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J. Biol. Chem., Vol. 278, Issue 35, 33150-33160, August 29, 2003

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A Critical Role for ATP in the Stimulation of Retinal Guanylyl Cyclase by Guanylyl Cyclase-activating Proteins^*

Akio Yamazaki   ¶ ||, Hao Yu  , Matsuyo Yamazaki  , Hanayo Honkawa  , Isao Matsuura  , Jiro Usukura ** and Russell K. Yamazaki ¶

From the Kresge Eye Institute and the Departments of Ophthalmology and ^¶Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan 48201 and the ^**Department of Anatomy and Cell Biology, Nagoya University, School of Medicine, Nagoya 466, Japan

It has been believed that retinal guanylyl cyclase (retGC), a key enzyme in the cGMP recovery to the dark state, is solely activated by guanylyl cyclase-activating proteins (GCAPs) in a Ca²⁺-sensitive manner. However, a question has arisen as to whether the observed GCAP stimulation of retGC is sufficient to account for the cGMP recovery because the stimulated activity measured in vitro is less than the light/GTP-activated cGMP phosphodiesterase activity. Here we report that the retGC activation by GCAPs is larger than previously reported and that a preincubation with adenine nucleotide is essential for the large activation. Under certain conditions, ATP is two times more effective than adenylyl imidodiphosphate (AMP-PNP), a hydrolysis-resistant ATP analog; however, this study mainly used AMP-PNP to focus on the role of adenine nucleotide binding to retGC. When photoreceptor outer segment homogenates are preincubated with AMP-PNP (EC₅₀ = 0.65 ± 0.20 mM), GCAP2 enhanced the retGC activity 10–13 times over the control rate. Without AMP-PNP, GCAP2 stimulated the control activity only 3–4-fold as in previous reports. The large activation is due to a GCAP2-dependent increase in V_max without an alteration of retGC affinity for GCAP2 (EC₅₀ = 47.9 ± 2.7 nM). GCAP1 stimulated retGC activity in a similar fashion but with lower affinity (EC₅₀ = 308 nM). In the AMP-PNP preincubation, low Ca²⁺ concentrations are not required, and retGC exists as a monomeric form. This large activation is accomplished through enhanced action of GCAPs as shown by Ca²⁺ inhibition of the activity (IC₅₀ = 178 nM). We propose that retGC is activated by a two-step mechanism: a conformational change by ATP binding to its kinase homology domain under high Ca²⁺ concentrations that allows large enhancement of GCAP activation under low Ca²⁺ concentrations.

Received for publication, April 9, 2003 , and in revised form, May 27, 2003.

^* This work was supported in part by National Institutes of Health Grant EY09631 and an unrestricted grant from Research to Prevent Blindness. 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.

^|| To whom correspondence should be addressed. Tel.: 313-577-2009; Fax: 313-577-0238; E-mail: ayamazak{at}med.wayne.edu.

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