The International Journal of Biochemistry & Cell Biology
ReviewStructure and dual function of vascular endothelial growth factor receptor-1 (Flt-1)
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 tumors in vivo [2]. Accumulating evidence strongly suggests that Vascular Endothelial Growth Factor (VEGF) and its receptor system are important for the regulation of neovascularization as well as for pathological angiogenesis [3], [4], [5], cooperating with other systems including Angiopoietin-Tie receptor and Ephrin2B-Eph4B receptor [6], [7], [8].
VEGF receptor family consists of three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), all of which belong to the receptor type tyrosine kinase superfamily (Fig. 1) [5], [9], [10], [11], [12]. Among these receptors, VEGFR-1 binds strongest to VEGF, VEGFR-2 binds much weaker, and VEGFR-3 shows essentially no binding to VEGF, although it binds other members of the VEGF family. Thus, these three structurally highly related receptors appear to have different biological functions in vivo. Here, I briefly summarize recent progress in the study of VEGFR-1 which has been found to have negative and positive roles in vertebrates (see Items Sections 8–11).
Section snippets
Structure of VEGFR-1 protein
The VEGFR-1 gene was originally isolated from a human placenta cDNA library and its product designated as Fms-like tyrosine kinase-1 (Flt-1), because of structural similarity to the Fms family [13]. This family, c-Fms (macrophage-colony stimulating factor receptor), c-Kit (stem cell factor receptor), Flt3/Flk2 and PDGFRs, have five Immunoglobulin (Ig)-like domains in the extracellular region, as compared to the seven Ig-like domains in VEGFR-1.
Human VEGFR-1 protein consists of 1338 amino acid
Intracellular region of VEGFR-1
The intracellular region of VEGFR-1 carries a tyrosine kinase domain and a carboxyl terminal region downstream of the kinase domain. VEGFR-1 tyrosine kinase domain is split into two by a long kinase insert similar to the Fms family receptors. However, the tyrosine-X-X-methionine motif, conserved in essentially all of the Fms family members and important for the binding of the PI3 kinase subunit and activation of the PI3 kinase pathway [22], is not present in any form of VEGFR-1 in mammals [13].
Soluble form of VEGFR-1
Human VEGFR-1 gene expresses two major short mRNAs of 3.0 and 2.4 kb [13]. The difference in the length of these two mRNAs is thought to result from a difference in the 3'-noncoding region, since a primer extension study showed a single transcriptional promoter site in the VEGFR-1 gene [23].
Short mRNAs encode a soluble form of VEGFR-1 which consists of the 1st–6th Ig-like domains together with an additional 31 amino acid residues derived from an intron sequence in the carboxyl terminal region
Genomic organization of VEGFR-1 gene
VEGFR-1 gene in mice consists of 30 exons spanning more than 150 kb [25]. Among Fms family receptors (five-Ig-type receptors), Fms, Kit and Flt3/Flk2 are essential for hematopoiesis, whereas seven-Ig-type receptors, VEGFR-1, -2 and -3, are closely involved in the formation of tube-like structures, blood vessels and lymphatic vessels. Therefore, the five-/seven-Ig receptor supergene family is considered to be phylogenetically established at a similar period, at the very early stage of vertebrate
Regulation of VEGFR-1 gene expression
The mRNA of VEGFR-1 gene is specifically expressed in most of the vascular endothelial cells with a few exceptions [29], [30], [31]. This is also true at the level of protein. Isotope-labeled VEGF detects its binding sites in most of the blood vessels from embryo to adult stages [32]. Based on competition experiments and the high affinity of VEGFR-1 for VEGF, more than half of these VEGF-binding sites in the blood vessels appear to be the VEGFR-1 protein.
Cell-type specific gene regulation of
Ligands for VEGFR-1
So far three different gene products, VEGF, PlGF and VEGF-B, are known to bind and activate VEGFR-1 [37], [38], [39], [40], [41]. Among these ligands, the affinity of VEGFR-1 is highest for VEGF (10–30 pM) and much weaker for PlGF (about 200 pM) and for VEGF-B. PlGF is expressed in placenta but very low or undetectable in other normal tissues. In some carcinomas such as renal cancer, however, PlGF is expressed at relatively high levels [42]. On the other hand, VEGF-B is expressed more
Endothelial cells
PlGF-2, a VEGFR-1-specific ligand, transduces positive signals for cell proliferation and vascular permeability, but these activities are one order of magnitude or more weaker than those of VEGF [39]. PlGF-1 is also reported to have some angiogenic activity in vivo [45]. When VEGFRs on endothelial cells are stimulated by the VEGFR-2-specific ligand VEGF-E, the degree of biological response is similar to that induced by VEGF [46], [47]. Therefore, the major positive signal of VEGF for
Gene targeting studies on VEGFR-1
Gene knockout studies to generate null mutations of VEGFR-1 gene were carried out by Breitman's group [52], [53]. Heterozygote mice for either VEGFR-1 or VEGFR-2 gene are healthy and essentially normal in blood vessel formation. However, the phenotypes of the homozygotes are quite different. VEGFR-1 (−/−) mice are embryonic lethal at E8.5–9.0 due to a disorganization of blood vessels and overgrowth of endothelial-like cells within the lumens of blood vessels. On the other hand, VEGFR-2 (−/−)
Signal transduction from VEGFR-1
The tyrosine kinase activity of VEGFR-1 is usually weak, about one tenth that of VEGFR-2 kinase. Without starving cells of serum, VEGF-dependent autophosphorylation is often difficult to detect [58], [59]. However, it is not a so called ‘dead kinase’ or inactivated kinase. When it is overexpressed in insect cells using the Baculovirus system, autophosphorylation of VEGFR-1 is easily detected [60]. This autophosphorylation is completely abolished by a point mutation from lysine to methionine at
Medical applications of soluble VEGFR-1
Since soluble VEGFR-1 has a strong affinity with VEGF and since this molecule is an endogenously expressed protein, several research groups have attempted to use it as a VEGF-blocking reagent in experimental animal models for carcinogenesis. Purified soluble VEGFR-1 protein itself or Adenovector-mediated soluble VEGFR-1 expression systems have been shown to be effective in the suppression of solid tumor growth in vivo [24], [66], [67], [68], [69]. Intratracheal administration of Adeno-soluble
Acknowledgements
I thank all the colleagues in my laboratory as well as those in other institutions for the continuous help to carry out the VEGF receptor research projects.
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