Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha-subunit, GtalphaGDPAlF4-, while its binding to GtalphaGTPgammaS or GtalphaGDP was weak. hRGSr and Pgamma did not compete for the interaction with GtalphaGDPAlF4-. Affinity of the Pgamma-GtalphaGDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtalphaGDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PgammaBC). Binding of hRGSr to GtalphaGDPAlF4- complexed with PgammaBC resulted in a maximal approximately 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtalphaGDPAlF4- (Kd 35 nM). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1. Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates exceeded 0.7 s-1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.
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PMID:Regulation of transducin GTPase activity by human retinal RGS. 921 88

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
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PMID:Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins. 955 60

In the current concept of phototransduction, the concentration of cGMP in retinal rod outer segments is controlled by the balance of two enzyme activities: cGMP phosphodiesterase (PDE) and guanylyl cyclase (GC). However, no protein directly mediates these two enzyme systems. Here we show that RGS9, which is suggested to control PDE activity through regulation of transducin GTPase activity (He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95-102), directly interacts with GC. When proteins in the Triton X-100-insoluble fraction of bovine rod outer segments were isolated by two-dimensional gel electrophoresis and binding of GC to these proteins was examined using a GC-specific antibody, proteins (55 and 32 kDa) were found to interact with GC. However, the activity of GC bound to the 55-kDa protein was not detected. This observation was elucidated by the finding that the 55-kDa protein inhibited GC activity in a dose-dependent manner. Amino acid sequence showed that five peptides derived from the 55-kDa protein were identical to corresponding peptides of RGS9. Together with other biochemical characterization of the 55-kDa protein, these observations indicate that the 55-kDa protein is RGS9 and that RGS9 inhibits GC. RGS9 may serve as a mediator between the PDE and GC systems.
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PMID:A possible role of RGS9 in phototransduction. A bridge between the cGMP-phosphodiesterase system and the guanylyl cyclase system. 971 27

The alpha subunit (Galpha) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Galpha structure essentially comprises a GTPase "Ras-like" domain (RasD) and a unique alpha-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Galpha (Galphat) and the closely related gustducin (Galphag), but not Galphai1, Galphas, or Galphaq synergistically enhance guanosine 5'-gamma[-thio]triphosphate bound Galphat (GalphatGTPgammaS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. GalphatGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Galphat with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Galphat within the PDE catalytic core in addition to the sites for the inhibitory Pgamma subunits. The HD moiety of GalphatGDP is an attenuator of the activated catalytic core, whereas in the presence of activated GalphatGTPgammaS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of Galphat activation enhances the PDE activation produced by subsaturating levels of Galphat, suggesting a HD-moiety synergism from a transient conformation of Galphat. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.
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PMID:The helical domain of a G protein alpha subunit is a regulator of its effector. 978 8

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.
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PMID:The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9. 1008 18

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin alpha-subunit (Gtalpha) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the gamma-subunit of the effector enzyme, cGMP-phosphodiesterase (Pgamma). In contrast, Pgamma attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the Pgamma subunit exerts its effects on the GTPase activity of the Gtalpha-RGS complex via the C-terminal domain, Pgamma-63-87. The structural determinants that control the direction of Pgamma effects on the RGS-Gtalpha system are localized within the RGS domains. The addition of Pgamma caused an increase in the maximal stimulation of Gtalpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gtalpha GTPase activity by chimeric RGS16 and RGS9 proteins and Pgamma has been investigated. This analysis suggests that in addition to the differences in primary structures, the overall conformations of the RGS fold in RGS9 and RGS16 are likely to be responsible for the opposite effects of Pgamma on the RGS9 and RGS16 GAP activity. The RGS9 alpha3-alpha5 region constituted the minimal insertion of the RGS9 domain into RGS16 that reversed the inhibitory effect of Pgamma. A model of the RGS9 complex with Gtalpha shows the alpha3-alpha5 helices in RGS9 facing the proximate Pgamma binding site on Gtalpha. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by Pgamma involves a more rigid stabilization of the Gtalpha switch regions when Gtalpha is bound to both RGS9 and Pgamma.
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PMID:Modulation of transducin GTPase activity by chimeric RGS16 and RGS9 regulators of G protein signaling and the effector molecule. 1021 94

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca(2+)-mediated, indirect connection. Using a retinal "regulator of G-protein signaling" (RGS9-1) and its fragments, we show that the N-terminus of RGS9-1 inhibits retGC activity. We also indicate that the GGL domain and/or the RGS domain function as an internal suppressor against the N-terminus, suggesting that proteins bound to these domains regulate the inhibitory activity of the N-terminus. Direct interaction of retGC with RGS9-1 and its N-terminus is also proved by immunoprecipitation and an overlay technique. Since RGS9-1 also controls the lifetime of transducin-activated PDE through regulating GTPase activity of transducin, this study strongly suggests that RGS9-1 mediates the direct interaction between PDE and retGC systems, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.
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PMID:Inhibition of retinal guanylyl cyclase by the RGS9-1 N-terminus. 1148 1

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.
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PMID:RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex. 1149 24

Vertebrate cone and rod photoreceptor cells use similar mechanisms to transduce light signals into electrical signals, but their responses to light differ in sensitivity and kinetics. To assess the role of G-protein GTP hydrolysis kinetics in mammalian cone photoresponses, we have characterized photoresponses and GTPase regulatory components of cones and rods from the cone-dominant retina of the eastern chipmunk. Sensitivity, based on the stimulus strength required for a half-maximum response, of the M-cone population was 38-fold lower than that of the rods. The relatively lower cone sensitivity could be attributed in part to lower amplification in the rising phase and in part to faster recovery kinetics. At a molecular level, cloning of chipmunk cDNA and expression of recombinant proteins provided standards for quantitative immunoblot analysis of proteins involved in GTPase acceleration. The ratio of the cGMP-phosphodiesterase inhibitory subunit gamma to cone pigment, 1:68, was similar to the levels observed for ratios to rhodopsin in bovine retina, 1:76, or mouse retina, 1:65. In contrast, the ratio to pigment of the GTPase-accelerating protein RGS9-1 was 1:62, more than 10 times higher than ratios observed in rod-dominant retinas. Immunoprecipitation experiments revealed that, in contrast to rods, RGS9-1 in chipmunk retina is associated with both the short and long isoforms of its partner subunit G(beta5). The much higher levels of the GTPase-accelerating protein complex in cones, compared with rods, suggest a role for GTPase acceleration in obtaining rapid photoresponse kinetics.
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PMID:GTPase regulators and photoresponses in cones of the eastern chipmunk. 1259 17

The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway.
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PMID:Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway. 1554 76


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