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 immunoglobulin-like 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 beta3-beta4 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 alpha-helix, and Pro-98 and Tyr-100 on the beta6 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.
Errico
Michela
M
Institute of Genetics and Biophysics Adriano Buzzati-Traverso, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy.
Riccioni
Teresa
T
Iyer
Shalini
S
Pisano
Claudio
C
Acharya
K Ravi
KR
Persico
M Graziella
MG
De Falco
Sandro
S
D000482:Allantois / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002642:Chick Embryo; D002823:Chorion / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D018698:Glutamic Acid; D005973:Glutamine; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010920:Placenta; D000072483:Placenta Growth Factor; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism*
D018076:DNA, Complementary; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D000072483:Placenta Growth Factor; D018698:Glutamic Acid; D040281:Vascular Endothelial Growth Factor Receptor-1