Functional analysis of protein tyrosine phosphatase in the formation of retinotectal projection

Eph受体是受体型酪氨酸激酶（RTK）中最大的家族，可分为A型（EphAs）和B型（EphBs）^[1]。Eph受体参与了许多事件，如细胞运动、轴突导向、突触形成、突触可塑性的维持、细胞边界的组织、心血管系统的形成和癌变^[2]。Eph受体与其配体肝配蛋白的相互作用需要体内细胞-细胞接触，因为受体和配体都是膜结合的。虽然存在例外^<3，4>，但EphA受体优先结合肝配蛋白-As，其通过糖基磷脂酰肌醇（GPI）锚附着于质膜，并且EphB受体优先结合跨膜肝配蛋白-Bs。在结合肝配蛋白配体后，Eph受体在几个酪氨酸残基处自磷酸化，随后激活下游的信号级联。然而，蛋白质酪氨酸磷酸化 ...更多信息 负责Eph负调节的ATP酶（PTP）尚未阐明。在这里，我确定了蛋白酪氨酸磷酸酶受体O型（Ptpro）作为PTP的Eph受体作为底物去磷酸化。在哺乳动物双杂交和免疫沉淀试验中，Ptpro的底物捕获突变体与EphA 4和EphB 2受体形成稳定的复合物。使用GST融合形式的Ptpro的体外测定显示Ptpro对Eph受体的直接去磷酸化。EphA 4和EphB 2的点突变构建体以及具有磷酸肽的构建体的实验表明，Ptpro使近膜区保守的磷酸酪氨酸残基去磷酸化，这是Eph受体的激活和信号传递所需的。使用小鸡retinotectal投影系统，我表明，Ptpro控制的敏感性视网膜轴突ephrins，从而有一个至关重要的作用，在建立地形投影。我的研究结果解释了决定Eph受体在体内对ephrin反应阈值的分子机制。少

Eph receptors are the largest family of receptor-type tyrosine kinases (RTKs) and are classified into two groups, A-type (EphAs) and B-type (EphBs)^1. The Eph receptors are involved in numerous events such as cell movements, axonal guidance, formation of synapses, maintenance of synaptic plasticity, organization of cell boundaries, formation of the cardiovasculature system and carcinogenesis^2. Interaction of the Eph receptors with their ligands, the ephrins, requires cell-cell contact in vivo because both the receptor and the ligand are membrane-bound. Although there exist exceptions^<3,4>, EphA receptors preferentially bind the ephrin-As, which are attached to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor, and EphB receptors preferentially bind the transmembrane ephrin-Bs. Upon binding the ephrin ligands, Eph receptors are autophosphorylated at several tyrosine residues and subsequently activate signaling cascades downstream. However, protein tyrosine phosph … More atases (PTPs) responsible for the negative regulation of Eph have not been elucidated. Here, I identified protein tyrosine phosphatase receptor type O (Ptpro) as a PTP that dephosphorylates Eph receptors as substrates. In mammalian two-hybrid and immunoprecipitation assays, a substrate-trapping mutant of Ptpro formed stable complexes with EphA4 and EphB2 receptors. In vitro assays using GST-fused forms of Ptpro showed direct dephosphorylation of Eph receptors by Ptpro. Experiments with point-mutant constructs of EphA4 and EphB2 as well as those with phosphopeptides revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Using the chick retinotectal projection system, I show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. My findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo. Less