Eps15R and clathrin regulate EphB2‐mediated cell repulsion

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.

A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

“…A large multi-domain protein, Eps15 binds physically to numerous other coat components, including AP-2 (Edeling et al, 2006; Benmerah et al, 1996; Traub et al, 1999; Schmid et al, 2006), Fcho1/2 (Henne et al, 2010; Reider et al, 2009; Uezu et al, 2011; Umasankar et al, 2012), Disabled-2 (Dab2) (Teckchandani et al, 2012), epsin 1 (Chen et al, 1998; McPherson et al, 1998), HIV-1 Rev-binding protein (Hrb) (Doria et al, 1999; Whitehead et al, 1999; Yamabhai et al, 1998), Numb (Santolini et al, 2000; Evergren et al, 2018), stonin 2 (Rumpf et al, 2008), and synaptojanin 1 (McPherson et al, 1998; Haffner et al, 1997), among other proteins including ubiquitin (Hofmann and Falquet, 2001). Because of this multiplicity of protein–protein contacts, residence of Eps15 at clathrin bud sites is not solely dependent on AP-2 (van Delft et al, 1997a).…”

Role of SHIP2 in cell repulsion regulated by Eph receptor and ephrin signaling

“…Previous studies have suggested that SHIP2 can inhibit clathrin-mediated endocytosis (Nakatsu et al, 2010; Zhuang et al, 2007) by dephosphorylating phosphoinositides that promote endocytosis (Posor et al, 2015), whereas it promotes endophilin-mediated endocytosis (Boucrot et al, 2015). EphB-ephrinB interactions can lead to endocytosis of the complex in the forward and/or reverse direction, and this influences the strength of cell repulsion responses (Evergren et al, 2018; Gaitanos et al, 2016; Gong et al, 2019; Marston et al, 2003; Pitulescu and Adams, 2010; Zimmer et al, 2003). Furthermore, SHIP2 binds to EphA2 and inhibits its forward endocytosis (Zhuang et al, 2007).…”

Endocytic Adaptor Proteins in Health and Disease: Lessons from Model Organisms and Human Mutations

“…Constitutive KO: lethal prior to gastrulation [247]; conditional ubiquitous KO: mild proteinuria, mild increase in serum cholesterol, reduced tumor incidence [231,232,247,256,264] (see also [256] [195] Somatic mutations/gene fusion in AML and lung cancer [370,375] Eps15R (EPS15L1) Ubiqui-tin-UIM EphB2/ ephrinB [180], TfR [170] AP-2, CHC, Dynamin, Epsins, Intersectin, Numb, Stonin2, Synaptoja-nin…”

“…The C-terminal region comprises a proline-rich domain characterized by the presence of multiple DPF (aspartate-proline-phenylalanine) repeats, essential for the interaction with AP-2 [179] (Figure 2). In addition, Eps15R was shown to directly bind the Clathrin terminal domain [180]. In vitro studies, supported by structural data, first suggested that Eps15/Eps15R can form complexes with multiple endocytic proteins including AP-2, Dynamin, Intersectin1/2 and Stonin2, underlining their involvement in endocytosis [181,182].…”

“…However, it is not clear yet whether these sorting events are physiologically relevant and whether Eps15R plays a role there as well. On the other hand, Eps15R was reported to act in the trans-endocytosis of EphB/ephrinB complexes [180]. The localisation of Eps15 at endosomal/TGN compartments [188,189] and the partial residence of Eps15R in the nucleus [190] suggest additional non-canonical functions for which the physiological importance is likewise unclear.…”