Eph (Erythropoietin-producing hepatoma) tyrosine kinase cell surface receptors comprise the largest group of receptor tyrosine kinases (Hirai et al., 1987
). These receptors with their membrane-bound ligands, the ephrins (Eph receptor interacting proteins), contribute to fundamental developmental processes such as boundary formation and cell migration in multicellular organisms ranging from sponges to vertebrates (Drescher, 2002; Kullander and Klein, 2002
). The signals resulting from Eph-ephrin complex formation can be bidirectional (forward: into the Eph-presenting cell, reverse: into the ephrin-presenting cell) (Cowan and Henkemeyer, 2002; Lim et al., 2008; Lu et al., 2001
There are fourteen Eph receptors and eight ephrin ligands in the human genome, each family divided into two classes (A and B) (Eph Nomenclature Committee, 1997
). The extracellular portions of ephrinA ligands are attached to the cell by a glycosylphosphatidylinositol anchor, while ephrinB ligands contain a transmembrane helix and a short intracellular region. Eph receptors are type 1 membrane proteins with an N-terminal extracellular region consisting of a ligand binding domain (LBD), a cysteine rich region, and two fibronectin type III domains. The extracellular region is followed by a single transmembrane span connecting to a cytoplasmic region comprising a protein tyrosine kinase domain that mediates autophosphorylation and phosphorylation of other proteins (Pasquale, 2005
), a sterile α motif (SAM), and a PDZ-binding motif.
Generally, it has been found that high affinity binding (nM to low μM KD
) of Eph receptors to ephrin ligands is restricted within classes (i.e., class A ligands bind class A receptors) (Gale et al., 1996
). However, this rule only holds to an extent. EphA4, for example, has been found to bind both to A and B class ligands and EphB2 has been found to bind to ephrinA5 (Gale et al., 1996; Himanen et al., 2004
EphA4 is associated with an extensive range of biological activity; for example, it is critical for the development of the nervous system (Canty et al., 2006; Coulthard et al., 2002; Dottori et al., 1998; Fu et al., 2007
), is also highly expressed during glioma cell proliferation (Fukai et al., 2008
), and has been linked with melanoma tumor suppression (Easty and Bennett, 2000
). The potential to target Eph receptors for cancer therapeutics is of growing interest. Structural studies of the LBD of EphB2 and EphA4, for example, have shown that these receptors may be useful targets for the design of small molecule cancer therapeutics (Chrencik et al., 2006b; Noberini et al., 2008; Qin et al., 2008
Knowledge of the molecular determinants that define Eph receptor cross-reactivity and specificity is a powerful prerequisite for the rational development of Eph receptor- and ephrin-based disease treatments. In this study, we sought to elucidate the mechanism by which the LBD of the EphA4 cell surface receptor binds to both A and B class ephrin ligands at the molecular level. We present binding affinity data of EphA4 with both A and B class ephrins and report the structure of the LBD of EphA4 alone and in complex with the receptor binding domain (RBD) of ephrinA2 and ephrinB2. From our analysis of these structures, we suggest that the LBD of EphA4 is structurally plastic and, as a consequence, has a bimorphic A and B class Eph receptor nature. These data provide a molecular basis for how differing patterns in Eph-ephrin binding define properties that influence class-dependent Eph receptor specificity and promiscuity.