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J. Biol. Chem., Vol. 282, Issue 30, 21645-21652, July 27, 2007

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Activation and Inhibition of Photoreceptor Guanylyl Cyclase by Guanylyl Cyclase Activating Protein 1 (GCAP-1)

THE FUNCTIONAL ROLE OF Mg²⁺/Ca²⁺ EXCHANGE IN EF-HAND DOMAINS^*

From the Hafter Research Laboratories, Pennsylvania College of Optometry, Elkins Park, Pennsylvania 19027

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca²⁺/Mg²⁺ sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg²⁺ in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg²⁺ binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg²⁺ binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg²⁺ binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca²⁺ binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca²⁺-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca²⁺ binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca²⁺, but did not prevent the inhibition. We conclude that 1) Mg²⁺ binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg²⁺ or Ca²⁺ binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca²⁺ binding in EF-hand 4.

Received for publication, March 20, 2007 , and in revised form, May 18, 2007.

^* This work was supported in part by National Institutes of Health Grant EY11522 and the Pennsylvania Lions Sight Conservation and Eye Research Foundation. 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 Martin and Florence Hafter Professor of Pharmacology. To whom correspondence should be addressed: 8360 Old York Rd., Elkins Park, PA 19027. Tel.: 215-780-1468; Fax: 215-780-1464; E-mail: adizhoor{at}pco.edu.

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