Abstract
CD44v6, a member of the CD44 family of transmembrane glycoproteins is a co-receptor for two receptor tyrosine kinases (RTKs), Met and VEGFR-2 (vascular endothelial growth factor receptor 2). CD44v6 is not only required for the activation of these RTKs but also for signalling. In order to understand the role of CD44v6in Met and VEGFR-2 activation and signalling we tested whether CD44v6 binds to their ligands, HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor), respectively. FACS analysis and cellular ELISA showed binding of HGF and VEGF only to cells expressing CD44v6. Direct binding of CD44v6 to HGF and VEGF was demonstrated in pull-down assays and the binding affinities were determined using MicroScale Thermophoresis, fluorescence correlation spectroscopy and fluorescence anisotropy. The binding affinity of CD44v6 to HGF is in the micromolar range in contrast with the high-affinity binding measured in the case of VEGF and CD44v6, which is in the nanomolar range. These data reveal a heparan sulfate-independent direct binding of CD44v6 to the ligands of Met and VEGFR-2 and suggest different roles of CD44v6 for these RTKs.
Highlights
Receptor tyrosine kinases (RTKs) are single-pass transmembrane proteins with an extracellular ligand binding domain and an intracellular catalytic domain
In order to understand the role of CD44v6 in Met and vascular endothelial growth factor receptor 2 (VEGFR-2) activation and signalling we tested whether CD44v6 binds to their ligands, hepatocyte growth factor (HGF) and VEGF, respectively
Binding of HGF and VEGF to cells detected only in CD44v6-expressing cells Since the CD44v6 ectodomain is required for Met activation, we hypothesize that its role in Met activation is linked to its ability to bind HGF
Summary
Receptor tyrosine kinases (RTKs) are single-pass transmembrane proteins with an extracellular ligand binding domain and an intracellular catalytic domain. Activation of Met and Erk ASs6 cells were serum-starved for 24 h, and induced with 20 ng/ml HGF for 5 min at 37 ◦C. For the HGF binding assay in presence of the CD44v6 and CD44s ectodomain, the T47D and T47D/Met cells were transfected with the corresponding expression vectors using Lipofectamine (Invitrogen) according to the manufacturer’s protocol. For blocking experiments serum-starved AS, ASs6, T47D and T47D/Met cells were incubated either with the respective species-specific CD44v6 antibody (100 μg/ml), v6 or control peptide (100 ng/ml) for 1 h at 4 ◦C and induced with 20 ng/ml biotinylated HGF for 1 h at 4 ◦C. For the VEGF binding assay serum-starved HUVEC and ASs6 cells were incubated with the respective species-specific v6 or control peptide as described above followed by induction with 20 ng/ml of VEGF165 for 1 h at 4 ◦C. As the intensity of fluorescence of the labelled HGF was found to be not affected by the binding process, the anisotropy r of HGF of can be expressed by: is the binding stoichiometry of the complex, rf and rb are the anisotropy of the free and fully bound HGF protein, respectively
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