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(Received for publication, October 3, 1996, and in revised form, December 26, 1996)
From the Institute of Molecular Medicine, Tumor Biology Center,
D-79106 Freiburg, Germany and The vascular endothelial growth factor (VEGF)
receptor FLT-1 has been shown to be involved in vasculogenesis and
angiogenesis. The receptor is characterized by seven Ig-like loops
within the extracellular domain. Upon VEGF binding FLT-1 becomes
phosphorylated, which has been thought to be preceded by receptor
dimerization. To further investigate high affinity binding of VEGF to
FLT-1 and ligand-induced receptor dimerization, we expressed in Sf9 cells the entire extracellular domain comprising all seven Ig-like loops: sFLT-1(7) and several truncated mutants consisting of loop one,
one and two, one to three, one to four, and one to five. The
corresponding proteins, named sFLT-1(1), (2), (3), (4), and (5) were
purified. Only mutants sFLT-1(3) to (7) were able to bind
125I-VEGF with high affinity. No binding of VEGF was
observed with sFLT-1(1) and sFLT-1(2), indicating that the first three
Ig-like loops are involved in high affinity binding of VEGF. The
binding of VEGF to sFLT-1(3) could be competed with placenta growth
factor (PlGF), a VEGF-related ligand, suggesting that high affinity
binding of VEGF and PlGF is mediated by the same or closely related
contact sites on sFLT-1. Deglycosylation of the sFLT-1(3), (4), (5),
and (7) did not abolish VEGF binding. Furthermore, unglycosylated sFLT-1(3), expressed in Escherichia coli, was able to bind
VEGF with similar affinity as sFLT-1(3) or sFLT-1(7), both expressed in
Sf9 cells. This indicates that receptor glycosylation is not essential
for high affinity binding. Dimerization of the extracellular domains of
FLT-1 upon addition of VEGF was detected with all mutants containing
the Ig-like loop four. Although sFLT-1(3) was able to bind VEGF,
dimerization of this mutant was inefficient, indicating that sites on
Ig-like loop four are essential to stabilize receptor dimers.
Volume 272, Number 16,
Issue of April 18, 1997
pp. 10382-10388
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
GSF-Forschungszentrum,
D-81377 München, Germany
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