FIt-I but not KDR/Flk-1 Tyrosine Kinase Is a Receptor for Placenta Growth Factor, Which Is Related to Vascular Endothelial Growth Factor1

Placenta Growth Factor (PIGF) is a new member of vascular endothelial growth factor (VEGF) family. Although VEGF binds FIt family Fit-I and KDR/FIk-I tyrosine kinases at high affinity for signal transduction, biological activities and the receptors of PIGF have not been extensively studied. Reverse transcription-PCR showed that P1GF-2, a subtype of PIGF-I that bears a basic amino acid-rich domain, is more abundant than P1GF-I and thus is the major subtype in human placenta. Using antibodies specific to PIGF-I or -2 as markers, we obtained large amounts of PIGFs in the baculovirus expression system. PIGF-2 had growthstimulatory activity on human umbilical vein endothelial cells and vascular permeability activity in the Miles assay at levels about 10-fold lower than those of VEGF. All PIGF-I activities were lower than those of P1GF-2. Both P1GFs competed for the binding of lasl labeled VEGF to Fit-I receptor but not to KDR/FIk-I expressed on NIH3T3 cells. Furthermore, lasI labeied PIGF bound to Fit-I at high affinity but not to KDR/Flk-1. Supporting the notion that PIGF can use only Fit-I as a receptor, PIGF activated Fit-I to autophosphorylate, whereas PIGF could not generate signals from KDR/ Fik-1. These results indicate that FIt-I, but not KDR/ Fik-1, is a receptor for PIGF, suggesting that the weak biological activities of PIGF are due to its use of only part of the available VEGF signaling. These mild characteristics of PIGF may be important for the appropriate development and maintenance of normal placental tissue. Received 9/22/95; accepted 11/17/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked advertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdicate this fact 1 This work was supported by a Grant-in-Aid for Special Project Research on Cancer-Bioscience 04253204 from the Ministry of Education, Science, Sports and Culture of Japan, and by Vakult Bioscience Foundation, The Naito Foundation, and The Foundation for Promotion of Cancer Research research grants. 2 To whom requests for reprints should be addressed. Phone: 81-3-54495550 (or 5551); Fax: 81 -3-5449-5425. Introduction Neovascularization including vasculogenesis and angiogenesis is crucial for embryonal development and the maintenance of the vertebrate body (1). Abnormal angiogenesis is involved in many pathological processes such as diabetes mellitus, rheumatoid arthritis, and the growth of solid tumors in vivo (2, 3). Accumulating evidence from a variety of biological and biochemical studies has revealed that VEGF3 and its receptor, FIt family, are key systems for the regulation of most neovascularization (4-6). During the very early stages of embryogenesis, the fit family, fit-i (7, 8), KDR/flk-i (9, 10), and flt-4 (1 1 , 12) receptor genes initiate their expression in the precursors of endothelial cells known as “hemangioblasts” in blood islands (1 3, 14). The expression of the fit gene family is restricted to endothelial cells from normal to pathological angiogenesis from embryo to adult stages (15-19). VEGF, a dimer of about Mr 23,000 subunits has two major biological activities, stimulation of endothelial cell growth and the enhancement of vascular permeability. The VEGF gene is expressed in a variety of mesenchymal and epithelial cells but not in endothelial cells, regulating the development and maintenance of the network in blood vessels in a paracrine manner. Furthermore, a variety of malignant tumors such as glioblastoma multiforme express VEGF mRNAs and secrete VEGF proteins, resulting in the stimulation of tumor angiogenesis for the supply of oxygen and nutrients to rapidly growing solid tumors (17, 18). VEGF binds two receptors of the Fit family, FIt-i and KDR/Fik-1 , and activates their tyrosine kinases for signaling into the cytoplasm (8, 15, 1 9, 20). Although the FIt and Fms/Kit/PDGF receptor families belong to a large supergene family based upon structural similarities, a unique signaling pathway from the Fit receptor family is considered to be present in endothelial cells. None of the cells expressing the Fit receptors other than endothelial cells can proliferate in response to VEGF (20-22). PIGF-i (PIGF131) has recently been molecularly cloned, and its amino acid sequence is highly homologous to that of VEGF (23). Furthermore, a new subtype of PIGF, called PIGF-2 (P1GF1S2), which carries a hepann-bmnding, basic amino acid-rich domain encoded in exon 6, has been isolated (24). A similar basic domain is also present in the subtypes of PDGF, which is distantly related to the VEGF/ PIGF family, indicating that the basic domain in PIGF-2 plays 3 The abbreviations used are: VEGF, vascular endothelial growth factor PIGF, placenta growth factor; RT-PCR, reverse transcriptase-PCR; HUVEC, human umbilical vain endothelial calls; VPF, vascular permeability factor.