Abstract
Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.
Highlights
Membrane receptor recycling is an important function in regulating animal physiology [1,2,3]. the machinery controlling recycling from intracellular endosomes has not been fully characterised, endosome-associated Ras-related (Rab) GTPases are implicated in regulating such pathways [4]
Previous work has shown that the VEGFR2 exhibits both plasma membrane and endosomal localisation [6,12,15,21]
Primary endothelial cells were first subjected to a block in new protein synthesis using cycloheximide: we detected a stable and distal pool of VEGFR2, which showed partial co-distribution with the transferrin receptor (TfR) present in early endosomes (Figure 1A, upper panels)
Summary
The machinery controlling recycling from intracellular endosomes has not been fully characterised, endosome-associated Ras-related (Rab) GTPases are implicated in regulating such pathways [4]. Two such Rab GTPases, Rab4a and Rab11a, regulate different recycling routes from early endosomes back to the plasma membrane [5]. Rab4a is functionally involved in membrane protein recycling from endosomes, exemplified by vascular endothelial growth factor receptors (VEGFRs) and αVβ3 integrin [5]. The vascular endothelial growth factor (VEGF) family of cytokines plays essential roles in vasculogenesis, angiogenesis and animal physiology [2,7,8].
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