Mechanisms of receptor protein tyrosine phosphatase signaling in Drosophila devel

描述(申请人提供)：受体蛋白酪氨酸磷酸酶(RPTPs)在神经系统发育中具有重要功能，并与代谢调节和癌症发生有关。然而，细胞外配体对它们的调控及其下游信号机制仍然存在许多问题。果蝇为研究RPTP在体内的作用机制提供了一个有吸引力的模型系统。RPTP的IIa型家族中的两个果蝇成员Lar和PTP69D，是R7光感受器选择正确的突触靶层所必需的，也是幼虫运动神经元在其靶肌肉上形成正确大小的突触所必需的。任何一种RPTP的突变都会产生一种强大的、可量化的表型。然而，R7光感受器和幼虫运动神经元对LAR特定结构特征的要求有很大的不同。这项提议将研究LAR用于指导R7靶向的新的信号机制与其在运动神经元突触生长中的作用模式以及与PTP69D信号的不同之处。一些RPTPs的磷酸酶活性受二聚化的负调控。初步数据表明，R7靶标的选择不需要LAR的磷酸酶活性，但确实需要一个介导LAR二聚的结构域。这项提议的第一个目的将检查PTP69D是否也有两种不同的信号机制，以及它是否可以与运动神经元中的LAR互换。它还将研究强制二聚对LAR功能的影响，并开发一种在体内可视化LAR二聚的方法。此外，还将研究PTP69D与LAR的均二聚或异二聚的重要性。RPTPs已被证明可以调节细胞与细胞和细胞与基质的黏附。在这项建议的第二个目标中，涉及这些功能的分子将被测试与LAR的遗传和物理相互作用，以确定R7光感受器中LAR的磷酸酶非依赖性功能是否使用其中一种机制。此外，遗传和生化方法都将用于无偏筛选潜在的新分子，这些分子需要二聚结构域与LAR相互作用，因此可能在R7中作用于LAR的下游。控制运动神经元中LAR活性的配体在R7中不调节它。该方案的最终目的是筛选跨膜蛋白和分泌蛋白对R7靶向的影响，以确定靶神经元表达的LAR或PTP69D的候选配体。然后，这些候选人将接受测试，以确定他们是否有能力与两个RPTP结合并调节其功能。综上所述，本方案中的实验将表征RPTP功能的非规范机制，并可能为这类重要但鲜为人知的受体识别新的配体和下游效应器。 公共卫生相关性：IIa家族的哺乳动物受体酪氨酸磷酸酶对于正常的神经元发育和再生以及代谢稳态是必不可少的；然而，这些受体发出信号的机制还不是很清楚。我们发现，其中一种受体在两个不同的发育过程中通过不同的机制发出信号；我们将使用果蝇这一提供强大遗传工具、速度和经济等优势的模型系统来表征这种新的机制。这些成分的进化保守性表明，我们的结果将适用于高等生物，并可能指导药物设计，以治疗由受体酪氨酸磷酸酶功能障碍引起的人类疾病，如癌症、糖尿病和溃疡性结肠炎。

DESCRIPTION (provided by applicant): Receptor protein tyrosine phosphatases (RPTPs) have important functions in nervous system development and are implicated in metabolic regulation and carcinogenesis. However, many questions remain regarding their regulation by extracellular ligands and their downstream signaling mechanisms. The fruit fly Drosophila offers an attractive model system in which to address the mechanisms of RPTP function in vivo. LAR and PTP69D, the two Drosophila members of the type IIa family of RPTPs, are required for R7 photoreceptors to select the correct synaptic target layer, and for larval motor neurons to form synapses of the correct size on their target muscles. Mutation of either RPTP produces a strong and quantifiable phenotype. However, R7 photoreceptors and larval motor neurons differ significantly in their requirements for specific structural features of LAR. This proposal will investigate how the novel signaling mechanism used by LAR to direct R7 targeting differs from its mode of action in motor neuron synapse growth and from PTP69D signaling. The phosphatase activity of some RPTPs is negatively regulated by dimerization. Preliminary data shows that R7 target selection does not require the phosphatase activity of LAR, but does require a domain that mediates LAR dimerization. The first aim of this proposal will examine whether PTP69D also has two distinct signaling mechanisms, and whether it is interchangeable with LAR in motor neurons. It will also study the effect of forced dimerization on LAR function and develop a method to visualize LAR dimerization in vivo. In addition, the importance of PTP69D homodimerization or heterodimerization with LAR will be investigated. RPTPs have been shown to regulate both cell-cell and cell-matrix adhesion. In the second aim of this proposal, molecules implicated in each of these functions will be tested for genetic and physical interactions with LAR to determine whether the phosphatase-independent function of LAR in R7 photoreceptors uses one of these mechanisms. In addition, both genetic and biochemical methods will be used in unbiased screens for potentially novel molecules that require the dimerization domain to interact with LAR and might therefore act downstream of LAR in R7. The ligands that control LAR activity in motor neurons do not regulate it in R7. The final aim of this proposal is to screen transmembrane and secreted proteins for an effect on R7 targeting, in order to identify candidate ligands for LAR or PTP69D expressed by the target neurons. These candidates will then be tested for their ability to bind to and regulate the function of both RPTPs. Taken together, the experiments in this proposal will characterize a non-canonical mechanism of RPTP function, and may identify new ligands and downstream effectors for this important but poorly understood class of receptors. PUBLIC HEALTH RELEVANCE: Mammalian receptor tyrosine phosphatases of the type IIa family are essential for normal neuronal development and regeneration, as well as for metabolic homeostasis; however, the mechanisms by which these receptors signal are not well understood. We have found that one such receptor signals through distinct mechanisms in two different developmental processes; using Drosophila, a model system that offers advantages such as powerful genetic tools, speed, and economy, we will characterize the novel mechanism. Evolutionary conservation of the components suggests that our results will be applicable to higher organisms, and may guide drug design to treat human diseases caused by receptor tyrosine phosphatase dysfunction such as cancer, diabetes and ulcerative colitis.