Ligand for EPH-related Kinase (LERK) 7 Is the Preferred High Affinity Ligand for the HEK Receptor*
- Martin Lackmann‡§,
- Richard J. Mann¶,
- Lucy Kravets‡,
- Fiona M. Smith‖,
- Tamara A. Bucci**,
- Kelly F. Maxwell¶,
- Geoffrey J. Howlett‡,
- Jane E. Olsson**§§,
- Tim Vanden Bos,
- Douglas Pat Cerretti and
- Andrew W. Boyd‖
- From the Cooperative Research Centre for Cellular Growth Factors,‡Growth Regulation Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), ¶Cancer Research Unit and **Lions Cancer Research Laboratory, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia, the ‡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
Abstract
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 FLAG™-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, FLAG™-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.
Footnotes
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↵* This work was supported by the Anti-Cancer Council of Victoria, the Leukemia Foundation of Queensland, the National Health and Medical Research Council, AMRAD Corp., Melbourne, and the Australian Government Cooperative Research Centers 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.
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↵§ To whom reprint requests should be addressed.
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↵§§ Present address: Laboritoire de Biologie Cellulaire et Moleculaire des Facteurs de Croissance CNRS, UPR 9044, 7, Rue Guy Moquet, BP8–94801, Villejuif Cedex, France.
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↵1 The abbreviations used are: RTK, receptor tyrosine kinase; Fc, hinge and Fc domain of human IgG1; FLAG™, refers to the amino acid sequence DYKDDDDK; LERK, ligand for EPH-related kinases; mAb, monoclonal antibody; PAGE, polyacrylamide electrophoresis; PBS, phosphate-buffered saline; SE, size exclusion; sHEK, soluble HEK extracellular domain; CHO Chinese hamster ovary; IL, interleukin.
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↵2 M. Lackmann, L. Kravets, T. Bucci, and A. Boyd, unpublished data.
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- Received December 26, 1996.
- Revision received February 24, 1997.
