Distinct Subdomains of the EphA3 Receptor Mediate Ligand Binding and Receptor Dimerization

doi:10.1074/jbc.273.32.20228

Distinct Subdomains of the EphA3 Receptor Mediate Ligand Binding and Receptor Dimerization*

From the ^‡Ludwig Institute for Cancer Research (Melbourne Branch), Post Office, Royal Melbourne Hospital, Victoria 3050 and the ^¶Queensland Institute for Medical Research, The Bancroft Centre, Post Office, Royal Brisbane Hospital, 4029 Queensland, Australia

Abstract

Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. In this study we make use of the observation that soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis to probe receptor function. Based on exon structure we have dissected the extracellular region of EphA3 receptor into evolutionarily conserved subdomains and used kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis to study their function. We show that ligand binding is restricted to the N-terminal region encoded by exon III, and we identify an independent, C-terminal receptor-dimerization domain. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. We propose a two-step mechanism of Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events.

Footnotes

↵* This work was supported in part by the National Health and Medical Research Council of Australia, the Leukaemia Foundation of Queensland, the Queensland Cancer Fund, and the Australian Government Cooperative Research Centres scheme.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Recipient of an Anti-cancer Councel of Victoria Postgraduate Research Scholarship.
↵‖ To whom correspondence should be addressed: Queensland Institute for Medical Research, The Bancroft Centre, Post Office, Royal Brisbane Hospital, 4029, Queensland, Australia. E-mail:andrewBo{at}qimr.edu.au.
↵2 M. Dottori and A. W. Boyd, unpublished observations.
↵3 M. Lackmann and L. Kravets, unpublished observations.
↵4 M. Lackmann, A. C. Oates, M. Dottori, F. M. Smith, C. Do, L. Kravets, C. Brennan, M. Power, N. Holder, and A. W. Boyd, manuscript submitted for publication.
↵5 J. M. Gad and H. M. Cooper, personal communication.
Abbreviations:

ECD

extracellular domain

DCC

deleted in colo-rectal cancer

FLAG^TM

refers to the amino acid sequence DYKDDDDK

mAb

monoclonal antibody

PAGE

polyacrylamide electrophoresis

RTK

receptor tyrosine kinase

sh-EphA3

soluble h-EphA3 extracellular domain

PDGFR

platelet-derived growth factor receptor

EGF

epidermal growth factor

CHO

Chinese hamster ovary

PCR

polymerase chain reaction

bp

base pair

oligos

oligonucleotides

hpf

hours post-fertilization

GFP

green fluorescent protein

DIG

digoxigenin.
- Received January 12, 1998.
- Revision received May 4, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.