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J Biol Chem, Vol. 274, Issue 16, 10823-10832, April 16, 1999

Mapping Functional Domains of the Guanylate Cyclase Regulator Protein, GCAP-2

Elena V. Olshevskaya, Sergei Boikov, Alexander Ermilov, Dmitri Krylov^§, James B. Hurley^§, and Alexander M. Dizhoor^¶

From the  Department of Ophthalmology/Kresge Eye Institute and the ^¶ Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201 and the ^§ Department of Biochemistry and HHMI, University of Washington, Seattle, Washington 98195

Guanylate cyclase regulator protein (GCAP)-2 is a Ca²⁺-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca²⁺-sensitive manner. GCAP-2 activates RetGC at free Ca²⁺ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca²⁺ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca²⁺-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe⁷⁸ and Asp¹¹³ determines whether GCAP-2 activates outer segment RetGC in low or high Ca²⁺ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca²⁺ concentrations. 2) The amino acid sequence of GCAP-2 between Lys²⁹ and Phe⁴⁸ that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca²⁺ and inhibition at high Ca²⁺ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val¹⁷¹-Asn¹⁸⁹, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca²⁺-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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