Probing the catalytic sites and activation mechanism of photoreceptor phosphodiesterase using radiolabeled phosphodiesterase inhibitors

Abstract

Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory gamma-subunits (Pgamma) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pgamma inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [(3)H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [(3)H]vardenafil binds stoichiometrically to both the alpha- and beta-subunits of the activated PDE6 heterodimer. [(3)H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pgamma, consistent with Pgamma blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [(3)H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [(3)H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pgamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.

FIGURE 1.

Histone II-AS stimulates binding of [³H]vardenafil to the active sites of PDE6 catalytic dimer. Purified Pαβ catalytic dimer (6 nm) lacking Pγ was incubated with 70 nm [³H]vardenafil and increasing amounts of histone II-AS at room temperature for 40 min. The amount of radiolabeled drug was determined by a membrane filtration assay (see “Experimental Procedures”). The data represent one of three similar experiments.

FIGURE 2.

Comparison of [³H]vardenafil binding to Pαβ stimulated by histones or Pγ. Purified Pαβ (5 nm) and 70 nm [³H]vardenafil were incubated with the following for 40 min at room temperature: 10 mm Tris (pH 7.5), 0.2 mg/ml histone II-AS, 0.2 mg/ml histone VIII-S, 10 μm Pγ, or 10 μm Pγ63-87. Vardenafil binding was quantified by the membrane filtration assay. The data represent the means ± range for duplicate measurements from one representative experiment; similar results were obtained in at least two other experiments (except the Pγ63-87 condition, which was tested one other time).

FIGURE 3.

[³H]Vardenafil binds stoichiometrically to each Pαβ catalytic subunit, but only in the absence of Pγ. Purified Pαβ (2.5 nm; filled circles) or Pαβ reconstituted with 10 μm Pγ (open circles) was incubated with 0.2 mg/ml histone II-AS and the indicated concentration of [³H]vardenafil. The samples were incubated for 40 min before membrane filtration. Vardenafil binding was normalized to the Pαβ concentration, as estimated by both [³H]cGMP binding assay, as well as hydrolytic activity measurements (which agreed to within 10%). The solid line represents the fit of the data for [³H]vardenafil binding to Pαβ assuming a single class of binding sites (apparent K_D = 7.8 nm and B_max = 2.0). The data are representative of three similar experiments.

FIGURE 4.

Transducin-activated PDE6 achieves only one-half of the extent of [³H]vardenafil binding or hydrolytic activity compared with Pαβ. Light-exposed ROS homogenates (containing 4 nm PDE6) were prepared (see “Experimental Procedures”), and portions were incubated with either buffer (nonactivated) or with 50 μm GTPγS (to activate transducin). A separate portion was exposed to trypsin to maximally activate PDE6 catalysis. The samples were incubated either with 74 nm [³H]vardenafil for 40 min before membrane filtration (black bars) or with cGMP for hydrolytic activity assays (gray bars). The data are the means ± S.D. of three experiments.

FIGURE 5.

A large excess of activated transducin α-subunit fully relieves Pγ inhibition of the active sites of the PDE6 catalytic dimer. The indicated concentrations of purified transducin α-subunits with GTPγS bound (Tα-GTPγS) were added to either 1 nm purified PDE6 holoenzyme (filled circles) or 2 nm ROS-PDE6 previously incubated with 50 μm GTPγS to activate endogenous transducin (open circles). In both cases, the hydrolytic rates were normalized by comparison with the catalytic activity of samples that had been treated with trypsin to fully activate PDE6 catalysis. The data are the means ± S.D. of two experiments.

FIGURE 6.

Model for PDE6 activation by transducin. The nonactivated PDE6 holoenzyme is a catalytic heterodimer (gray) in which each catalytic subunit binds an inhibitory Pγ subunit (black S-shaped rod) at multiple sites within the GAF domains and the catalytic domain (reviewed in Refs. and 15). The depiction of the catalytic dimer (based on the structure reported in Ref. 18) shows an active site (ellipse) within each catalytic domain and a cGMP-binding site (circle) within the GAF domain. Proteolytic removal of the Pγ subunits (model A) relieves inhibition of catalysis and permits vardenafil binding to each catalytic site. Two alternative models (models B and C) are presented to explain how activation of ROS membrane-associated PDE6 holoenzyme by activated transducin α-subunit (Tα*; dark gray oval) might occur in a biphasic manner. In model B, Tα* has one high affinity binding site on PDE6 holoenzyme that efficiently binds Pγ and thereby relieves inhibition at one active site, with a second, low affinity binding site that requires high concentrations of Tα* to relieve Pγ inhibition at the second catalytic site. In model C, binding of one Tα* to PDE6 holoenzyme (thereby activating PDE6 to one-half of its full catalytic potential) reduces the effectiveness of a second Tα* to displace the second Pγ from its inhibitory sites of interaction within the PDE6 catalytic domain. In both models, subsequent dissociation of the Tα*-Pγ complex from PDE6 catalytic dimer is not depicted but does occur in certain circumstances (54–56).