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(Received for publication, December 26, 1996, and in revised form, February 24, 1997)
From the Cooperative Research Centre for Cellular Growth Factors,
HEK is a member of the EPH-like receptor tyrosine
kinase family, which appear to have roles in development and
oncogenesis. Recently, we purified a soluble HEK ligand which is also a
ligand (AL1) for the HEK-related receptor EHK1. Promiscuity appears to be a characteristic feature of interactions between the EPH-like receptors and their ligands, termed ligands for EPH-related kinases (LERKs). This prompted us to analyze the interactions between the HEK
exodomain and fusion proteins comprising candidate LERKs and the Fc
portion of human IgG1 (Fc) or a
FLAGTM-peptide tag by surface plasmon resonance, size
exclusion high performance liquid chromatography, sedimentation
equilibrium, and transphosphorylation. Our results indicate that
AL1/LERK7 is the preferred high-affinity ligand for HEK, forming a
stable 1:1 complex with a dissociation constant of 12 nM.
As expected the apparent affinities of bivalent fusion proteins of
LERKs and the Fc portion of human IgG1 had significantly
reduced dissociation rates compared with their monovalent,
FLAGTM-tagged derivatives. High-avidity binding of
monovalent ligands can be achieved by antibody-mediated cross-linking
of monovalent ligands and with LERK7 results in specific
phosphorylation of the receptor. By extrapolation, our findings
indicate that some of the reported LERK-receptor interactions are a
consequence of the use of bivalent ligand or receptor constructs and
may be functionally irrelevant.
Volume 272, Number 26,
Issue of June 27, 1997
pp. 16521-16530
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,
,
,
Growth Regulation Laboratory, Russell Grimwade School of
Biochemistry and Molecular Biology, University of Melbourne, Parkville,
Victoria 3052, Australia, §§ Immunex
Corporation, Seattle, Washington 98101, and the 
Queensland
Institute for Medical Research, The Bancroft Centre, Post Office, Royal
Brisbane Hospital, Queensland 4029, Australia
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