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J. Biol. Chem., Vol. 279, Issue 48, 50342-50349, November 26, 2004

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Ca²⁺-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis^*

Igor V. Peshenko, Elena V. Olshevskaya, and Alexander M. Dizhoor, Martin and Florence Hafter professor of pharmacology

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

Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca²⁺ but activate retGC when Ca²⁺ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca²⁺. We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotide-dependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser²⁰¹, had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca²⁺ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca²⁺-insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca²⁺-sensitive manner. The Ca²⁺-dependent conformational changes in GCAP-2 affect the areas around Glu⁶² residue in the entering helix of EF-hand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu¹³⁶–Glu ¹³⁸ between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser²⁰¹ from the constraint caused by the Ca²⁺-bound conformation.

Received for publication, July 30, 2004 , and in revised form, September 15, 2004.

^* This work was supported by National Institutes of Health Grant EY11522 from the NEI. 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: Pennsylvania College of Optometry, 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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