Identification of placenta growth factor determinants for binding and activation of Flt-1 receptor

Placenta growth factor (PlGF) belongs to the vascular
endothelial growth factor (VEGF) family and represents
a key regulator of angiogenic events in pathological conditions.
PlGF exerts its biological function through the
binding and activation of the seven immunoglobulinlike
domain receptor Flt-1, also known as VEGFR-1.
Here, we report the first detailed mutagenesis studies
that provide a basis for understanding molecular recognition
between PlGF-1 and Flt-1, highlighting some of
the residues that are critical for receptor recognition.
Mutagenesis analysis, performed on the basis of a structural
model of interaction between PlGF and the minimal
binding domain of Flt-1, has led to the identification
of several PlGF-1 residues involved in Flt-1 recognition.
The two negatively charged residues, Asp-72 and Glu-73,
located in the 3-4 loop, are critical for Flt-1 binding.
Other mutations, which bring about a significant decrease
in PlGF binding activity, are Gln-27, located in
the N-terminal -helix, and Pro-98 and Tyr-100 on the 6
strand. The mutation of one of the two glycosylated residues
of PlGF, Asn-84, generates a PlGF variant with
reduced binding activity. This indicates that, unlike in
VEGF, glycosylation plays an important role in Flt-1
binding. The double mutation of residues Asp-72 and
Glu-73 generates a PlGF variant unable to bind and
activate the receptor molecules on the cell surface. This
variant failed to induce in vitro capillary-like tube formation
of primary endothelial cells or neo-angiogenesis
in an in vivo chorioallantoic membrane assay.