Diversity of calcium sensor signaling in zebrafish rod and cone photoreceptors

斑马鱼杆状和锥状光感受器中钙传感器信号的多样性

• 批准号: 322057463
• 负责人: Professor Dr. Karl-Wilhelm Koch
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/322057463?language=en
• 关键词: Diversity; calcium; sensor; signaling; zebrafish

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.

脊椎动物视网膜的有效光传导需要反馈机制，该机制使光感受器在光刺激后恢复其响应性。几个主要的反馈回路是由细胞质钙浓度的变化触发或控制的。这些事件停止光激发，细胞可以回到黑暗状态，并可以调整其对环境光强度的灵敏度。失活的一个主要步骤是特定G蛋白偶联受体激酶（GRKs）磷酸化视紫红质和视锥蛋白。这些酶在c末端磷酸化发光视紫红质或视锥蛋白，并通过与钙传感器恢复蛋白的相互作用进行调节。斑马鱼视网膜光感受器细胞表达杆状GRK1和锥状GRK7的2个同源基因和4个恢复基因。在钙结合、构象变化和靶相互作用方面，恢复蛋白同种异构体对钙的反应不同。这些发现表明斑马鱼恢复形态在照明期间对不同中间水平的细胞质钙浓度有一个逐步的反应。然而，recoverin变体的不同表达水平、它们与GRK靶点的共定位以及它们的相互作用特性表明，recoverin以及一种GRK变体（zGRK7b）的结合靶点未知。本项目的工作假设是，恢复和GRK变体的未知结合伙伴存在于光感受器细胞中。我们的目标是测试和识别未知的交互伙伴。我们将进一步验证这一假设，即非视觉视蛋白，视网膜G蛋白偶联受体（RGR）是否与GRK7b相互作用，以及RGR的光异构酶活性是否受这一相互作用过程的控制。该提案的第三个目标是使用基于荧光的报告试验将斑马鱼GRK活性与八种不同的视蛋白底物联系起来。视蛋白特异性c -末端将作为底物。通过这种方法，我们想要澄清，是否视蛋白激酶磷酸化其底物特异性或更非特异性的方式。