Phosphorylation of bovine rod photoreceptor cyclic GMP phosphodiesterase.
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ABSTRACT:
SUBMITTER: Udovichenko IP
PROVIDER: S-EPMC1134818 | biostudies-other | 1993 Oct
REPOSITORIES: biostudies-other
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Project description:Knowledge of the kinetics of the rod cyclic GMP phosphodiesterase is essential for understanding the kinetics and gain of the light response. Therefore, the interactions between Mg2+, cyclic GMP, and purified, trypsin-activated bovine rod cyclic GMP phosphodiesterase (EC 3.1.4.17) were examined. The effects of Mg2+ and of cyclic GMP on the rod phosphodiesterase activity were mutually concentration-dependent. Formation of a free Mg-cyclic GMP complex is unlikely due to its high dissociation constant (Kd = 19 mM). Plots of 1/velocity versus 1/[cyclic GMP] as a function of [Mg2+] and 1/velocity versus 1/[Mg2+] as a function of [cyclic GMP] intersected to the left of the 1/velocity axis. This is consistent with the formation of a ternary complex between the phosphodiesterase, Mg2+, and cyclic GMP. A competitive inhibitor of the phosphodiesterase relative to cyclic GMP, 3-isobutyl-1-methylxanthine, non-competitively inhibited the enzyme relative to Mg2+, Pb2+, a competitive inhibitor of the phosphodiesterase relative to Mg2+ [D. Srivastava, R.L. Hurwitz and D. A. Fox (1995) Toxicol. Appl. Pharmacol, in the press] non-competitively inhibited the enzyme relative to cyclic GMP. Collectively these results are suggestive of a rapid equilibrium random binding order of Mg2+ and cyclic GMP to the rod phosphodiesterase.
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Project description:Recently, the retinitis pigmentosa 3 (RP3) gene has been cloned and named retinitis pigmentosa GTPase regulator (RPGR). The amino-terminal half of RPGR is homologous to regulator of chromosome condensation (RCC1), the nucleotide exchange factor for the small GTP-binding protein Ran. In a yeast two-hybrid screen we identified the delta subunit of rod cyclic GMP phosphodiesterase (PDEdelta) as interacting with the RCC1-like domain (RLD) of RPGR (RPGR392). The interaction of RPGR with PDEdelta was confirmed by pull-down assays and plasmon surface resonance. The binding affinity was determined to be 90 nM. Six missense mutations at evolutionary conserved residues within the RLD, which were found in RP3 patients, were analyzed by using the two-hybrid system. All missense mutations showed reduced interaction with PDEdelta. A non-RP3-associated missense substitution outside the RLD, V36F, did not abolish the interaction with PDEdelta. PDEdelta is widely expressed and highly conserved across evolution and is proposed to regulate the membrane insertion or solubilization of prenylated proteins, including the catalytic subunits of the PDE holoenzyme involved in phototransduction and small GTP-binding proteins of the Rab family. These results suggest that RPGR mutations give rise to retinal degeneration by dysregulation of intracellular processes that determine protein localization and protein transport.
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Project description:The activity of many proteins orchestrating different biological processes is regulated by allostery, where ligand binding at one site alters the function of another site. Allosteric changes can be brought about by either a change in the dynamics of a protein, or alteration in its mean structure. We have investigated the mechanisms of allostery induced by chemically distinct ligands in the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. PDE5 is the target for catalytic site inhibitors, such as sildenafil, that are used for the treatment of erectile dysfunction and pulmonary hypertension. PDE5 is a multidomain protein and contains two N-terminal cGMP-specific phosphodiesterase, bacterial adenylyl cyclase, FhLA transcriptional regulator (GAF) domains, and a C-terminal catalytic domain. Cyclic GMP binding to the GAFa domain and sildenafil binding to the catalytic domain result in conformational changes, which to date have been studied either with individual domains or with purified enzyme. Employing intramolecular bioluminescence resonance energy transfer, which can monitor conformational changes both in vitro and in intact cells, we show that binding of cGMP and sildenafil to PDE5 results in distinct conformations of the protein. Metal ions bound to the catalytic site also allosterically modulated cGMP- and sildenafil-induced conformational changes. The sildenafil-induced conformational change was temperature-sensitive, whereas cGMP-induced conformational change was independent of temperature. This indicates that different allosteric ligands can regulate the conformation of a multidomain protein by distinct mechanisms. Importantly, this novel PDE5 sensor has general physiological and clinical relevance because it allows the identification of regulators that can modulate PDE5 conformation in vivo.
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Project description:A light-activated phosphodiesterase (PDE) from retinal rod outer segments (ROS) has been strongly implicated as a possible mediator in the propagation of the visual response. In view of the probable importance of this enzyme in the visual system, a comparison of the PDE proteins from ROS of different evolutionary species was made. Partial purification of the PDE, as measured by enzymic activity, followed by resolution of the protein components on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicated that the major Coomassie Blue-staining species in the ROS of all species studied is a doublet of 84000-88000 Da. After radioiodination, this doublet was converted in all but the frog proteins into a single band of 85000 Da. Two-dimensional tryptic peptide mapping of the radioiodinated peptides indicated that at least six major peptides of the putative PDE have been conserved in all of the species studies. If this protein is indeed associated with PDE activity, such conserved peptides may play an important role in the catalytic and/or regulatory functions of the enzyme.
| S-EPMC1153190 | biostudies-other
Project description:Light-activated cyclic GMP-phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells which regulates the level of the internal transmitter cyclic GMP. PDE consists of catalytic P alpha and P beta subunits, and two copies of inhibitory P gamma subunit. The two P gamma subunits block the enzyme's activity in the dark and are removed by the alpha-subunit of transducin (alpha 1) upon light-activation of photoreceptor cells. Here we have examined the role of various regions of P gamma, the N-terminal, the central cationic and the C-terminal regions, in interaction with the catalytic subunits of PDE. N-Terminal truncation of P gamma (12-87-P gamma) did not change the potency of PDE inhibition, and thus we conclude that the P gamma N-terminal region is not critical for P gamma-P alpha beta interaction. The central region, 24-46-P gamma, participates in interaction with the catalytic P alpha beta subunits. A synthetic peptide corresponding to this site inhibited approximately 50% of trypsin-activated PDE (tPDE) (Ki approximately 15 microM) and competed with P gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that 125I-Tyr-24-46-P gamma peptide bound with high affinity to tPDE, but not to P alpha beta gamma 2. The C-terminal region of 46-87-P gamma was found to be the major region involved in inhibition of PDE. It fully inhibited tPDE with a Ki of approximately 0.8 microM. It also bound to tPDE, but not P alpha beta gamma 2, in h.p.l.c. gel-filtration experiments. In addition, P gamma was cross-linked by p-phenylenedimaleimide to both P alpha and P beta, as was shown by using subunit-specific anti-P alpha, -P beta and -P gamma antibodies. Cys68 of P gamma, which presumably participates in cross-linking, is located near the P gamma C-terminus. These data provide evidence for two regions of P gamma that interact with, and inhibit, P alpha beta. The central region, 24-46 P gamma, is important in binding, but inhibits PDE only weakly, whereas the C-terminal region is most important for PDE inhibition. These results help to explain the well-known fact that P gamma trypsin-activation and C-terminal truncation both lead to PDE activation. Furthermore, our findings on the mechanism of PDE inhibition of P gamma are relevant for understanding the mechanism of PDE activation by transducin.
| S-EPMC1131025 | biostudies-other
Project description:The vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light-dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors.We used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas.As in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil.We show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light-dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.
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Project description:Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.
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Project description:Motility, chemotaxis, and the acrosome reaction of animal sperm are all regulated by cyclic nucleotides and protein phosphorylation. One of the cyclic AMP-dependent protein kinase (PKA) substrates in sea urchin sperm is a member of the phosphodiesterase (PDE) family. The molecular identity and in vivo function of this PDE remained unknown. Here we cloned and characterized this sea urchin sperm PDE (suPDE5), which is an ortholog of human PDE5. The recombinant catalytic domain of suPDE5 hydrolyzes only cyclic GMP (cGMP) and the activity is pH-dependent. Phospho-suPDE5 localizes mainly to sperm flagella and the phosphorylation increases when sperm contact the jelly layer surrounding eggs. In vitro dephosphorylation of suPDE5 decreases its activity by approximately 50%. PDE5 inhibitors such as Viagra block the activity of suPDE5 and increase sperm motility. This is the first PDE5 protein to be discovered in animal sperm. The data are consistent with the hypothesis that suPDE5 regulates cGMP levels in sperm, which in turn modulate sperm motility.
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Project description:As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory ?-subunit, the prenyl-binding protein (PrBP/?), and activated transducin. In solution, the PDE6 catalytic dimer (P??) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory P? subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when P?? was reconstituted with P? truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/? bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of P? with the catalytic dimer.
| S-EPMC3370194 | biostudies-literature
Project description:Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.
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