Diversity of calcium sensor signaling in zebrafish rod and cone photoreceptors

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Effective phototransduction in vertebrate retinae requires feedback mechanisms, which enable the photoreceptors to regain their responsiveness after light stimulation. Several of the main feedback loops are triggered or controlled by changes in the cytoplasmic calcium concentration. These events stop photoexcitation, the cell can return to the dark state and it can adjust its sensitivity to ambient light intensities. One major step of deactivation is the phosphorylation of rhodopsin and cone opsins by specific G protein-coupled receptor kinases (GRKs). These enzymes phosphorylate illuminated rhodopsin or cone opsins at their C-termini and are regulated by interaction with the calcium sensor recoverin. Zebrafish retinae express two orthologs of rod GRK1 and cone GRK7 and four recoverin genes in zebrafish photoreceptor cells. Recoverin isoforms respond differently to calcium with respect to calcium binding, changes in conformation and target interaction. These findings indicate a step-by-step response of zebrafish recoverin forms to different intermediate levels of cytoplasmic calcium-concentration during illumination. However, different expression levels of recoverin variants, their co-localization with GRK targets and their interaction properties indicate unknown binding targets for recoverin as well as for one GRK variant (zGRK7b).The working hypothesis of the present project is that unknown binding partners for recoverin and GRK variants exist in photoreceptor cells. We aim to test for and identify unknown interaction partners. We will further test the hypothesis, whether a non-visual opsin, the retinal G protein-coupled receptor (RGR) interacts with GRK7b, and whether the photo-isomerase activity of RGR is controlled by this interaction process. A third aim of the proposal is to relate zebrafish GRK activities to the eight different opsin substrates using a fluorescence based reporter assay. Opsin specific C-termini will serve as substrates. By this approach, we want to clarify, whether Opsin kinases phosphorylate their substrates in a specific or more non-specific manner.