Heparin Binding and Oligomerization of Hepatocyte Growth Factor/Scatter Factor Isoforms

doi:10.1074/jbc.272.14.9457

Heparin Binding and Oligomerization of Hepatocyte Growth Factor/Scatter Factor Isoforms

HEPARAN SULFATE GLYCOSAMINOGLYCAN REQUIREMENT FOR Met BINDING AND SIGNALING*

From the Laboratory of Cellular and Molecular Biology, NCI, the
^‡ Protein Expression Laboratory, NIAMSD, and the
^§ Glycobiology Program, NIDR, National Institutes of Health, Bethesda, Maryland 20892

^¶ To whom correspondence should be addressed:
NCI/DBS/LCMB, Bldg. 37, Rm. 1E24, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255.
Tel.: 301-496-4265; Fax: 301-496-8479; E-mail: rubinj{at}dc37a.nci.nih.gov

Abstract

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide that stimulates cell proliferation, motility, and morphogenesis by activation of its receptor, the c-Met tyrosine kinase. HGF/SF consists of a series of structural units, including an amino-terminal segment with a hairpin loop, four kringle domains, and a serine protease-like region. In this study, we demonstrate that the amino-terminal (N) domain retains the heparin-binding properties of full-length HGF/SF. In contrast to a previous hypothesis, selected basic amino acid residues in the hairpin loop are not critical for heparin binding, although alanine substitution at a subset of these sites markedly reduced the biological activity of the HGF/SF isoform, HGF/NK1. Covalent cross-linking experiments performed with wild-type and heparan sulfate glycosaminoglycan (HSGAG)-deficient Chinese hamster ovary (CHO) cells revealed that Met-HGF/NK1 binding was strongly dependent on HSGAG. Addition of heparin to HSGAG-deficient CHO cells not only restored ligand binding, but also increased ligand-dependent Met tyrosine phosphorylation and c-fos expression. Moreover, our results showed that heparin stimulated ligand oligomerization through an interaction with the N domain. These findings establish the importance of the N domain for heparin-ligand and ligand-ligand interactions, and demonstrate a crucial role for HSGAG in receptor binding and signal transduction.

Footnotes

↵* 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*.
↵¹ The abbreviations used are:

HSGAG

heparan sulfate glycosaminoglycan

HGF/SF

hepatocyte growth factor/scatter factor

HGF/NK1

truncated HGF/SF isoform containing amino-terminal (N) domain and kringle 1

HGF/NK2

truncated HGF/SF isoform containing N domain and kringles 1 and 2

K2

second kringle domain in HGF/SF

CHO-WT

wild type Chinese hamster ovary (CHO) cell line

MDCK

Madin-Darby canine kidney

2A/NK1, 3A/NK1, and 5A/NK1

alanine substitution mutants in HGF/NK1 with 2, 3, or 5 substitutions as described in the text

PBS

phosphate-buffered saline

PAGE

polyacrylamide gel electrophoresis

IGF

insulin-like growth factor
↵²S. J. Stahl, P. T. Wingfield, J. D. Kaufman, L. K. Pannell, V. Cioce, H. Sakata, W. G. Taylor, J. S. Rubin, and D. P. Bottaro, submitted for publication.
↵³H. Sakata, W. G. Taylor, J. M. Rosenberg, and J. S. Rubin, unpublished observations.
- Received December 11, 1996.