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. Author manuscript; available in PMC: 2006 Jun 1.
Published in final edited form as: Oncogene. 2004 Jul 15;23(32):5523–5531. doi: 10.1038/sj.onc.1207712

Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation

Rosana D Meyer 1, Amrik Singh 1, Fredric Majnoun 1, Catharina Latz 1, Kameran Lashkari 2, Nader Rahimi 1,*
PMCID: PMC1472702  NIHMSID: NIHMS9541  PMID: 15107818

Abstract

VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.

Keywords: VEGFR-1, VEGFR-2, angiogenesis, tyrosine kinase activation, tyrosine phosphorylation, endothelial cells

Introduction

Vascular endothelial growth factor (VEGF) and its two high-affinity tyrosine kinase receptors (VEGFR-1/Flt-1) and VEGFR-2/FLK-1 initiate signaling pathways that play a pivotal role during embryonic development and pathological angiogenesis (Breier, 2000; Yancopoulos et al., 2000). VEGF activates a broad spectrum of biological responses in endothelial cells, including cell proliferation, migration, survival, differentiation and permeability to macromolecules (Waltenberger et al., 1994; Neufeld et al., 1996; Rahimi et al., 2000; Suarez and Ballmer-Hofer, 2001). Although VEGFR-1 and VEGFR-2 are structurally highly similar, their mechanisms of angiogenic signal transduction relay appear to differ drastically from each other. Targeted deletion of VEGFR-2 has revealed an essential requirement for VEGFR-2 in embryogenesis. Homozygous VEGFR-2 null mice exhibited early embryonic lethality due to the absence of endothelial cells (Shalaby et al., 1995). In contrast, targeted deletion of VEGFR-1 resulted in early embryonic lethality due to overgrowth of endothelial cells, suggesting a negative role for VEGFR-1 in angiogenesis (Fong and Rossant, 1995; Fong et al., 1999; Kearney et al., 2002). The molecular mechanisms that account for these differences between the two highly related receptors are not fully understood.

VEGFR-1, like other RTKs, possesses all the known signatures of RTKs such as GXGXXG, an ATP-binding motif, HRDLA, a motif essential for catalysis and many potential tyrosine phosphorylation sites within its kinase domain and carboxyl tail (Hanks and Quinn, 1991). Despite having these similarities with other RTKs, the ligand-dependent stimulation of VEGFR-1 results in no significant autophosphorylation of VEGFR-1 itself or induces proliferation of endothelial cells (Waltenberger et al., 1994; Rahimi et al., 2000). However, VEGFR-1 may stimulate biological responses in endothelial cells potentially by heterodimerizing with VEGFR-2 (Kanno et al., 2000; Rahimi et al., 2000; Autiero et al., 2003). VEGFR-1 activation in some nonendothelial cells is reported to stimulate cell migration and proliferation (Clauss et al., 1996; Athanassiades et al., 1998). VEGF binds to multiple endothelial cell surface receptors, including VEGFR-1, VEGFR-2 and Neuropilin-1 and 2 (Soker et al., 1998; Neufeld et al, 1999; Lee et al., 2002). We have recently constructed a chimeric receptor containing the extracellular domain of human CSF-1R/c-fms, fused with the transmembrane and the cytoplasmic domains of human VEGFR-1 (Rahimi et al., 2000). This model permitted us to dissect the function of VEGFR-1 in endothelial cells by selectively stimulating the receptor with CSF-1. In this report, we demonstrate that the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to activate MAPK in endothelial cells. Specifically, we show that substitution of C-terminus of VEGFR-2 with that of VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation.

Results

VEGFR-1 is devoid of ligand-dependent autophosphorylation in vivo and in vitro

In this study, we have used chimeric VEGFR-1 containing the extracellular domain of human CSF-1R/c-fms, fused with the transmembrane and the cytoplasmic domains of murine VEGFR-1 (herein called CTR) to analyse the ligand-dependent autophosphorylation of VEGFR-1 and activation of signaling proteins (Rahimi et al., 2000). Stimulation of pig aortic endothelial (PAE) cells expressing individual chimeric VEGFR-1 (CTR) and VEGFR-2 (CKR) with CSF-1 demonstrated that under comparable experimental conditions of expression and activation, CTR and CKR were strikingly different in their ability to undergo autophosphorylation and kinase activation (Figure 1a and c). Stimulation of CKR but not CTR resulted in robust tyrosine phosphorylation. Only a long exposure of film detected a trace amount of ligand-dependent tyrosine phosphorylation of CTR. Pretreatment of cells expressing CTR with sodium-orthovanadate, a general tyrosine phosphatase inhibitor, also did not increase the ligand-dependent autophosphorylation of CTR (data not shown). As shown in Figure 1b, CKR but not CTR underwent autophosphorylation in an in vitro kinase assay.

Figure 1.

Figure 1

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Ligand-dependent activation of chimeric VEGFR-1 (CTR) and chimeric VEGFR-2 (CKR). Serum-starved semi-confluent PAE cells expressing CKR or CTR were stimulated with CSF-1 (40 ng/ml) for 0, 10 and 30 min, lysed and immunoprecipitated with an anti-CSF-1R antibody. The immunoprecipitated proteins were collected and subjected to Western blot analysis using antiphosphotyrosine antibody (a). To determine the protein levels in each lane, the same membrane was reprobed with an anti-CSF-1 antibody (b). Immunoprecipitated proteins derived from 0 and 10 min CSF-1 stimulated cells were subjected to an in vitro kinase assay (b). Serum-starved semiconfluent PAE cells expressing CKR or CTR were stimulated with CSF-1 lysed and total cell lysates were subjected to Western blot analysis using anti-phospho-PLCγ1 antibody (d) or anti-phospho-MAPK antibody (f). The same membranes were reprobed with anti-PLCγ1 antibody and anti-p42 (MAPK) antibody, respectively (e and g)

To test the ability of CTR to activate the downstream signaling proteins, we also analyzed activation of PLCγ1 and MAPK using phosphospecific antibodies generated against the active forms of PLCγ1 and MAPK, respectively. The results show that MAPK but not PLCγ1 is activated by CTR stimulation (Figure 1d and f). As previously shown, stimulation of cells expressing CKR results in the activation of both PLCγ1 and MAPK (Rahimi et al., 2000; Meyer et al., 2002, and Figure 1d and f). Collectively, these results suggest that VEGFR-1 is not fully autophosphorylated in response to ligand stimulation and does not activate PLCγ1. VEGFR-1 stimulation, however, activates limited signaling pathways, which leads to MAPK activation.

Selective activation of VEGFR-1 is not associated with endothelial cell migration, intracellular calcium release and angiogenesis in vitro

Activation of VEGFR-1 is not associated with endothelial cell proliferation (De Vries et al., 1992; Keyt et al., 1996; Fong et al., 1999; Rahimi et al., 2000; Gille et al., 2001). To test whether VEGFR-1 activation modulates other cellular functions, we subjected cells expressing CTR to cell migration, intracellular calcium release and in vitro angiogenesis. Cells expressing chimeric VEGFR-2 (CKR) were used as a control. Figure 2 shows that stimulation of cells expressing CTR results in no significant endothelial cell migration or intracellular calcium release. CTR activation induced only scanty sprouting of endothelial cells in an in vitro angiogenesis assay (Figure 2c). In contrast to CTR, stimulation of cells expressing CKR induced cell migration, intracellular calcium release and angiogenesis in vitro (Figure 2a-c). These results suggest that VEGFR-1 is unable to stimulate endothelial cell migration, intracellular calcium release and angiogenesis in vitro.

Figure 2.

Figure 2

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Activation of VEGFR-1 is not associated with cell migration, intracellular calcium response or tubulogenesis. Serum-starved PAE cells expressing empty vector, pLXSN2 (N2), CKR or CTR were treated with CSF-1 and cell migration was measured as described in Materials and methods (a). Serum-starved PAE cells expressing CKR or CTR were grown on glass coverslips, stimulated with CSF-1 and intracellular calcium was measured with confocal microscopy using Fluo-3AM probe as described in Materials and methods (b). PAE cells expressing CKR or CTR were prepared as spheroid and subjected to in vitro angiogenesis assay with or without CSF-1. Sprouting and tubulogenesis were observed after 2 days under an inverted phase contrast microscope (Nikon) and pictures were taken using the SPOT camera system (c)

We have recently shown that ligand-dependent autophosphorylation of VEGFR-2 is highly influenced by its carboxyl terminus (Meyer et al., 2002, 2004). Based on these observations, we postulated that the carboxyl terminus of VEGFR-1 might also play a role in the ligand-dependent autophosphorylation of VEGFR-1. In order to test this hypothesis, we initially deleted the entire carboxyl-terminus consisting of 176 amino acids just after its kinase domain boundary of VEGFR-1 chimera, CTR (Figure 3a). The truncated receptor was expressed in PAE cells by retroviral system and was denoted ΔCTR. To allow for easy detection of ΔCTR, we also introduced a Myc tag at its carboxyl tail. CTR contains the extracellular domain of human CSF-1 receptor/c-Fms fused with the transmembrane and the cytoplasmic domains of human VEGFR-1 (Rahimi et al., 2000). In this study, we used CTR to avoid crosstalk between VEGFR-1 and the other endothelial receptors such as VEGFR-2, Neuropilin-1 and Neuropilin-2. As presented in Figure 3b, equal numbers of PAE cells expressing either empty vector, wild-type CTR or ΔCTR were lysed and subjected to Western blot analysis using an anti-Myc antibody. Both CTR and ΔCTR were expressed in PAE cells at comparable levels.

Figure 3.

Figure 3

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Effect of deletion of carboxyl tail of CTR on its tyrosine phosphorylation. Schematic representation of CTR and C-tail truncated CTR (ΔCTR). ΔCTR was constructed as described in Materials and methods (a). Equal numbers of PAE cells expressing empty vector (pLXSN2), CTR or ΔCTR were lysed and total cell lysates were subjected to Western blot analysis using anti-Myc antibody (b). PAE cells expressing CKR, CTR or ΔCTR were stimulated with CSF-1 (40 ng/ml, lysed and immunoprecipitated with anti-CSF-1R antibody and were subjected to Western blot analysis using antiphosphotyrosine antibody (c). Cells expressing CKR, CTR, ΔCTR or Kinase dead CTR (CTR/R861) were stimulated with CSF-1 (40 ng/ml), and lysed and total cell lysates were subjected to Western blot analysis using phospho-MAPK antibody (e). The same membranes were reprobed with anti-Myc or anti-p42 MAPK antibodies, respectively (d, f)

As presented in Figure 1c and d, VEGFR-1 was significantly devoid of the ligand-dependent autophosphorylation and kinase activation. To test the contribution of C-terminus to this nature of VEGFR-1, we first analysed the ligand-dependent autophosphorylation of CTR and ΔCTR. PAE cells expressing chimeric VEGFR-2 (CKR) were used as a positive control. To this end, equal numbers of serum-starved cells were stimulated with CSF-1 and lysed, then immunoprecipitated with anti-CSF-1R antibody and subjected to Western blot analysis using an antiphosphotyrosine antibody. As Figure 3c shows, no significant ligand-dependent autophosphorylation of either CTR or ΔCTR was observed. In contrast, ligand-dependent autophosphorylation of CKR was readily detectable. Collectively, these results suggest that deletion of carboxyl-tail of VEGFR-1 does not rescue its poor ligand-dependent autophosphorylation. To test the contribution of C-tail and kinase activity of CTR to its ability to stimulate MAPK activation, we subjected PAE cells expressing CTR, ΔCTR and R861/CTR to MAPK activation. Lysine 861 (R861), which is located in the kinase domain of VEGFR-1 and is predicted to bind ATP, was replaced with arginine. As shown in Figure 3e, deletion of the C-tail and mutation of the ATP-binding site of VEGFR-1 (lysine to arginine) significantly reduced, but it did not abolish, the ability of CTR to stimulate MAPK activation. This observation suggests that the basal kinase activation of VEGFR-1 and the presence of its carboxyl tail are required for its ability to stimulate MAPK activation.

Exchanging the carboxyl terminus of VEGFR-2 with that of VEGFR-1 promotes ligand-dependent autophosphorylation of VEGFR-1 and cell proliferation

Our results show that deletion of the carboxyl tail of VEGFR-1 does not rescue the poor autophosphorylation of receptor. One interpretation for the observed finding is that the carboxyl tail of VEGFR-1 is not involved in its ligand-induced activation. However, other possibilities are also feasible. For instance, if the C-tail of VEGFR-1 is lacking certain amino acids or motifs necessary for its active participation in receptor activation, deletion of the C-tail of VEGFR-1 thus is not expected to overcome its poor ligand-dependent autophosphorylation. One way to test this possibility is to replace the C-tail of VEGFR-1 with that of another receptor and test whether this replacement can rescue the poor ligand-dependent autophosphorylation of VEGFR-1. To this end, we exchanged the carboxyl terminus of VEGFR-1 with that of VEGFR-2 and tested its impact on the ligand-dependent activation of VEGFR-1. The carboxyl terminus of VEGFR-1 and VEGFR-2 represents the most divergent region between the two receptors, suggesting a potential regulatory role in the activation of VEGFR-1 and VEGFR-2. Figure 4a shows the strategy used to generate the carboxyl tail-swapped VEGFR-1 (herein denoted as CTR/c-FLK-1). The resultant engineered receptor was subsequently expressed in PAE cells. Equal numbers of PAE cells expressing CTR, CKR and CTR/cFLK-1 were lysed, and total cell lysates were subjected to Western blot analysis using anti-CSF-1R antibody, which specifically recognizes the extracellular domain of human CSF-1R (Figure 4b). In addition, we reblotted the same membrane with anti-VEGFR-2 antibody (1412) that specifically recognizes the carboxyl domain of VEGFR-2 (Figure 4c). Thus, all the engineered receptors are expressed at relatively equal levels in PAE cells.

Figure 4.

Figure 4

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Exchanging the carboxyl tail of CTR with that of VEGFR-2 promotes ligand-dependent tyrosine phosphorylation of CTR. Schematic representation of the carboxyl tail exchanged CTR (CTR/cFLK-1). CTR/cFLK-1 was constructed using PCR as described in Materials and methods (a). Equal numbers of PAE cells expressing CTR, CKR or CTR/cFLK-1 were lysed and subjected to Western blot analysis using CSF-1R antibody (b). The same membrane was reprobed with anti-VEGFR-2 antibody, which specifically recognizes the carboxyl tail of VEGFR-2 (c). Serum-starved PAE cells expressing CKR, CTR or CTR/cFLK-1 were stimulated with CSF-1 for 10 min, lysed and immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with anti-phosphotyrosine antibody (d). The same membrane reprobed with anti-VEGFR-2 antibody, which specifically recognizes the carboxyl tail of VEGFR-2 (e). Cells expressing CTR/cFLK-1 or CTR/F1212/cFLK-1 were stimulated with CSF-1 for 0, 10 or 30 min, and total cell lysates were subjected to Western blot analysis using an antiphosphotyrosine antibody (f). The same membrane reprobed with anti-VEGFR-2 antibody, which recognizes the C-tail of VEGFR-2 (g). Alignment of tyrosine residues of carboxyl tail of VEGFR-1 with that of VEGFR-2 (h)

To test the ligand-dependent autophosphorylation of the swapped receptor, cells expressing either chimeric VEGFR-2 (positive control), CTR (chimeric VEGFR-1) or CTR/cFLK-1 (C-tail swapped CTR) were stimulated with CSF-1 for 10 min, lysed and immunoprecipitated with antiphosphotyrosine antibody. The immunoprecipitated proteins were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected to Western blot analysis using an antiphospho tyrosine antibody (Figure 4d). The same membrane was reprobed with anti-VEGFR-2 antibody (Figure 4e). The result shows that replacement of C-tail of VEGFR-1 with that of VEGFR-2 is able to rescue the lack of ligand-dependent autophosphorylation of VEGFR-1. The ability of C-tail of VEGFR-2 to make VEGFR-1 undergo ligand-dependent tyrosine autophosphorylation suggests that the carboxyl tail of VEGFR-2 but not VEGFR-1 contains certain amino acid residues, in particular, tyrosine that allows VEGFR-2 to undergo tyrosine autophosphorylation in response to ligand stimulation. Alignment of the C-tail of VEGFR-1 with that of VEGFR-2 shows that the C-tails of both VEGFR-1 and VEGFR-2 contain equal numbers of tyrosine residues (Figure 4h). At face value, this may imply that whether the poor ligand-dependent tyrosine phosphorylation of VEGFR-1 is not associated with the absence of tyrosine residues in its C-tail or these tyrosines are not phosphorylated. We recently have shown that the ligand-dependent activation of VEGFR-2, in part, is mediated by phosphorylation of tyrosine 1212, located within its carboxyl tail (Meyer et al., 2002). To test whether the ligand-dependent tyrosine phosphorylation of CTR/cFLK-1 (C-tail swapped CTR) also is influenced by phosphorylation of tyrosine 1212, we first evaluated phosphorylation of tyrosine 1212 in both chimeric VEGFR-2 and CTR/cFLK-1 (C-tail swapped CTR), using a phosphospecific tyrosine 1212 antibody. The result showed that tyrosine 1212 is phosphorylated on both CKR and CTR/cFLK-1 in a ligand-dependent manner (data not shown and Meyer et al., 2004). To demonstrate the importance of tyrosine 1212 to the ligand-dependent autophosphorylation of CTR/cFLK-1, we mutated the tyrosine 1212 to phenylalanine and evaluated its contribution to its activation. As presented in Figure 4g, the lack of tyrosine 1212 on CTR/cFLK-1 reduces the ligand-dependent autophosphorylation of the receptor. This highlights the importance of tyrosine 1212 not only on the activation of VEGFR-2 but also suggest that the lack of tyrosine 1212 on the carboxyl tail of VEGFR-1 may be associated with the poor ligand-dependent autophosphorylation of VEGFR-1.

Selective activation of VEGFR-1 in endothelial cells is not associated with endothelial cell proliferation (De Vries et al., 1992; Waltenberger et al., 1994; Rahimi et al., 2000; Kearney et al., 2002). To test whether the C-tail-swapped VEGFR-1 is able to simulate biological responses, we subjected cells either expressing CTR or CTR/cFLK-1 to proliferation assay. As presented in Figure 5, stimulation of cells expressing the C-tail-swapped receptor (CTR/cFLK-1) was able to induce cell proliferation. In contrast, stimulation of cells expressing CTR resulted in no significant cell proliferation. Altogether, these results suggest that the C-tail of VEGFR-1 in part might contribute to the inferior kinase activation and its inability to stimulate cell proliferation.

Figure 5.

Figure 5

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Exchanging the carboxyl tail of CTR with that of VEGFR-2 promotes ligand-dependent proliferation of PAE cells. PAE cells expressing CTR or CTR/cFLK-1 were treated with the indicated concentrations of CSF-1 and DNA synthesis was measured by [3H] tymidine uptake as described in Materials and methods. The data are expressed as a ratio of stimulated over unstimulated samples

VEGFR-1 is impaired in its ability to undergo ligand-dependent downregulation

Ligand binding to RTKs is associated with their activation leading to subsequent downregulation and removal from cell surface (Gruenberg, 2001; Sorkin and Von Zastrow, 2002). Ligand-dependent downregulation of RTKs including their recruitment to clathrin-coated pits and endosomal sorting compartments require their kinase activation (Honegger et al., 1987; Felder et al., 1990). Since VEGFR-1 is not fully kinase active receptor, it is possible that the ability of this receptor to undergo ligand-dependent downregulation is also impaired. To test this possibility, we tested the ability of chimeric VEGFR-1 (CTR) to undergo ligand-dependent downregulation. To this end, we first measured the ligand-dependent downregulation of chimeric CTR by pulse-chase analysis using 35S-labeled-methionine. Stimulation of cells expressing CTR with CSF-1 promoted no significant degradation and downregulation of receptor. After 30 min of ligand stimulation, only a small percentage of CTR underwent downregulation. Surprisingly, after 60 min of ligand stimulation no apparent downregulation of CTR was observed (Figure 6a). In addition, we measured the ligand-dependent downregulation of CTR by stimulating PAE cells expressing CTR with CSF-1 for 30, 60 and 120 min. Finally, cells were lysed and total cell lysates were subjected to Western blot analysis using VEGFR-1 antibody. The result showed that ligand stimulation is not associated with robust downregulation of VEGFR-1 (data not shown). In contrast, in a similar experiment, VEGFR-2 rapidly underwent ligand-dependent down-regulation after 30 min of stimulation with ligand. The presence of C-terminus of VEGFR-2 is required for its ligand-dependent downregulation (Meyer et al., 2004). Altogether, these results demonstrate that VEGFR-1 is defective in its ability to undergo ligand-dependent downregulation and degradation. In addition, the data suggest that VEGFR-1 may undergo ligand-dependent recycling rather than downregulation and degradation. To test the influence of carboxyl tail of VEGFR-2 on the ligand-dependent downregulation of VEGFR-1, we also analysed the ligand-induced downregulation of C-tail-swapped VEGFR-1 (CTR/c-FLK-1). As presented in Figure 6b, stimulation of cells expressing CTR/c-FLK-1 with CSF-1 dramatically reduced the half-life of CTR/cFLK-1. The ligand-induced downregulation of CTR/cFLK-1 is more similar to that of ligand-dependent downregulation of VEGFR-2 (data not shown). Interestingly, when the carboxyl terminus of VEGFR-1 was introduced to VEGFR-2, the ligand-dependent autophosphorylation and downregulation of the C-tail-swapped VEGFR-2 was not impaired (Meyer et al., 2004).

Figure 6.

Figure 6

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VEGFR-1 fails to undergo ligand-dependent down-regulation. PAE cells expressing chimeric VEGFR-1 (CTR) (a) or C-tail-swapped CTR (CTR/cFLK-1) (b) were starved in serum-free and methionine/cystine-free medium and then were labeled with [35S]methionine/cystine (75 μCi/ml) for 3 h. Cells were stimulated with CSF-1 for indicated period of times (−60 corresponds to cells that are not stimulated with ligand, but were lysed at the same time as 60 min stimulation with ligand)

Discussion

VEGFR-1 and VEGFR-2 are high-affinity receptors for VEGF (Neufeld et al., 1996; Yancopoulos et al., 2000). Although VEGFR-1 and VEGFR-2 are structurally highly similar, their angiogenic signal transduction appears to differ drastically from each other. Activation of VEGFR-2 positively regulates angiogenesis (Breier 2000; Yancopoulos et al., 2000; Gille et al., 2001) and it is highly autophosphorylated in response to ligand stimulation (Waltenberger et al., 1994; Shalaby et al., 1995; Rahimi et al., 2000). VEGFR-1 is poorly autophosphorylated in response to ligand stimulation in endothelial cells and appears to regulate angiogenesis negatively (Waltenberger et al., 1994; Fong et al., 1999; Rahimi et al., 2000; Kearney et al., 2002). Despite its poor autophosphorylation, VEGFR-1 can heterodimerize with VEGFR-2 and transphosphorylates VEGFR-2 (Rahimi et al., 2000; Autiero et al., 2003). VEGFR-1 also has a much greater affinity to VEGF than does the VEGFR-2 (Shibuya, 2001).

In some RTKs such as colon carcinoma kinase-4 (CCK4), ErbB3/HER3, KLG and Ror1, abnormalities in the kinase domain have been suggested to contribute to the kinase-defective phenotype of these RTKs (Chou and Hayman, 1991; Guy et al., 1994; Mossie et al., 1995). VEGFR-1 has no aberrant amino-acid replacement in these regions, suggesting that its kinase-defective phenotype is not associated with structural abnormality within the kinase domain. Replacement of the JM (Juxtamembrane) region of VEGFR-1 with that of VEGFR-2 also does not rescue its poor ligand-dependent kinase activation (Gille et al., 2000). Our current study shows that the kinase-defective phenotype of VEGFR-1 is linked to its carboxyl tail. Deletion of the carboxyl tail of VEGFR-1 does not overcome the poor tyrosine phosphorylation. However, replacement of the carboxyl tail of VEGFR-1 with that of VEGFR-2 facilitated the ligand-dependent autophosphorylation and its activation induced cell proliferation. More importantly, our data demonstrate that VEGFR-1 is defective in its ability to undergo ligand-dependent downregulation and degradation. Surprisingly, replacement of carboxyl tail of VEGFR-1 with that of VEGFR-2 not only facilitated the ligand-induced receptor activation, it also caused the receptor to undergo ligand-dependent downregulation and degradation. The regulation of angiogenesis is controlled by cooperative and antagonistic signals generated by VEGFRs (Hanahan and Folkman 1996; Breier 2000; Yancopoulos et al., 2000). It appears that during the evolution, VEGFR-1 lost its ability to undergo VEGF-dependent downregulation and kinase activation. However, its extracellular domain is evolved in which it can bind to VEGF more efficiently than VEGFR-2 (Hiratsuka et al., 1998; Shibuya, 2001). These two unique phenotypes of VEGFR-1 in part may contribute to its negative role in angiogenesis.

The carboxyl tails of receptor tyrosine kinases are involved in both positive and negative regulation of receptor tyrosine kinases. For instance, the deletion of carboxyl tails of the EGF receptor and Tie-2 promotes their activation (Walton et al., 1990; Li et al., 1991; Niu et al., 2002), whereas deletion of carboxyl tail of the PDGF receptor (Seedorf et al., 1992) and VEGFR-2 impairs their ligand-dependent activation (Meyer et al., 2004). How does the carboxyl tail of VEGFR-2 rescue the kinase-defective phenotype of VEGFR-1? One possible explanation is that the carboxyl tail of VEGFR-2 contains more tyrosine residues than that of VEGFR-1. Thus, upon ligand stimulation the C-tail-swapped VEGFR-1 is autophosphorylated in additional tyrosine residues. This possibility, however, appears not to be the case. The alignment of the amino acids corresponding to the carboxyl tail of VEGFR-1 with that of VEGFR-2 shows that the carboxyl tails of both receptors contain equal tyrosine residues (see Figure 5 in this manuscript). One can envision that the carboxyl tail of VEGFR-1 is required for its ligand-dependent autophosphorylation and downregulation; however, it lacks critical amino acids in its carboxyl tail. Consequently, the lack of critical residues in its C-tail tampers the overall ligand-dependent activation and downregulation of VEGFR-1. This phenotype subsequently causes VEGFR-1 to act as a kinase-defective and decoy-type receptor (Hiratsuka et al., 1998). The carboxyl domain of VEGFR-2 is required for its ligand-dependent autophosphorylation (Meyer et al., 2004), and substitution of carboxyl domain of VEGFR-2 with that of VEGFR-1 promotes tyrosine phosphorylation and kinase activation of VEGFR-2 (data presented in this manuscript). Thus, it is highly possible that in the carboxyl tail of VEGFR-1 certain critical amino acids are altered. The lack of these critical residues in part may contribute to the poor kinase activation of VEGFR-1.

The information presented in this manuscript also demonstrates that although VEGFR-1 is not autophosphorylated after ligand stimulation, nevertheless, it stimulates MAPK phosphorylation. This observation suggests that VEGFR-1 is a functional protein and thus it is not entirely a decoy receptor as previously suggested (Hiratsuka et al., 1998). More importantly, the results suggest that activation of MAPK by VEGFR-2 is not associated with endothelial cell proliferation. Similar results using different endothelial systems have shown that activation of VEGFR-1 is not associated with endothelial cell proliferation (De Vries et al., 1992; Keyt et al., 1996; Fong et al., 1999; Rahimi et al., 2000; Gille et al., 2001). VEGFR-1 activation even in some nonendothelial cells, such as hepatic stellate cells, is reported to attenuate contraction of these cells without effecting DNA synthesis (Mashiba et al., 1999). How VEGFR-1 stimulates MAPK activation and what is the biological importance of MAPK in this system remains to be addressed. Our data show that activation of VEGFR-1 also fails to stimulate endothelial cell migration, intracellular calcium release and tubular formation in vitro. How VEGFR-1 activation contributes to angiogenesis remains unclear and further studies are needed to address its role in angiogenesis. It is possible that VEGFR-1 plays an auxiliary role in angiogenesis by modulating signals transmitted by other RTKs such as VEGFR-2 (Rahimi et al., 2000; Zeng et al., 2003). A similar role for other RTKs such as HER3/ErbB3 and CCK-4 has been suggested (Guy et al., 1994; Alimandi et al., 1995; Mossie et al., 1995). VEGFR-1 activation in part may achieve this role in angiogenesis by trapping VEGF (Hiratsuka et al., 1998). Our results show that VEGFR-1 is able to activate a limited set of signaling proteins such as MAPK, suggesting that VEGFR-1 may elicit its auxiliary role at the signal transduction level by amplifying or antagonizing a certain signaling pathway.

Altogether, these results suggest that the carboxyl tail of VEGFR-1 is responsible for its inferior kinase activation, downregulation and ability to stimulate cell proliferation. Our results also show that the carboxyl terminus of VEGFR-2 is able to accommodate the VEGFR-1 ligand-dependent autophosphorylation and couple VEGFR-1 to mitogenic signals that VEGFR-1 inherently lacks these phenotypes. The carboxyl terminus of VEGFR-2 but not VEGFR-1 may contain a motif that directs the receptor to endocytosis. This pathway may facilitate activation of VEGFR-1 and its ability to stimulate cell proliferation. Recent studies from Drosophila support this possibility. For instance, endocytosis-defective delta proteins failed to mediate activation of Notch receptor and Drosophila development (Parks et al., 2000).

Angiogenesis is a complex process necessitating a tightly regulated temporal and spatial control. In the past years, VEGF and its receptors have emerged as key players in angiogenesis. Delineation of distinct functions of VEGFR-1 and VEGFR-2 may facilitate therapies for diseases associated with angiogenesis.

Materials and methods

Reagents and antibodies

Rabbit anti-CSF-1R antibody and mouse antiphosphotyrosine (4G10) were purchased from UBI. Mouse antiphosphotyrosine (PY-20) antibody was purchased from Transduction Laboratories. Rabbit anti-VEGFR-1 and VEGFR-2 antibodies are made to amino acids corresponding to kinase insert or C-tail of VEGFR-2 (Rahimi et al., 2000). Anti-c-myc antibody was purchased from Sigma. Phospho-MAPK antibody was purchased from Cell signaling. Anti-p42 MAPK antibody and PLCγ1 antibody was purchased from Santa Cruz. Phospho-PLCγ1 antibody was purchased from Biosurce Int.

Cell lines

PAE cells expressing the chimeric VEGFR-1 and chimeric VEGFR-2 (herein referred to as CTR and CKR, respectively) were established by retroviral system as described before (Rahimi et al., 2000). The other mutant and truncated receptors were established in a similar manner.

Construction of cDNAs

Construction of chimeric VEGFR-1 (CTR) and chimeric VEGFR-2 (CKR) was established by polymerase chain reaction (PCR)-based site-directed mutagenesis as described before (Rahimi et al., 2000). To generate ΔCTR, we deleted the entire carboxylic tail of CTR just after the kinase domain. The downstream primer for making ΔCTR was synthesized corresponding to the nucleotides 3420–3450 of human VEGFR-1 cDNA sequence. Also, we introduced a c-myc sequence (GAA-CAA-AAA-CTC-ATC-TCA-GAA-GAGGAT-CTG) at c-tail of CTR for its detection. Following c-myc sequence a stop code (TAG) was also added. To create CTR/cFLK-1 (C-tail swapped CTR), we PCR amplified the entire carboxylic tail (630 bp) of murine VEGFR-2 (nucleotides 3470–4100) and the entire CTR but without its carboxyl tail. The two PCR products were ligated in frame using Bcl I site and subsequently was cloned into pLNCX2 vector. The sequence of all the constructs was verified by sequencing.

Immunoprecipitation and Western blot analysis

PAE cells expressing CTR or CKR were grown in sparse condition in 10% fetal bovine serum (FBS), and serum starved overnight in Dulbecco's modified Eagle's medium (DMEM). Cells were left either resting or stimulated with 40 ng/ml CSF-1. Cells were washed twice with H/S buffer (25 mm HEPES, pH 7.4, 150 mm NaCl, 2 mm Na3VO4) and lysed in lysis (EB) buffer (10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 50 mm NaCl, 50 mm NaF, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, and 20 μg/ml aprotinin), and VEGF receptors were immunoprecipitated. Immunoprecipitates were resolved on a 7.5% SDS–PAGE gel, and were subjected to Western blot analysis. On some occasions, the membranes were stripped by incubating them in a buffer containing 6.25 mm Tris-HCl, pH 6.8, 2% SDS and 100 mm β-mercaptoethanol in 50°C for 30 min and reprobed.

In vitro kinase assay

Kinase activities of CTR and CKR were analysed exactly as described before (Rahimi et al., 2000). Briefly, CTR and CKR immunoprecipitates were incubated in 10 μCi of [γ−32P]ATP for 15 min at 30°C. The reaction was stopped by adding 2 × sample buffer and samples were resolved by SDS–PAGE, radio-labeled CKR and CTR were detected by autoradiography.

Metabolic labeling

The ligand-dependent downregulation of VEGFR-1, VEGFR-2 and chimeric receptors was evaluated by pulse-chase analysis using a mixture of [35S]-labeled l-methionine/l-cysteine as described elsewhere (Meyer et al., 2004). In brief, equal numbers of PAE cells were plated in tissue culture dishes containing DMEM supplemented with 10% FBS. Cells were then starved for approximately 16 h in serum-free medium. The medium was removed and cells were rinsed with phosphate-buffered saline solution (PBS) before an additional 2 h of starvation at 37°C in l-methionine/l-cystine-free DMEM (Life Technologies, Inc.) supplemented with l-glutamine (Life Technologies, Inc.). Cells were pulse-labeled by adding 75 μCi/ml [35S]methionine/cystine (NEN, Beverly, MA, USA) for 3 h at 37°C and then chased with DMEM supplemented with 100-fold excess l-methionine and l-cysteine. Cells were stimulated with CSF-1 (40 ng/ml) at 37°C for the indicated times. Finally, cells were washed and lysed. Equal amounts of protein from each lysate were immunoprecipitated using appropriate antibodies as indicated. Immunocomplexed proteins were resolved on SDS–PAGE. Gels were prepared for autoradiography, dried and exposed to film (Eastman Kodak Co.) for 2 h at −70°C. Autoradiograms were quantified by using the Kodak 1D Image Analysis Software (Eastman Kodak Co.).

Cell proliferation

CSF-1 -stimulated cell proliferation was measured by uptake of [3H]thymidine. Cells were plated at 2 × 104 cells/ml in DMEM containing 10% FBS in 24-well plates and incubated at 37°C for 12 h as described before (Rahimi et al., 2000). Cells were then washed 2 × in PBS and growth arrested in DME containing 1 mg/ml BSA and 0.1% calf serum for 24 h at 37°C. Various amounts of CSF-1 were added and incubated for 18–20 h at 37°C. Cells were pulsed for 4 h with [3H]thymidine (0.2 μCi/ml) and harvested. Triplicate samples were performed for each group. Three independent experiments were performed and essentially the same results were obtained. The data are presented as fold increases over control.

Tubulogenesis/in vitro angiogenesis assay

Endothelial cell spheroids were generated as previously described (Korff and Augustin, 1999; Meyer et al., 2003). A defined number of cells was suspended in DMEM containing 1% FBS and 0.24% (w/v) carboxymethylcellulose, 4000 centipoise, in nonadherent round-bottom 96-well plates under standard cell culture conditions. After 24 h all cells formed one single spheroid per well (750 cells/spheroid). Spheroids were cultured for 2 days before using them in the in vitro angiogenesis assay in the following manner. Spheroids containing 750 cells were embedded in collagen gels. Collagen (8 vols) were mixed with 1 vol 10 × Hank's balanced salt solution containing 10% 10 × DMEM with phenol red. The pH was adjusted to 7.4 with 0.1 n NaOH. Spheroids were centrifuged and suspended in 9 ml of DMEM, containing 0.96% carboxymethylcellulose. Collagen and spheroids were mixed and transferred to prewarmed 24-wellplates, and the gels were allowed to polymerize in the incubator. After 30 min, 100 μl DMEM containing various concentrations of CSF-1 was added on top of the gel. Sprouting and tubulogenesis was observed after 2 days under an inverted phase contrast microscope (Nikon) and pictures were taken, using the SPOT camera system.

Migration assay

Migration of PAE cells expressing CKR and CTR was assessed using the Boyden chamber (Neuro Probe, Gaithersburg, MD, USA). CSF-1 was diluted in DMEM to a concentration of 5 ng/ml and placed in the bottom wells of the chamber. Semiconfluent cells were trypsinized and resuspended in DMEM to make a concentration of 1.5 × 106 cells/ml, and 50 μl of this suspension was loaded into each upper well. Chambers were incubated at 37°C for 8 h. After incubation, the membranes were removed and fixed and stained with Quick Diff (Dade International, Miami, FL, USA), washed with water and mounted on 75 mm × 50 mm glass slides (Fisher Scientific, Pittsburgh, PA, USA) bottom side down. The top cell layer was wiped off with a cotton-tipped applicator leaving only cells that had crossed through the membrane. Representative areas were counted at 20 × magnification. A total of 12 wells were used for a given concentration of test substance in each independent experiment. The experiment was repeated three times and essentially the same results were obtained. The data are presented as mean of cells ±s.d.

Calcium flux assay

PAE cells expressing either CKR or CTR were grown on 25 mm round glass coverslips and serum-starved for 12–18 h. Cells were incubated in an HEPES-buffered saline solution with 4 μm Fluo-3AM, supplemented with 0.02% pluronic acid for 30 min at 37°C. The live cells were placed in an open chamber (Molecular Probes, Inc., Eugene, OR, USA) with 500 μl of HEPES solution and positioned on the stage of a Zeiss LSM 510 Axiovert confocal laser scanning microscope equipped with an argon laser. For each experiment, cells were scanned for at least 5–10 s before the addition of CSF-1 to establish a baseline fluorescence reading. All the readings were made while continuously scanning the cells every 789 ms (Klepeis et al., 2001; Meyer et al., 2003).

Acknowledgements

This work was supported in part grants from National Institute of Health (NIH) EY 0137061, EY012997, RPB Career Development Award (to NR) and Massachusetts Lions Eye Research Fund.

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