Mapping Functional Domains of the Guanylate Cyclase Regulator Protein, GCAP-2
| Autor: | James B. Hurley, Alexander M. Dizhoor, Dmitri M. Krylov, Elena V. Olshevskaya, Sergei G. Boikov, Alexander Ermilov |
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| Rok vydání: | 1999 |
| Předmět: |
Molecular Sequence Data
Regulator Biology Biochemistry Cell Line law.invention law Recoverin Animals Humans Photoreceptor Cells Amino Acid Sequence Molecular Biology Peptide sequence Neurocalcin Sequence Homology Amino Acid Calcium-Binding Proteins HEK 293 cells Cell Biology Photoreceptor outer segment membrane Fusion protein Guanylate Cyclase-Activating Proteins Recombinant Proteins Cell biology Mutagenesis Site-Directed biology.protein Recombinant DNA Cattle |
| Zdroj: | Journal of Biological Chemistry. 274:10823-10832 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.274.16.10823 |
| Popis: | 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 nmlevel, 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. |
| Databáze: | OpenAIRE |
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