VEGF-A splice variants bind VEGFRs with differential affinities

Abstract

Vascular endothelial growth factor A (VeGf-A) and its binding to VeGfRs is an important angiogenesis regulator, especially the earliest-known isoform, VeGf-A 165a. Yet several additional splice variants play prominent roles in regulating angiogenesis in health and in vascular disease, including VeGf-A 121 and an anti-angiogenic variant, VeGf-A 165b. few studies have attempted to distinguish these forms from their angiogenic counterparts, experimentally. previous studies of VeGf-A:VeGfR binding have measured binding kinetics for VeGfA 165 and VeGf-A 121 , but binding kinetics of the other two pro-and all anti-angiogenic splice variants are not known. We measured the binding kinetics for VeGf-A 165 ,-A 165b , and-A 121 with VEGFR1 and VEGF-R2 using surface plasmon resonance. We validated our methods by reproducing the known affinities between VEGF-A 165a :VEGFR1 and VEGF-A 165a :VEGFR2, 1.0 pM and 10 pM respectively, and validated the known affinity VEGF-A 121 :VEGFR2 as K D = 0.66 nM. We found that VeGf-A 121 also binds VEGFR1 with an affinity K D = 3.7 nM. We further demonstrated that the anti-angiogenic variant, VeGf-A 165b selectively prefers VEGFR2 binding at an affinity = 0.67 pM while binding VEGFR1 with a weaker affinity-K D = 1.4 nM. These results suggest that the − A 165b anti-angiogenic variant would preferentially bind VEGFR2. These discoveries offer a new paradigm for understanding VEGF-A, while further stressing the need to take care in differentiating the splice variants in all future VeGf-A studies. The vascular endothelial growth factors have been extensively studied as signaling molecules in angiogenesis 1,2 , and their signaling comprises several components. The mammalian VEGF family includes five homodimeric ligands: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) 3. VEGF-A signal transduction unfolds following the pattern common to other tyrosine kinase receptors (RTKs), like the fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) families: (1) ligands bind a receptor monomer, promoting dimerization with another free receptor; (2) phosphorylation occurs at specific tyrosine residues depending on conformational changes allowed by the ligand-i.e. signaling is not directly coupled to binding, but dependent on ligand structure 4,5 ; (3) adaptor proteins bind these tyrosine residues and undergo phosphorylation 6 , and (4) phosphorylated adaptor proteins initiate effector signaling cascades 7 that can ultimately mediate cell-level responses such as cell migration, proliferation, and cell survival 8. The most well-studied ligand is VEGF-A 9. VEGF-A has a wide array of isoforms produced through alternative mRNA splicing 10,11 , including: VEGF-A 121 , VEGF-A 121 b, VEGF-A 145 , VEGF-A 145 b, VEGF-A 165 , VEGF-A 165 b, VEGF-A 183 , VEGF-A 189 , and VEGF-A 206 (Fig. 1), in addition to VEGF-A 111 , an abnormal splice variant induced by genotoxic stressors 12. Members of the VEGF-A xxx family are pro-angiogenic, whereas those designated as VEGF-A xxx b have been desc...

New insights on the role of vascular endothelial growth factor in biliary pathophysiology

“…Secreted VEGF isoforms bind to specific receptor tyrosine kinases (RTKs), i.e. VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1) and VEGFR-3, but can also interact with non-tyrosine kinase co-receptors, such as the members of the Neuropilin receptors family (Nrp-1 and -2) and J o u r n a l P r e -p r o o f the heparan sulfate proteoglycans (HSPGs) [11,12]. Upon ligand binding, tyrosine residues present in the intracellular domain of VEGFRs undergo autophosphorylation thus triggering the transduction of signals through different intracellular mediators.…”

Enhanced Microvasculature Formation and Patterning in iPSC–Derived Kidney Organoids Cultured in Physiological Hypoxia

“…VEGF-A165b is a low efficacy agonist, binding VEGFR2 with a stronger affinity and thereby reducing binding of VEGF-A165, resulting in strongly decreased signal transduction via the VEGFR2 receptor. ( Peach et al, 2018 ; Mamer et al, 2020 ) VEGF-A165b is upregulated in quiescent vessels and in adult kidneys ( Li et al, 2004 ; Jagannathan et al, 2016 ), inhibiting endothelial cell migration ( Li et al, 2004 ; Jagannathan et al, 2016 ), and is downregulated in nephrogenesis during capillary loop formation ( Bevan et al, 2008 ; Stolz and Sims-Lucas, 2015 ). Downregulation of the VEGF-A165b isoform in hypoxia at day 7 + 25 could indicate increased binding of VEGF-A165 to the VEGFR2 receptor, enhanced signal transduction and consequently the initiation of angiogenesis.…”

Characterization of the Expression of Angiogenic Factors in Cutaneous Squamous Cell Carcinoma of Domestic Cats

“…These isoforms correspond to the well-characterized splice variants described in humans, mice, and other species [ 60 , 61 ]. The protein variants, which differ in their inclusion of key polypeptide domains, exhibit distinct biological behaviors associated with their differential sites and timing of expression, affinity for extracellular matrix, and liberation by proteolysis [ 62 , 63 , 64 , 65 ]. Larger VEGF and PLGF isoforms interact with the extracellular space due to the presence of heparin/heparan sulfate binding domains; they accumulate in the extracellular matrix forming a reservoir of growth factor that can be mobilized via simple dissociation or proteolysis [ 66 ].…”