title: Functional EF-hands in neuronal calcium sensor GCAP2 determine its phosphorylation state and subcellular distribution in vivo, and are essential for photoreceptor cell integrity.
abstract: 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.
authors: Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors: Natalia López-Del
authors: NL
authors: Hoyo
authors: Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
authors: Santiago
authors: S
authors: López-Begines
authors: Department of Physiological Sciences II, University of Barcelona-Bellvitge Health Science Campus, Barcelona, Spain.
authors: Jose Luis
authors: JL
authors: Rosa
authors: Department of Cell and Neurobiology, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
authors: Jeannie
authors: J
authors: Chen
authors: Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain; Department of Pathology and Experimental Therapeutics, University of Barcelona-Bellvitge Health Science Campus, Barcelona, Spain.
authors: Ana
authors: A
authors: Méndez
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D020832:EF Hand Motifs / Q000235:genetics; D051604:Guanylate Cyclase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; C496740:Guca1b protein, mouse; D006152:Cyclic GMP; D002118:Calcium