Mechanisms of receptor protein tyrosine phosphatase signaling in Drosophila devel

描述（由申请人提供）：受体蛋白酪氨酸磷酸酶（RPTP）在神经系统发育中具有重要功能，并参与代谢调节和致癌作用。然而，许多问题仍然存在关于他们的调控细胞外配体和他们的下游信号传导机制。果蝇提供了一个有吸引力的模型系统，在其中解决RPTP功能在体内的机制。LAR和PTP 69 D是RPTP的IIa型家族的两个果蝇成员，R7光感受器需要选择正确的突触靶层，幼虫运动神经元需要在其靶肌肉上形成正确大小的突触。任一RPTP的突变产生强的和可量化的表型。然而，R7光感受器和幼虫运动神经元的LAR的特定结构特征的要求显着不同。该提案将研究LAR用于指导R7靶向的新型信号传导机制如何不同于其在运动神经元突触生长中的作用模式和PTP 69 D信号传导。 一些RPTP的磷酸酶活性受二聚化负调控。初步数据显示，R7靶选择不需要LAR的磷酸酶活性，但需要介导LAR二聚化的结构域。本提案的第一个目的是研究PTP 69 D是否也有两种不同的信号传导机制，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化对LAR功能的影响，并开发一种在体内可视化LAR二聚化的方法。此外，将研究PTP 69 D同二聚化或异二聚化与LAR的重要性。 RPTP已显示调节细胞-细胞和细胞-基质粘附。在本提案的第二个目标中，将测试与这些功能中的每一个有关的分子与LAR的遗传和物理相互作用，以确定R7光感受器中LAR的磷酸酶独立功能是否使用这些机制之一。此外，遗传和生物化学方法将用于无偏筛选潜在的新分子，这些分子需要二聚化结构域与LAR相互作用，因此可能在R7中的LAR下游起作用。 在运动神经元中控制LAR活性的配体在R7中不调节它。该建议的最终目的是筛选跨膜和分泌蛋白对R7靶向的影响，以鉴定靶神经元表达的LAR或PTP 69 D的候选配体。然后测试这些候选物结合两种RPTP并调节其功能的能力。两者合计，本提案中的实验将表征RPTP功能的非经典机制，并可能为这类重要但知之甚少的受体鉴定新的配体和下游效应物。 公共卫生相关性：哺乳动物受体酪氨酸磷酸酶的IIa型家庭是必不可少的正常神经元的发育和再生，以及代谢的稳态，然而，这些受体的信号的机制还没有很好地理解。我们已经发现，一个这样的受体信号通过不同的机制，在两个不同的发展过程中;使用果蝇，一个模型系统，提供的优势，如强大的遗传工具，速度和经济，我们将表征新的机制。组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.