Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5

Abstract

Eph receptor tyrosine kinases and their ephrin ligands mediate cell signaling during normal and oncogenic development. Eph signaling is initiated in a multistep process leading to the assembly of higher-order Eph/ephrin clusters that set off bidirectional signaling in interacting cells. Eph and ephrins are divided in two subclasses based on their abilities to bind and activate each other and on sequence conservation. EphA4 is an exception to the general rule because it can be activated by both A-and B-class ephrin ligands. Here we present high-resolution structures of the complete EphA4 ectodomain and its complexes with ephrin-A5. The structures reveal how ligand binding promotes conformational changes in the EphA4 ligand-binding domain allowing the formation of signaling clusters at the sites of cell-cell contact. In addition, the structural data, combined with structure-based mutagenesis, reveal a previously undescribed receptor-receptor interaction between the EphA4 ligand-binding and membrane-proximal fibronectin domains, which is functionally important for efficient receptor activation.crystallography | phosphorylation | protein | transmembrane E ph receptors, the largest family of receptor tyrosine kinases, and their ephrin ligands regulate a variety of cell-cell interactions during development and in the adult organism (1-3). Because both receptors and ligands are membrane-bound, their interactions at sites of cell-cell contact initiate unique bidirectional signaling cascades (4). The signaling downstream of the Eph is referred to as "forward," and the signaling downstream of the ephrins is referred to as "reverse."The 16 Eph receptors and 9 ephrins are divided into two subclasses based on sequence homology and binding affinities. Receptor-ligand binding within each subclass is fairly promiscuous, whereas cross-subclass signaling happens rarely (5). The best-known exception to this general rule is EphA4, which has long been known to bind and be activated by both A-and B-class ligands (6, 7). Because of its unique properties, EphA4 is an attractive target for studying the structural details of subclass specificity (8).Eph and ephrins were originally identified as axon guidance molecules; they have now been implicated in a vast array of cell communication events. Those include bone morphogenesis and homeostasis, immunological and inflammatory host responses, stem cell plasticity, learning and memory, and Alzheimer's disease (9). However, currently, the most intensely studied function of the Eph/ephrin system is that during development and progression of different cancers. Many A-and B-class receptors were shown to be overexpressed in various tumor types (10-13) and to regulate critical steps of blood vessel formation (vasculogenesis) and remodeling (angiogenesis) and hence tumor growth.Structural studies on the minimal binding domains of Eph receptors and ephrins revealed important details of receptorligand recognition (2, 14). The initial binding force comes from a penetration of a long, hydrophobic ephrin...

Angiopoietin receptor TEK mutations underlie primary congenital glaucoma with variable expressivity

“…28 and the Human Protein Reference Database [HPRD], http://www.hprd.org), sheds light on its novel role in ligand-mediated TEK receptor activation. The FN3 domain in the Eph family tyrosine kinase receptors directly stimulates intermolecular interactions to cluster the receptor into higher-order multimers at the plasma membrane (56). These Eph clusters act as efficient signaling centers to trigger cell responses and propagate phosphotyrosine signals.…”

Structural basis of Tie2 activation and Tie2/Tie1 heterodimerization

“…Although the nature of the impaired interactions remains elusive, the disease-causing mutation supports the evidence for the Fn2-mediated Tie2 clustering. Similarly, in the Eph family tyrosine kinase receptors, the membrane-proximal Fn-like domains are known to stimulate intermolecular interactions (45).…”

The Neural Cell Adhesion Molecule (NCAM) Promotes Clustering and Activation of EphA3 Receptors in GABAergic Interneurons to Induce Ras Homolog Gene Family, Member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated Growth Cone Collapse

“…Crystallographic studies of closely related EphA2 and EphA4 receptors identified a leucine zipper-like interface in the CRD comprised of hydrophobic residues that mediates EphA clustering in the plasma membrane (31,41,47,53) and an additional LBD interface that promotes large-scale multimerization of EphA2 (31, 47, 53). EphA3 has not been crystallized, but the LBD interface involved in EphA2 dimerization is highly conserved in EphA3 (31).…”

“…To more finely map the interaction determinants at the amino acid level, molecular modeling was performed using the ClusPro server (40) to analyze docking between rat NCAM Ig1-3 (PDB code 1QZ1) (38) and mouse EphA3 LBD/CRD modeled on human EphA4 (PDB code 4M4P) (41). Docking poses were screened for compatibility with interaction on the surface of a membrane in a cis conformation, lack of steric hindrance between additional domains of the molecules, and the number of hydrogen bonds within the interaction interface.…”