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J. Biol. Chem., Vol. 283, Issue 44, 29699-29705, October 31, 2008

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Direct Allosteric Regulation between the GAF Domain and Catalytic Domain of Photoreceptor Phosphodiesterase PDE6^*

Xiu-Jun Zhang, Karyn B. Cahill, Arye Elfenbein¹, Vadim Y. Arshavsky, and Rick H. Cote²

From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824 and the Departments of Ophthalmology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory subunit (P), known to directly inhibit PDE6 catalysis, was increased 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of P to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of P1–60, P lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. P1–60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although P-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.

Received for publication, May 22, 2008 , and in revised form, August 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant EY-05798. This is Scientific Contribution Number 2361 from the New Hampshire Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755.

² To whom correspondence should be addressed. Tel.: 603-862-2458; Fax: 603-862-4013; E-mail: rick.cote{at}unh.edu.

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