Abstract LB-332: A novel TIMP-2-mediated cytoplasmic mechanism for the regulation of receptor tyrosine kinase, VEGFR-2

Abstract

Abstract The vascular endothelial growth factors (VEGFs) and their receptors (receptor tyrosine kinases) are critical for angiogenesis. The binding of VEGF-A induces dimerization and autophosphorylation of its receptor, VEGFR-2. This initiates a signaling cascade that results in increased cell proliferation, migration, and permeability. The introduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) protein or a dominant negative mutant that has no MMP inhibitory activity (Ala+TIMP-2) intercepts this signaling cascade and affects these cellular functions. To elucidate the mechanisms of this disruption in VEGFR-2 signaling, we explored direct (competition binding, substrate affinity, total binding capacity) and indirect (receptor degradation, receptor dimerization by cross-linking, and FRET) mechanisms that could facilitate the inhibition of growth factor signaling by TIMP-2. Our studies showed that TIMP-2 did not directly compete for binding to VEGFR-2 as previously reported for TIMP-3 (Qi JH. Nature Medicine 2003;9:407-15). Further, TIMP-2 did not increase the internalization and degradation of VEGFR-2, nor did it decrease the affinity of the VEGF-A ligand. In contrast, TIMP-2 decreased the total cellular binding capacity for VEGF-A. These data suggest a novel mechanism by which TIMP-2 may regulate the binding conformation or state of VEGFR-2 dimerization, thereby influencing the cell sensitivity to VEGF-A stimulation. To study the conformation or state of VEGFR-2 dimerization, we conducted cross-linking analysis of VEGFR-2, and the results showed that TIMP-2 pretreatment prior to VEGF-A treatment significantly decreased VEGFR-2 dimer formation. In order to study this quantitatively, we designed a fluorescence resonance energy transfer (FRET) system that would detect the intracellular dimers of VEGFR-2. We fused Cerulean or Venus to the C-terminus of human VEGFR-2, transfected porcine aortic endothelial (PAE) cells, and selected stable cells expressing the constructs. Sensitized emission FRET analysis of the transfected PAE cells showed that Ala+TIMP-2 pretreatment prior to VEGF-A treatment decreased the VEGFR-2 dimerization to basal levels. This demonstrated that TIMP-2 regulates the binding capacity of VEGFR-2 by modulating the monomer-dimer equilibrium of VEGFR-2. This is the first demonstration of a cytoplasmic signaling mechanism influencing the monomer-dimer equilibrium of a receptor tyrosine kinase. This may potentially be a common mechanism for cellular regulation of growth factor sensitivity for this class of receptor tyrosine kinases.