An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly

Abstract

Erythropoetin-producing hepatoma (Eph) receptors are cell surface protein tyrosine kinases mediating cell-cell communication. Upon activation they form signalling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2), alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD ). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across Aclass Ephs. eEphA2-ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but re-arranged to accommodate ephrinA5 RBD . Cell surface expression of mutant EphA2s demonstrated that these interfaces are critical for localization at cellcell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions seed an arrangement of receptors which can propagate into extended signalling arrays.Ephs constitute a quarter of all known human receptor tyrosine kinases. They direct key processes during development and repair of the nervous system, blood vessel formation, insulin secretion, immune system function, intestinal homeostasis and bone tissue integrity 1 . They are grouped into two classes, A and B 2 , but domain composition is conserved across the family (Fig. 1a). The extracellular region comprises an N-terminal ligand-binding domain (LBD), a cysteine-rich region and two fibronectin type III domains (FN1 and FN2). FN2 connects to the transmembrane helix (TM), followed by an intracellular region comprising a juxtamembrane region (JM), a tyrosine kinase domain and a sterile-alpha motif (SAM) domain often linked to a C-terminal PDZ binding motif. Structures have been determined for examples of all Eph domains in isolation, with the exception of the cysteinerich region 3,4 .Activation of Eph receptors depends on the presence of their ligands (ephrins) and involves the packing of Ephs into signalling clusters 1,4 . Ephrins consist of an N-terminal extracellular receptor-binding domain (ephrin RBD ) and a C-terminal extension linked to the plasmamembrane by a lipid anchor (class A ephrins) or transmembrane helix (class B ephrins). Binding affinities vary for different Eph-ephrin pairs, but in general binding within classes is favoured 5 . Crystal structures for 1:1 complexes of Eph LBDs bound to ephrin RBD s show a hydrophobic ephrin-binding groove on the receptor and provide insight into ligand-receptor specificity, but do not define a mechanism for ligand-dependent signalling 6 . Members of the Correspondence and request for materials should be addressed to A.R.A (radu@strubi.ox.ac.uk) or E.Y.J. (yvonne@strubi.ox.ac.uk).. Author contributions E.S. conducted crystallographic and cellular studies. K.H. performed crystal mounting and oversaw X-ray data collection. G.S. led the MALS analysis. A.R.A. and E.Y.J. participated in study design and oversaw all aspects of the work. Author informationAtomic coordinates and structur...

Architecture of Eph receptor clusters

“…While this manuscript was under consideration, Y. Jones and colleagues published similar structure findings (23).…”

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila

“…6F, panel iv). In vertebrates, the trans Eph-Ephrin interaction leads to the formation of higher signaling clusters (Seiradake et al 2010;Kania and Klein 2016). Oligomers of Ephs and Ephrins are terminated by bidirectional transendocytosis or its cleavage by ADAMtype proteases, leading to the activation of Rho family GTPases.…”

Structural basis for LAR-RPTP/Slitrk complex-mediated synaptic adhesion

“…Previously, a hypothetical model was postulated wherein the higher-order assemblies of the neurexin/neuroligin synaptic adhesion complex are required for the presynaptic differentiation activity of neuroligins 23,24 . In addition, functional forward and reverse Eph/Ephrin synaptic adhesion signalling requires formation of higher-order clusters, initiated by initial contact between them in trans, followed by formation of larger signalling clusters and ultimately leading to lateral recruitment of additional Eph clusters 25,26 . Similar adhesionpromoted lateral clustering has been reported for cadherins 27 .…”