Abstract

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans‐endocytosis of the inter‐cellular receptor‐ligand EphB‐ephrinB complex is required. The molecular mechanism underlying trans‐endocytosis is poorly defined. Here we show that the process is clathrin‐ and Eps15R‐mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co‐cultures of EphB2‐ and ephrinB1‐expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2‐mediated cell repulsion as shown in a rescue experiment in the EphB2 co‐culture assay where wild type Eps15R but not the clathrin‐binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans‐endocytosis and thereby cell repulsion.

Highlights

  • EphB receptors are a large family of receptor tyrosine kinases that interact with the transmembrane ephrinB ligands

  • We overexpressed full-length Eps[15] and showed that it cannot compensate for the loss of Eps15R (Fig. 6C, D). This demonstrated that Eps15R has a distinct function that is not redundant in Eps[15]. Taken together these results show that the interaction between Eps15R and clathrin is critically important to maintain functional endocytosis of the EphB2-ephrinB1 complex resulting in cellcell repulsion in cell culture

  • In this study we instead used a targeted approach and employed the EphB2 interaction partner Numb[18] as a starting point to show that it directly interacted with endocytic proteins Eps[15] and Eps15R

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Summary

Introduction

EphB receptors are a large family of receptor tyrosine kinases that interact with the transmembrane ephrinB ligands. The adhesive interaction between EphB receptors and ephrinB ligands activates intracellular signalling pathways that regulate cell-cell repulsion, migratory behaviour, adhesion and cell polarity. The EphB receptor and the ephrinB ligands are not expressed in the same cell and their interaction can lead to cell-cell repulsion. There are two mechanisms described to date: transendocytosis (trans-cellular internalisation event) of the receptor-ligand complex into one of the cells[10,11], or cleavage of the extracellular domain by a protease[12,13,14,15]. The endocytosis of EphB/ephrinB complexes is an unusual type of endocytosis where two trans-membrane proteins from neighbouring cells are internalised into one cell, forming vesicles from two plasma membranes[11].

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