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

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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