may not be coordinately controlled.     

Reduced rhodopsin phosphorylation during retinal dystrophy

During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week-old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd-mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy.     

In vivo differential prenylation of retinal cyclic GMP phosphodiesterase catalytic subunits.

A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.     

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.     

Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.     

cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its gamma and delta subunits.

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.     

Phosphodiesterase of Cone Photoreceptors from the Lizard, Anolis carolinensis

Cone and rod photoreceptors utilize cyclic guanosine monophosphate (cGMP) in the light regulation of membrane polarization. The prototype for visual transduction is established for rod photoreceptors, which utilize a cascade of reactions to regulate a cyclic nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) and thereby control the intracellular concentration of cGMP. Although cones appear to utilize a comparable cGMP cascade for their phototransduction, evidence exists that the PDE from cone photoreceptors may be different from that of rods. Dissociated cone photoreceptors, isolated retinas, and cone outer segments from the lizard, Anolis carolinensis, have been used to identify and characterize a PDE enzyme complex that shares several features in common with the rod outer segment (ROS) PDE complex. Immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis have identified a subunit of lizard cone PDE that has an apparent electrophoretic mobility of 84 kDa and a subunit of lizard rod PDE that migrates at approximately 90 kDa. The lizard cone PDE complex is similar in size, extraction, activation, and immunological characteristics to the PDE complex of rod photoreceptors from lizard, bovine, and human retinas. The lizard cone PDE complex, and perhaps that from cone photoreceptors in general, differs from that of ROS in its chromatographic properties on anion-exchange resins. The sharing of physical and activation properties of the rod and cone PDE complex is compatible with the phototransduction process occurring by a similar mechanism in both cell types. The differences in light sensitivity and speed of response may be attributable to features of the individual proteins that form the PDE complexes of rods and cones or to other undisclosed features of the respective cascades.     

Crystal structure of the GAF-B domain from human phosphodiesterase 10A complexed with its ligand, cAMP

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.     

Identification of the gamma-subunit interaction sites in the retinal cyclic-GMP phosphodiesterase beta-subunit.

Using synthetic peptides, the identification of the retinal cyclic-GMP phosphodiesterase (cGMP PDE) interaction sites for the inhibitory gamma-subunit in the catalytic alpha-subunit were recently localized to residues #16-30 and 78-90 in the alpha-subunit (1). In this study, a binding radioimmunoassay (RIA) showed a weak interaction between PDE gamma and PDE beta subunits in PDE beta residues #15-34, and stronger interaction sites were found in residues #91-110 and 211-230. Sequence comparison between PDE alpha and PDE beta illustrate some differences in these regions, particularly in PDE alpha 16-30 and PDE beta 15-34 regions. Differences in interaction sites in PDE alpha and PDE beta for PDE gamma may account for the differences in affinities observed between PDE gamma and the catalytic subunits.     

An early decrease in interphotoreceptor retinoid-binding protein gene expression in Abyssinian cats homozygous for hereditary rod-cone degeneration

Levels of interphotoreceptor retinoid-binding protein (IRBP) protein and message in retinas of Abyssinian cats homozygous for progressive rod-cone degeneration were determined at early ages, well before the onset of clinical retinal degeneration. IRBP gene expression was assessed by immunochemical quantitation of IRBP protein, and by Northern blotting and slot-blotting of total RNA using a human IRBP cDNA probe. Morphology was assessed by electron microscopy and immunocytochemistry. Levels of both IRBP protein and message in affected Abyssinian cat retinas were significantly reduced below normal as early as 4 weeks of age at the earliest stage of retinal disorientation. Opsin mRNA was more abundant in affected Abyssininian cat retinas than in control retinas. This was at least 1 year before the onset of clinical symptoms. The reduction in IRBP gene expression to levels significantly below normal well before the onset of retinal degeneration in affected Abyssinian cat retinas indicates that this represents a primary defect or at least an early problem that could itself cause adverse effects.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

Altered activities of cyclic nucleotide phosphodiesterases and soluble guanylyl cyclase in cultured RFL-6 cells

We utilized rat fetal lung fibroblasts (RFL-6) to evaluate our PDE5 inhibitors at cellular level and observed a decrease in cGMP accumulation induced by sodium nitroprusside (SNP) and PDE5 inhibitors with passage. To further investigate this observation, we examined cGMP synthesis via soluble guanylyl cyclase (sGC) and degradation via phosphodiesterases (PDEs) at different passages. At passage (p)4, p9, p14, major cGMP and cAMP degradation activities were contributed by PDE5 and PDE4, respectively. The PDE5 activity decreased 50% from p4 to p14, while PDE4 activity doubled. The cGMP accumulation was evaluated in the presence of sodium nitroprusside (SNP) and/or PDE inhibitors in p4 and p14 cells. SNP together with sildenafil, a PDE5 inhibitor, induced dose-dependent increase in cGMP levels in cells at p4, but showed little effect on cells at p14. The possible down regulation of sGC at mRNA level was explored using real-time RT-PCR. The result showed the mRNA level of the alpha1 subunit of sGC decreased about 98% by p9, while the change on beta1 mRNA was minimal. Consistently, sGC activities in cell lysate decreased by 94% at p9. Forskolin stimulated a dramatic increase in cAMP levels in cells at all passages examined. Our results show that sGC activity decreased significantly and rapidly with passage due to a down regulation of the alpha1 subunit mRNA, yet the adenylyl cyclase activity was not compromised. This study further emphasized the importance of considering passage number when using cell culture as a model system to study NO/cGMP pathway.     

Reconstitution of the vertebrate visual cascade using recombinant heterotrimeric transducin purified from Sf9 cells

For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (T alpha beta gamma) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant T alpha with GTP gamma S. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTP gamma S-loaded recombinant transducin (T(*)) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T(*) was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T(*) suggested that only the complex of PDE with two T(*) molecules, PDE. T(2)(*), was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T(*) in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin.     

The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.     

Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy.

In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic Expression of Cyclic GMP Phosphodiesterase Activity Defines Abnormal Photoreceptor Differentiation in Neurological Mutants of Inherited Retinal Degeneration

We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death.     

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.     

The gamma subunit of the rod photoreceptor cGMP phosphodiesterase can modulate the proteolysis of two cGMP binding cGMP-specific phosphodiesterases (PDE6 and PDE5) by caspase-3

We have investigated whether the proteolysis of members of the cGMP binding phosphodiesterases (PDE6, PDE5A1, and PDE10A2) by caspase-3 is modulated by the gamma inhibitor subunit of PDE6. We show here that purified caspase-3 proteolyses PDE6, an enzyme composed of two nonidentical catalytic subunits (termed alpha and beta) with molecular mass of 88 and 84 kDa. The proteolysis of PDE6 produced a single fragment with a molecular mass of 78 kDa. This corresponds to the possible cleavage of the caspase-3 consensus DFVD site (amino acids: 164-168) in the alpha subunit and leads to a 50% decrease in the cGMP hydrolysing activity of the enzyme. The addition of rod PDEgamma to the incubation completely blocked the cleavage of PDE6 by caspase-3. In contrast, rod PDEgamma converted PDE5A1 (molecular mass of 98 kDa) to a better substrate for caspase-3. This resulted in the formation of four major fragments with molecular mass of 82-83, 67, 43, and 34 kDa. In addition, caspase-3 induced an approximately 80% reduction in the activity of a partially purified preparation of PDE5A1 in the presence of rod PDEgamma. Caspase-3 also cleaved PDE10A2 (molecular mass of 95 kDa) to a single 48-kDa fragment. This was consistent with cleavage of the DLFD site (amino acids: 312-315) in PDE10A2. In contrast with both PDE6 and PDE5A1, rod PDEgamma was without effect on this enzyme. These data show that rod PDEgamma interacts with at least two members of the cGMP binding PDE family (PDE5A1 and PDE6) and can exert differential effects on the cleavage of these enzymes by caspase-3.     

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.