Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation - PubMed (original) (raw)
Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation
Rosana D Meyer et al. Oncogene. 2004.
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.
Figures
Figure 1
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)
Figure 2
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)
Figure 3
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)
Figure 4
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)
Figure 5
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
Figure 6
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)
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