Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity

• Published in: PLoS genetics Vol. 10; no. 7; p. e1004480
• Main Authors: Hoyo, Natalia López-Del, López-Begines, Santiago, Rosa, Jose Luis, Chen, Jeannie, Méndez, Ana
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-07-2014; Public Library of Science (PLoS)
• Subjects: Animals; Biology and Life Sciences; Calcium; Calcium - metabolism; Calcium Signaling; Calcium-Binding Proteins - metabolism; Colleges & universities; Cyclic GMP - metabolism; EF Hand Motifs - genetics; Experiments; Fotoreceptors; Genetic disorders; Genetic research; Genetic transformation; Guanylate Cyclase-Activating Proteins - genetics; Guanylate Cyclase-Activating Proteins - metabolism; Humans; Light; Medicine and Health Sciences; Mice; Mice (Laboratory animals); Neurons; Neurons - metabolism; Phosphorylation; Photoreceptor Cells - metabolism; Photoreceptors; Physiological aspects; Proteins; Ratolins (Animals de laboratori); Research and Analysis Methods; Retina; Retina - metabolism; Retina - pathology; Retinal Degeneration - genetics; Retinal Degeneration - metabolism; Rodents; Sensors; Transformació genètica; Values; Massachusetts; California
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1004480

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca2+-free and Ca2+-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca2+ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca2+. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF-GCAP2), we show that GCAP2 locked in its Ca2+-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca2+-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF-GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca2+-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca2+ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in "equivalent-light" scenarios.