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.
Highlights
The superfamily of phosphodiesterase (PDE)2 enzymes plays a critical role in maintaining the cellular levels of cAMP and cGMP [1]
Histone II-AS Stabilizes [3H]Vardenafil Binding to the Catalytic Sites of PDE6 Catalytic Dimer—Previous work has shown that most of the so-called PDE5-selective inhibitors inhibit the catalytic activity of the closely related photoreceptor PDE6 [23,24,25]
Consistent with previous studies of PDE5 binding to radiolabeled inhibitors [35], we observed that histone II-AS stabilized [3H]vardenafil binding to PDE6 catalytic dimer (P␣) in a concentration-dependent manner (Fig. 1)
Summary
The superfamily of phosphodiesterase (PDE)2 enzymes plays a critical role in maintaining the cellular levels of cAMP and cGMP [1]. During the first steps in vision, photoisomerized rhodopsin activates transducin, which binds GTP and releases its activated ␣-subunit (T␣-GTP) to activate membrane-associated rod PDE holoenzyme by displacing the inhibitory ␥-subunit (P␥) from the active sites of the PDE6 catalytic dimer (P␣).
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