Regulation of Drosophila arrestins in light adaptation

果蝇视紫红质在光适应中的调控

• 批准号: 8247060
• 负责人: BIH-HWA SHIEH
• 金额: $ 37.07万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247060
• 关键词: ARRB1 gene; Affect; Antibodies; Arr2; Arrestins; Binding; Biochemical; Biological Models; Catalytic Domain; Cations; Coupled; Data; Drosophila arrestin; Drosophila eye; Drosophila genus; Drug Delivery Systems; Endocytosis; Event; Functional disorder; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic; Health; Heterozygote; Homologous Gene; Human; Hydrolysis; In Vitro; Intervention; Knowledge; Light; Light Adaptations; Mediating; Membrane; Okadaic Acid; Output; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Photoreceptors; Physiology; Play; Protein Dephosphorylation; Protein phosphatase; Regulation; Retina; Retinal Degeneration; Rhodopsin; Role; Signal Pathway; Signal Transduction; Stream; Testing; Vision; Visual; Visual Acuity; base; calmodulin-dependent protein kinase II; fly; improved; in vivo; insight; metarhodopsin; metarhodopsins; mutant; prevent; protein complex; response

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.

描述（由申请人提供）：本提案的目的是研究磷酸化如何调节抑制蛋白活性以控制视紫红质的输出，从而微调视觉信号，以果蝇眼睛为模型系统。视觉信号是一种g蛋白偶联机制，其中光激活视紫红质启动一系列生化事件，导致光感受器去极化。目前已经证实，视紫红质的活性是由阻断其与下游g蛋白相互作用的抑制蛋白调控的。在果蝇的光感受器中，有两种不同的抑制蛋白同源物，Arr1和Arr2。研究支持Arr1促进视紫红质的内吞作用，而Arr2则需要通过解偶联激活的视紫红质的视觉反应快速失活。关于Arr1和Arr2在体内是如何被调节的，我们知之甚少。Arr1和Arr2都经历光依赖性磷酸化，Arr2在体外被Ca2+/钙调素依赖性蛋白激酶II （CaMKII）磷酸化。为了研究CaMKII如何调节Arr2，我们首先通过Ca2+依赖性自磷酸化在视网膜中鉴定了CaMKII。我们发现，冈田酸极大地增强了CaMKII的磷酸化，这表明CaMKII的去磷酸化是由蛋白磷酸酶2A （PP2A）调节的。值得注意的是，我们发现PP2A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，我们证明了在果蝇中抑制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Arr2的磷酸化，对视觉反应产生负调控作用。相反，在mts突变体中，PP2A活性降低导致视觉反应降低，但不影响光适应，这表明PP2A参与了Arr2和/或CaMKII的调控。基于这些发现，我们提出CaMKII和PP2A催化Arr1和Arr2的可逆磷酸化来调节视觉反应。此外，CaMKII和PP2A在视网膜中形成稳定的蛋白复合物，从而对CaMKII活性进行时间控制。为了验证这些假设，我们将(1)表征Arr2磷酸化的功能贡献，(2)研究CaMKII和PP2A在Arr1可逆磷酸化中的参与，(3)探索PP2A和CaMKII是否在光感受器中形成稳定的蛋白质复合物，以动态调节CaMKII活性。我们对果蝇体内阻滞蛋白调控的见解将拓宽我们对视觉阻滞蛋白和¿-阻滞蛋白在调节多种信号通路中参与的g蛋白偶联受体（gpcr）的多种功能的认识。考虑到gpcr在人体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活动，以改善或优化gpcr的输出。在这方面，视觉抑制可能为未来的药物干预提供一个切实的药物靶点，以防止视网膜变性和提高视力。公共卫生相关性：我们希望通过遗传学、生化和电生理综合分析来研究可逆性磷酸化对视觉骤停的调节。我们认为CaMKII通过促进视觉抑制蛋白的磷酸化，对视觉反应的负调控至关重要。此外，CaMKII的活性可能受到PP2A的紧密调控。