Regulation of Drosophila arrestins in light adaptation

描述（由申请人提供）：本提案的目的是使用果蝇眼作为模型系统，研究磷酸化如何调节arrestin活性以控制视紫红质的输出，从而微调视觉信号。视觉信号传导是G蛋白偶联的机制，其中光激活视紫红质以启动导致光感受器去极化的级联生化事件。已经确定视紫红质的活性受抑制蛋白调节，抑制蛋白阻断与下游G蛋白的相互作用。在果蝇的光感受器中，有两种不同的arrestin同系物，Arr 1和Arr 2。研究支持Arr 1促进视紫红质的内吞作用，而Arr 2是通过解偶联激活的视紫红质来快速失活视觉反应所必需的。关于Arr 1和Arr 2在体内是如何调节的知之甚少。Arr 1和Arr 2都经历光依赖性磷酸化，并且Arr 2在体外被Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）磷酸化。为了研究CaMKII如何调节Arr 2，我们首先通过Ca 2+依赖性自磷酸化在视网膜中鉴定了CaMKII。我们发现冈田酸大大增强了CaMKII的磷酸化，这表明CaMKII的去磷酸化受蛋白磷酸酶2A（PP 2A）的调节。值得注意的是，我们发现PP 2A的催化亚基可以被抗CaMKII抗体共免疫沉淀。为了深入了解CaMKII在体内的作用，我们证明了在苍蝇中抑制CaMKII会导致视觉反应增强和异常的光适应。这些数据表明CaMKII可能通过催化Arr 2的磷酸化来对视觉反应进行负调节。相反，在mts突变体中PP 2A活性降低导致视觉反应降低而不影响光适应，表明PP 2A参与Arr 2和/或CaMK II的调节。基于这些发现，我们提出CaMKII和PP 2A催化Arr 1和Arr 2的可逆磷酸化来调节视觉反应。此外，CaMKII和PP 2A在视网膜中形成稳定的蛋白质复合物，用于暂时控制CaMKII活性。为了验证这些假设，我们将（1）表征Arr 2磷酸化的功能贡献，（2）研究CaMK II和PP 2A在Arr 1可逆磷酸化中的参与，（3）探索PP 2A和CaMK II是否在光感受器中形成稳定的蛋白质复合物以动态调节CaMK II活性。我们对果蝇中arrestins的体内调节的见解将拓宽我们对视觉arrestins和<$-arrestins在调节参与不同信号通路的G蛋白偶联受体（GPCR）中的不同功能的认识。考虑到GPCR在人类生理学和病理生理学的许多方面的贡献，人们可能能够微调抑制蛋白的活性以改善或优化GPCR的输出。在这方面，视觉抑制蛋白可以为未来的药物干预提供切实的药物靶点，以预防视网膜变性并提高视敏度。公共卫生相关性：我们希望通过遗传学、生物化学和电生理学的综合分析来研究可逆磷酸化对视觉抑制蛋白的调节作用。我们认为CaMKII通过促进视觉抑制蛋白的磷酸化，对视觉反应的负调节至关重要。此外，CaMKII的活性可能受到PP 2A的紧密调节。

DESCRIPTION (provided by applicant): The objective of this proposal is to investigate how phosphorylation modulates arrestin activity to control the output of rhodopsin thereby fine tuning the visual signaling, using Drosophila eye as a model system. Visual signaling is a G-protein coupled mechanism in which light activates rhodopsin to initiate a cascade of biochemical events leading to depolarization of photoreceptors. It is well established that the activity of rhodopsin is regulated by arrestin that blocks the interaction with the down-stream G-protein. In Drosophila photoreceptors, there are two distinct arrestin homologues, Arr1 and Arr2. Studies support that Arr1 promotes endocytosis of rhodopsin, whereas Arr2 is required for fast deactivation of the visual response by uncoupling activated rhodopsin. Little is known regarding how Arr1 and Arr2 are modulated in vivo. Both Arr1 and Arr2 undergo light-dependent phosphorylation, and Arr2 was shown phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vitro. To investigate how CaMKII modulates Arr2, first we identified CaMKII in the retina by the Ca2+ dependent autophosphorylation. We show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, suggesting that dephosphorylation of CaMKII is regulated by protein phosphatase 2A (PP2A). Significantly, we found that the catalytic subunit of PP2A can be co-immunoprecipitated by anti- CaMKII antibodies. To gain insights into the role of CaMKII in vivo, we demonstrate that suppression of CaMKII in flies leads to enhanced visual response and abnormal light adaptation. These data indicate that CaMKII exerts a negative regulation of the visual response, possibly by catalyzing phosphorylation of Arr2. In contrast, a reduced activity of PP2A in the mts mutants results in reduced visual response without affecting light adaptation, suggesting that PP2A participates in the regulation of Arr2 and/or CaMKII. Based on these findings, we propose that CaMKII and PP2A catalyze the reversible phosphorylation of both Arr1 and Arr2 to modulate the visual response. Moreover, CaMKII and PP2A form a stable protein complex in the retina for a temporal control of the CaMKII activity. To test these hypotheses, we will (1) characterize the functional contribution of Arr2 phosphorylation, (2) investigate the involvement of CaMKII and PP2A in the reversible phosphorylation of Arr1, (3) explore whether PP2A and CaMKII form a stable protein complex in photoreceptors for a dynamic modulation of the CaMKII activity. Our insights into the in vivo regulation of arrestins in Drosophila will broaden our knowledge on the diverse functions that visual arrestins and ¿-arrestins serve in modulating G-protein coupled receptors (GPCRs) involved in diverse signaling pathways. Considering the contribution of GPCRs in many aspects of human physiology and pathophysiology, one may be able to fine-tune the activity of arrestins for improving or optimizing the outputs of GPCRs. In this regard, visual arrestins may present a tangible drug target for future pharmacological intervention to prevent retinal degeneration and to enhance visual acuity. PUBLIC HEALTH RELEVANCE: We would like to investigate regulation of visual arrestins by reversible phosphorylation by a combined genetics, biochemical, and electrophysiological analysis. We propose that CaMKII, by promoting phosphorylation of visual arrestins, is critical for the negative regulation of the visual response. Moreover, the activity of CaMKII may be tightly modulated by PP2A.