Regulation of RPE phagocytosis via avb5 integrin

描述（由申请人提供）：本研究项目的长期目标是确定视网膜色素上皮细胞（RPE）吞噬哺乳动物眼睛中昼夜节律脱落的感光细胞外段片段（POS）的分子机制，并了解这些机制中因衰老和突变导致的特定变化如何导致视网膜变性。有丝分裂后的RPE细胞是已知的最活跃的吞噬细胞。RPE吞噬作用的减弱直接导致影响数百万人的不可治愈的人类致盲疾病，包括年龄相关性黄斑变性和色素性视网膜炎。因此，识别RPE对外段更新的基本视网膜过程的贡献与我们对人类视网膜疾病的理解直接相关。最近，我们开发和探索了新的实验方法，允许第一次直接和定量地确定基因突变和年龄相关的变化如何改变体内和培养中RPE吞噬蛋白的活性。脱落的POS需要通过其视网膜配体MFG-E8连接RPE的α ν β 5受体，以刺激RPE细胞中的细胞质信号传导途径，该途径经由粘着斑激酶朝向酪氨酸激酶受体MerTK，这对于有效的POS吞噬是必不可少的。通过aV <$5受体的同步POS清除对于长期视网膜健康是必不可少的：在老化的aV <$5缺陷小鼠中缺乏吞噬节律导致促氧化剂RPE脂褐素的过度积累和视力丧失。在目标1中，我们提出鉴定aV <$5和MerTK表面受体之间缺失的分子连接，并阐明Src激酶在RPE吞噬作用中的作用。一种新的底物筛选将鉴定特异于POS吞噬作用的直接Src底物，并确定Src活性是否足以促进MerTK活化和POS吞噬。我们的初步数据表明，在POS结合的初始爆发后，RPE细胞保留了充足的表面aV 5和MerTK受体，但未能促进延长的POS结合或吞噬信号。因此，在目标2和3中，我们提出确定RPE细胞在完成昼夜POS清除后主动和特异性抑制aV 5和MerTK功能的分子机制。我们将检验这样的假设，即RPE细胞通过切断α V β 5受体对肌动蛋白细胞骨架的锚定的整联蛋白相关蛋白的特异性蛋白水解，以及通过刺激特异性蛋白酪氨酸磷酸酶来终止POS吞噬作用，所述特异性蛋白酪氨酸磷酸酶抑制MerTK。长期以来，人们一直认为存在防止过早或过度RPE吞噬活性的机制，但据我们所知从未进行过研究。我们建议，这种抑制机制可能会确保POS脱落和吞噬视网膜的健康平衡。我们已经获得了合适的体内模型，比活性测定和检测试剂用于鉴定

DESCRIPTION (provided by applicant): It is the long-term goal of this research project to identify molecular mechanisms used by the retinal pigment epithelium (RPE) to phagocytose photoreceptor outer segment fragments (POS) shed in a circadian rhythm in the mammalian eye and to understand how specific changes in these mechanisms due to aging and mutations cause retinal degeneration. Digesting several thousand POS disks every day for life, post-mitotic RPE cells are the most active phagocytes known. Abnormalities in RPE phagocytosis directly contribute to incurable human blinding diseases affecting millions, including age-related macular degeneration and retinitis pigmentosa. Identifying the contributions of the RPE to the fundamental retinal process of outer segment renewal is thus directly relevant to our understanding of human retinal disease. Recently, we developed and explored novel experimental approaches that allow for the first time to directly and quantitatively determine how genetic mutations and age-related changes alter activity of phagocytic proteins by the RPE in vivo and in culture. Shed POS require ligation of aV¿5 receptors of the RPE by their retinal ligand MFG-E8 to stimulate a cytoplasmic signaling pathway in RPE cells via focal adhesion kinase towards the tyrosine kinase receptor MerTK that is indispensable for efficient POS engulfment. Synchronized POS clearance via aV¿5 receptors is essential for long-term retinal health: lack of phagocytic rhythm in aging aV¿5 - deficient mice causes excessive accumulation of pro-oxidant RPE lipofuscin and loss of vision. In aim 1 we propose to identify the missing molecular links between aV¿5 and MerTK surface receptors and unravel the role of Src kinase in RPE phagocytosis. A novel substrate screen will identify direct Src substrates specific to POS phagocytosis and determine if Src activity is sufficient to promote MerTK activation and POS engulfment. Our preliminary data show that RPE cells following an initial burst of POS binding retain ample surface aV¿5 and MerTK receptors but fail to promote prolonged POS binding or engulfment signaling. In aims 2 and 3 we therefore propose to identify molecular mechanisms used by RPE cells to actively and specifically inhibit aV¿5 and MerTK functions following completion of diurnal POS clearance. We will test the hypothesis that RPE cells terminate POS phagocytosis by specific proteolysis of integrin associated proteins that severs anchorage of aV¿5 receptors to the actin cytoskeleton, and by stimulating specific protein tyrosine phosphatases that inactivate MerTK. Mechanisms that prevent untimely or excess RPE phagocytic activity have long been assumed to exist but to our knowledge never been studied. We propose that such inhibitory mechanisms may ensure the healthy balance of POS shedding and engulfment in the retina. We have procured appropriate in vivo models, specific activity assays and detection reagents for their identification