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，分为a型（EphAs）和b型（EphBs）^1。Eph受体参与许多事件，如细胞运动、轴突引导、突触形成、突触可塑性维持、细胞边界组织、心血管系统形成和癌变^2。由于受体和配体都是膜结合的，因此Eph受体与其配体（ephrin）的相互作用需要在体内进行细胞间接触。尽管存在例外^<3,4 bb0，但EphA受体优先结合通过糖基磷脂酰肌醇（GPI）锚点附着在质膜上的ephrin- a，而EphB受体优先结合跨膜ephrin- b。在与ephrin配体结合后，Eph受体在几个酪氨酸残基上进行自磷酸化，随后激活下游的信号级联。然而，酪氨酸磷酸化蛋白（protein tyrosine phosph, ptp）对Eph的负调控作用尚不清楚。在这里，我确定了蛋白酪氨酸磷酸酶受体O型（Ptpro）作为一种PTP去磷酸化作为底物的Eph受体。在哺乳动物双杂交和免疫沉淀实验中，一种捕获底物的Ptpro突变体与EphA4和EphB2受体形成稳定的复合物。使用gst融合形式的Ptpro进行的体外实验显示，Ptpro可直接使Eph受体去磷酸化。对EphA4和EphB2的点突变构建体以及磷酸化肽构建体进行的实验表明，Ptpro使保存在膜旁区域的磷酸酪氨酸残基去磷酸化，这是Eph受体激活和信号传递所必需的。使用小鸡视网膜顶投影系统，我发现Ptpro控制视网膜轴突对ephrin的敏感性，因此在建立地形投影中起着至关重要的作用。我的发现解释了在体内决定Eph受体对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