title: Dissecting the EphA3/Ephrin-A5 interactions using a novel functional mutagenesis screen.
abstract: The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes.
authors.affiliation: Leukaemia Foundation of Queensland Laboratory, Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Queensland 4029, Australia.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Smith
authors.forename: Christopher
authors.initials: C
authors.lastname: Vearing
authors.forename: Martin
authors.initials: M
authors.lastname: Lackmann
authors.forename: Herbert
authors.initials: H
authors.lastname: Treutlein
authors.forename: Juha
authors.initials: J
authors.lastname: Himanen
authors.forename: Ke
authors.initials: K
authors.lastname: Chen
authors.forename: Allan
authors.initials: A
authors.lastname: Saul
authors.forename: Dimitar
authors.initials: D
authors.lastname: Nikolov
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Boyd
mesh_terms: D001665:Binding Sites / Q000235:genetics; D036386:Ephrin-A5 / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D036121:Receptor, EphA3
chemical_list: D036386:Ephrin-A5; C491718:EPHA3 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D036121:Receptor, EphA3