Chimeric VEGFRs are activated by a small-molecule dimerizer and mediate downstream signalling cascades in endothelial cells.
Despite much interest in vascular endothelial growth factor (VEGF) and its receptors (VEGFRs -1 and -2), VEGF-induced signalling cascades remain incompletely defined. Attempts to assign individual responses to a particular receptor have used either transfected cell lines, receptor-specific growth factors or antisense oligonucleotides. Such studies have attributed the majority of VEGF-induced responses to activation of VEGFR-2. As a consequence of poor growth factor-induced VEGFR-1 autophosphorylation however, observations from these studies may instead reflect the relative activation of the two receptors. We have generated novel chimeric VEGF receptors in which the dimerization domain of the B subunit of DNA gyrase is fused to the cytoplasmic domain of VEGFRs -1 and -2. When expressed in porcine aortic endothelial cells, both chimeric VEGFR-1 and -2 autophosphorylate in response to addition of the small-molecule dimerizing agent, coumermycin. Once activated, both receptors induce downstream signalling cascades, exemplified here by the activation of MAPK, PLCgamma and PKB/Akt. Furthermore, we demonstrate that the Y1175 residue of VEGFR-2 is essential for the activation of PLCgamma mediated by this chimeric receptor. In contrast to previous reports which show a limited ability of VEGFR-1 to mediate signalling cascades, we show that once sufficiently activated, VEGFR-1 signals in a similar manner to VEGFR-2 in endothelial cells.
Knight
E L
EL
Department of Biochemistry, University of Leicester, UK.
Warner
A J
AJ
Maxwell
A
A
Prigent
S A
SA
D049933:Aminocoumarins; D000818:Animals; D001011:Aorta / Q000166:cytology; D001665:Binding Sites; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine; D059005:Topoisomerase II Inhibitors; D010738:Type C Phospholipases / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1
D049933:Aminocoumarins; D003374:Coumarins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D011993:Recombinant Fusion Proteins; D059005:Topoisomerase II Inhibitors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C004628:coumermycin