Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits

Sitaramayya, Ari; Harkness, Julia; Parkes, John H.; Gonzalez-Oliva, Clara; Liebman, Paul A.

doi:10.1021/bi00351a021

Cited by 91 publications

(59 citation statements)

References 29 publications

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“…Biochemical evidence indicates that these proteins and PDE6␣␤ bind PDE6␥ simultaneously (56,60), so this compact structure is likely compatible with tight binding to PDE6␣␤. In this scenario, nearly the entire N-terminal half of the molecule must adopt an extended chain conformation to reach from the catalytic base of PDE6 to the GAFa domains.…”

Section: Discussionmentioning

confidence: 96%

“…Trypsinization of PDE6 causes degradation and/or dissociation of the entire PDE6␥ peptide (58). In contrast, when activated by transducin, PDE6␥ is displaced from the active site but may remain bound to the complex (50,51,56,59,60). In addition, the C-terminal cleavages of PDE6␣ and -␤ caused by trypsin (49), although not sufficient for the rearrangement, may contribute to structural instability.…”

Section: Discussionmentioning

confidence: 99%

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Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

Zhang

Constantine

et al. 2015

Journal of Biological Chemistry

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Background: PDE6, the rod photoreceptor phosphodiesterase, is the key effector enzyme in phototransduction. Results: EM reconstructions of PDE6 complexed with various probes are presented. Conclusion:Fitting of x-ray structures yielded an atomic model of the catalytic subunits, and the locations of other structural features are indicated. Significance: These data offer the most complete view to date of the PDE6 holoenzyme.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

Zhang

Constantine

et al. 2015

Journal of Biological Chemistry

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“…The sonicated vesicles were stored on ice until use. Bovine membrane fractions were prepared as described elsewhere and washed to remove extrinsic membrane proteins (17 were added to the cis chamber, and the chamber was briefly stirred. In the presence of an osmotic gradient across the bilayer and 50 ,aM cGMP in the cis chamber, conductance events were generally seen in 5-15 min.…”

Section: Methodsmentioning

confidence: 99%

“…The sonicated vesicles were stored on ice until use. Bovine membrane fractions were prepared as described elsewhere and washed to remove extrinsic membrane proteins (17 cGMP to ensure that no residual nucleotide was present for the initial measurements; in these experiments the ROS vesicles were exposed to the bilayer for 10-20 min, after which the cis chamber was perfused with buffer. Incorporations were seen in >90% of all experiments.…”

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confidence: 99%

Incorporation of a retinal rod cGMP-dependent conductance into planar bilayers.

Tanaka¹,

Furman²,

Cobbs³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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The light-modulated current of vertebrate retinal rods flows through a 3',S'-cyclic GMP-dependent conductance located in the outer segment plasma membrane. We report the incorporation into planar bilayers of a conductance derived from vertebrate rod outer segment membranes specifically activated by cGMP but not by cAMP, 5'-GMP, GTP, or 5'-AMP. When the mean currents were measured as a function of increasing cGMP concentration, maximal activation occurred at concentrations <50 AMM. Washout of cGMP rapidly reversed the effect. The apparent half-saturating concentrations were between 12 and 27 ,AM. Sodium, lithium, cesium, and potassium supported current in the presence of low concentrations of Ca21, Mg2', and 100 ,.M cGMP; choline did not. Removal of the divalent cations reversibly increased the currents. When calcium was the only current-carrying cation, attenuated currents were seen. These experiments support the hypothesis that calcium is a permeant blocker of the conductance. At low concentrations of cGMP in solutions also containing 0.5 mM EDTA, brief current spikes occurred with amplitudes from 0.5 to 4 pA at 50 mV. These spikes differed from the well-defined, unitary conductance steps usually associated with the opening and closing of ion channels. Occasionally we saw longer-lasting channel-like events; however, amplitude histograms did not resolve discrete conductance levels.The cGMP-dependent conductance of the retinal rod outer segment (ROS) plasma membrane has been identified with the light-modulated conductance (1-8). The relation between current activation and cGMP concentration is reversible and cooperative. In addition, cGMP-dependent cation fluxes have been reported in rod disc membranes (9-12).Molecular details about the cGMP-dependent ion transport in photoreceptor plasma membranes have undergone significant clarification in the past 2 years (for review, see ref. 13). Initial measurements of the light-sensitive conductance, which failed to detect single channels, were interpreted as supporting either a carrier or pore mechanism with <100 fS maximal conductance (1,8). Recently, however, single channels of -8 and =24 pS have been recorded in excised patches at low agonist concentrations in the absence of divalent cations (4, 5). Rapid, intermittent blocking by divalent cations, proposed to account for the low conductance estimates from noise measurements on photoreceptors (14), was also seen in these excised patches (4, 5).We report the incorporation into planar lipid bilayers of a cGMP-dependent conductance from vertebrate photoreceptor membranes. Activation of this conductance shows a specific, cooperative dependence on cGMP that is reversible. The conductance is cation selective, preferring monovalent over divalent cations. In the presence of divalent cations, the cGMP-dependent current shows bursts of noise consisting of brief-current spikes; without divalent cations, longer-lasting channel-like events are observed with conductances similar to those seen in the aforementioned patch reco...

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“…The Toe subunit may bind to the oe-and fl-subunits of PDE, preventing the 7-subunit from reassociating [11].…”

Section: Introductionmentioning

confidence: 99%

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase γ‐subunit

1987

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Synthetic peptides corresponding to various regions of the light‐activated guanosine 3′,5′‐cyclic monophosphate phosphodiesterase (PDE) γ‐subunit (PDEγ) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDEγ residues 31–45, inhibited PDE activity and GTPase activity in a dose‐dependent manner. The GTPase activity was inhibited by PDEγ‐3 non‐competitively. This region of the PDEγ subunit may be involved in the direct interaction of transducin and PDEαβ with PDEγ.

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Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits

Cited by 91 publications

References 29 publications

Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

Domain Organization and Conformational Plasticity of the G Protein Effector, PDE6

Incorporation of a retinal rod cGMP-dependent conductance into planar bilayers.

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase γ‐subunit

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