Title

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 (P gamma) 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 P gamma 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 P gamma, consistent with P gamma 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 P gamma inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.

Publication Date

11-13-2009

Journal Title

Journal of Biological Chemistry

Publisher

American Society for Biochemistry and Molecular Biology

Digital Object Identifier (DOI)

10.1074/jbc.M109.018606

Scientific Contribution Number

2392

Document Type

Article

Rights

© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.