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Molecular and Cellular Biology, September 2001, p. 5946-5957, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.5946-5957.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Receptor and Heparin Binding
Sites in Fibroblast Growth Factor 4 by Structure-Based
Mutagenesis
Paola
Bellosta,1,
Akiyo
Iwahori,1
Alexander N.
Plotnikov,2
Anna V.
Eliseenkova,2
Claudio
Basilico,1 and
Moosa
Mohammadi2,*
Departments of
Microbiology1 and
Pharmacology,2 New York University
School of Medicine, New York, New York 10016
Received 26 March 2001/Returned for modification 22 May
2001/Accepted 11 June 2001
Fibroblast growth factors (FGFs) comprise a large family of
multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that
are subject to alternative splicing. FGFR binding specificity is an
essential mechanism in the regulation of FGF signaling and is achieved
through primary sequence differences among FGFs and FGFRs and through
usage of two alternative exons, IIIc and IIIb, for the second half of
immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and
activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it
shows little activity towards the IIIb splice forms of FGFR1 to -3 as
well as towards FGFR4. To begin to explore the structural determinants
for this differential affinity, we determined the crystal structure of
FGF4 at a 1.8-Å resolution. FGF4 adopts a
-trefoil fold similar to
other FGFs. To identify potential receptor and heparin binding sites in
FGF4, a ternary FGF4-FGFR1-heparin model was constructed by
superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin
structure. Mutation of several key residues in FGF4, observed to
interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based
on the modeling and mutational data, we propose that FGF4, like FGF2,
but unlike FGF1, engages the
C'-
E loop in D3 and thus can
differentiate between the IIIc and IIIb splice isoforms of FGFRs for
binding. Moreover, we show that FGF4 needs to interact with both the
2-O- and 6-O-sulfates in heparin to exert
its optimal biological activity.
*
Corresponding author. Mailing address: Department of
Pharmacology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-2907. Fax: (212) 263-7133. E-mail:
mohammad{at}saturn.med.nyu.edu.

Present address: Department of Zoology, University of Zurich,
Zurich,
Switzerland.
Molecular and Cellular Biology, September 2001, p. 5946-5957, Vol. 21, No. 17
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.17.5946-5957.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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