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• W2018309862 startingPage "10140" @default.
• W2018309862 abstract "Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca(2+) binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the binding patch prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94." @default.
• W2018309862 created "2016-06-24" @default.
• W2018309862 creator A5003668929 @default.
• W2018309862 creator A5022732606 @default.
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• W2018309862 creator A5057047177 @default.
• W2018309862 creator A5059222843 @default.
• W2018309862 date "2014-04-01" @default.
• W2018309862 modified "2023-10-12" @default.
• W2018309862 title "Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)" @default.
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• W2018309862 doi "https://doi.org/10.1074/jbc.m113.540716" @default.
• W2018309862 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3974984" @default.
• W2018309862 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24567338" @default.
• W2018309862 hasPublicationYear "2014" @default.
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