Flt1 and Flk1KDR Tyrosine Kinases

5.1.1. Binding Characteristics

Two VEGF receptor tyrosine kinases (RTKs) have been identified. The Flt-1 (fms-like tyrosine kinase) (80) and kinase domain region (KDR) (81) receptors bind VEGF with high affinity. The murine homolog of KDR, Flk-1 (fetal liver kinase-1), shares 85% sequence identity with human KDR (82). Both Flt-1 and KDR/Flk-1 have seven immunoglobulin (Ig)-like domains in the extracellular domain (ECD), a single transmembrane region, and a consensus tyrosine kinase sequence which is interrupted by a kinase-insert domain (82-84). Flt-1 has the highest affinity for rhVEGF165, with a Kd of approx 10-20 pM(80). KDR has a somewhat lower affinity for VEGF: The Kd has been estimated to be approx 75-125 pM (81).

A cDNA coding an alternatively spliced soluble form of Flt-1 (sFlt-1), lacking the seventh Ig-like domain, transmembrane sequence, and the cytoplasmic domain, has been identified in human umbilical vein endothelial cells (85). This sFlt-1 receptor binds VEGF with high affinity (Kd 10-20 pM), and is able to inhibit VEGF-induced mitoge-nesis, and may be a physiological negative regulator of VEGF action (85).

An additional member of the family of RTKs with seven Ig-like domains in the ECD is Flt-4 (86-88), which, however, is not a receptor for VEGF, but rather binds a newly identified ligand called VEGF-C or VEGF-related peptide (VRP) (89,90). VEGF-C/VRP has been shown to be a regulator of lymphatic angiogenesis (91).

Recent studies have mapped the binding s ite for VEGF to the second immunoglobulin-like domain of Flt-1 and KDR. Deletion of the second domain of Flt-1 completely abol ished the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant, lacking the homologous domain, restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. To further test this hypothesis, chimeric receptors, in which the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, were created. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF (92).

One application of these structure-function studies is the generation of inhibitors of VEGF activity. The first three Ig-like domains of Flt-1, fused to a heavy-chain Fc, potently inhibits VEGF bioactivity across species. The Fc may confer sufficient half-life and stability when injected systemically (93). Therefore, this agent may a useful tool to determine the role of endogenous VEGF in several in vivo models.

5.1.2. Signal Transduction

VEGF has been shown to induce the phosphorylation of at least 11 proteins in bovine aortic endothelial cells (94). PLC-y, and two proteins that associate with PLC-g, were phosphorylated in response to VEGF. Furthermore, immunoblot analysis for mediators of signal transduction, which contain SH2 domains, demonstrated that VEGF induces phosphorylation of phosphatidylinositol 3-kinase, ras GTPase activating protein (GAP), and several others. These findings suggest that VEGF promotes the formation of multimeric aggregates of VEGF receptors with proteins that contain SH2 domains. These studies, however, did not identify which VEGF receptor(s) are involved in these events. Recently, it has been suggested that NO mediates, at least in part, the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary venules (95). The proliferative effect of VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. These findings suggest that VEGF stimulates proliferation of postcapillary endothelial cells, through the production of NO and cGMP accumulation.

Several studies have indicated that Flt-1 and KDR have different signal transduction properties (96,97). Porcine aortic endothelial cells, lacking endogenous VEGF receptors, display chemotaxis and mitogenesis in response to VEGF, when transfected with a plas-mid coding for KDR (96). In contrast, transfected cells expressing Flt-1 lack such responses (96,97). Flk-1/KDR undergoes strong ligand-dependent tyrosine phosphorylation in intact cells, but Flt-1 reveals a weak or undetectable response (96,97). Also, VEGF stimulation results in weak tyrosine phosphorylation that does not generate any mitogenic signal in transfected NIH 3T3 cells expressing Flt-1 (97). These findings agree with other studies showing that placenta growth factor (PlGF), which binds with high affinity to Flt-1, but not to Flk-1/KDR, lacks direct mitogenic or permeability-enhancing properties, or the ability to effectively stimulate tyrosine phosphorylation in endothelial cells (98). Therefore, interaction with Flk-1/KDR is a critical requirement to induce the full spectrum of VEGF biologic responses. In further support of this conclusion, VEGF mutants, which bind selectively to Flk-1/KDR, are fully active endothelial cell mitogens (99). These findings cast doubt on the role of Flt-1 as a truly signaling receptor. However, more recent evidence indicates that Flt-1 indeed signals, although understanding of these events is fragmentary. C unningham et al. (100) have demonstrated an interaction between Flt-1 and the p85 subunit of phosphatidylinositol 3-kinase, suggesting that p85 couples

Flt-1 to intracellular signal transduction systems, and implicating elevated levels of PtdIns(3,4,5)P3 levels in this process (100). Also, members of the Src family, such as Fyn and Yes, show an increased level of phosphorylation following VEGF stimulation in transfected cells expressing Flt-1, but not KDR (96). Furthermore, it has been shown that a specific biological response, the migration of monocytes in response to VEGF (or PlGF), is mediated by Flt-1 (101).

5.1.3. Regulation

The expression of Flt-1 and Flk-1/KDR genes is mostly restricted to the vascular endothelium. The promoter region of Flt-1 has been cloned and characterized, and a 1-kb fragment of the 5' flanking region, essential for endothelial-specific expression, was identified (102). Likewise, a 4-kb 5' flanking sequence has been identified in the promoter of KDR that confers endothelial cell specific activation (103).

Similar to VEGF, hypoxia has been proposed to play an important role in the regulation of VEGF receptor gene expression. Exposure of rats to acute or chronic hypoxia led to pronounced upregulation of both Flt-1 and Flk-1/KDR genes in the lung vasculature (104). Also, Flk-1/KDR and Flt-1 mRNAs were substantially upregulated throughout the heart, following myocardial infarction in the rat (105). However, in vitro studies have yielded unexpected results. Hypoxia increases VEGF receptor number by 50% in cultured bovine retinal capillary endothelial cells, but the expression of KDR is not induced, but paradoxically shows an initial downregulation (106). Brogi et al. (107) have proposed that the hypoxic upregulation of KDR observed in vivo is not direct, but requires the release of an unidentified paracrine mediator from ischemic tissues. Recent studies have provided evidence for a differential transcriptional regulation of the Flt-1 and KDR genes by hypoxia (108). When human umbilical vein endothelial cells (HUVEC) were exposed to hypoxic conditions in vitro, increased levels of Flt-1 expression were observed. In contrast, Flk-1/KDR mRNA levels were unchanged or slightly repressed. Promoter deletion analysis demonstrated a 430-bp region of the Flt-1 promoter to be required for transcriptional activation in response to hypoxia. This region includes a heptamer sequence matching the HIF-1 consensus binding site previously found in other hypoxia inducible genes. The element mediating the hypoxia response was further defined as a 40-bp sequence, including the putative HIF-1 binding s ite. S uch element was not found in the Flk-1/KDR promoter. These findings indicate that, unlike the KDR/Flk-1 gene, the Flt-1 receptor gene is directly upregulated by hypoxia via a hypoxia-inducible enhancer element located at position -976 to -937 of the Flt-1 promoter (108). Also, recent studies have shown that both TNF-a (109) and TGF-|3 (110) have the ability to inhibit the expression of the KDR gene in cultured endothelial cells.

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