Identification and Characterization of a Fibroblast Growth Factor (FGF) Binding Domain in the Cysteine-rich FGF Receptor

doi:10.1074/jbc.272.8.5167

Identification and Characterization of a Fibroblast Growth Factor (FGF) Binding Domain in the Cysteine-rich FGF Receptor*

From the ^∥ Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and
** Walther Cancer Institute, Indianapolis, Indiana 46208

^‡‡ To whom correspondence should be addressed:
Dept. of Molecular, Cellular, and Developmental Biology, Campus Box 347, University of Colorado, Boulder, CO 80309.
Tel.: 303-492-6816; Fax: 303-492-1587; E-mail: bradley.olwin{at}colorado.edu

↵^‡ Present address: Dept. of Genetics, Stanford University School of Medicine, Stanford, CA 94305.
↵^§ Present address: Dept. of Biology, University of California, La Jolla, CA 92093-0366.
↵^¶ Present address: Dept. of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138.

Abstract

Three distinct transmembrane glycoproteins bind fibroblast growth factor (FGF) family members. These include heparan sulfate proteoglycans, the tyrosine kinase-containing FGF receptors (FGFRs), and a cysteine-rich FGF receptor (CFR). The four FGFRs are thought to mediate FGF-signaling events but require the participation of the heparan sulfate proteoglycans to bind FGFs and transduce intracellular signals. However, a number of groups have proposed that FGF action requires events independent of FGFR activation. CFR, a high affinity FGF-binding protein, was first isolated from chicken embryos. To better understand the interactions between CFR and FGFs, we have constructed a series of CFR deletion mutants and CFR fragments. Analysis of these has identified a ∼200-amino acid domain that constitutes a CFR FGF binding site. A CFR fragment of 450 residues, CFR_290-740, binds FGF-2 with an affinity indistinguishable from the full-length molecule, whereas smaller fragments display greatly reduced FGF binding. Although CFR binds heparin with high affinity, an analysis of the heparin-CFR interaction failed to identify a linear sequence containing a heparin binding site. Two types of FGF binding sites were identified: an ionic strength and heparin-independent site that represents FGF binding to CFR_290-740 and an additional FGF binding site that is heparan sulfate-dependent and sensitive to high ionic strength. This latter site is likely to bind FGF indirectly via heparan sulfate binding to CFR. FGF-2 peptides that encompass a sequence implicated in FGF-2 binding to FGFRs also block FGF-2 binding to CFR. Our data suggest that binding of FGFs to CFR and FGFRs is mutually exclusive, since the CFR FGF binding site does not require heparan sulfate, and similar regions on FGF-2 interact with both FGFRs and CFR.

Footnotes

↵* This work was supported in part by grants from the National Institutes of Health (to B. B. O.). 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:

FGF

fibroblast growth factor

CFR

cysteine-rich FGF receptor

FGFR

FGF receptor

PAGE

polyacrylamide gel electrophoresis

RISA

radioimmunosorbent assay

wt

wild type

HA1

influenza hemagglutinin epitope

LA

CFR monoclonal antibody epitope

TBST

Tris-buffered saline with Triton X-100

HBT

homogenization buffer with Triton X-100

CHO

Chinese hamster ovary.
↵² Z. Zhou and B. B. Olwin, unpublished data.
- Received June 12, 1996.
- Revision received November 27, 1996.