Rab GTPase Regulation of VEGFR2 Trafficking and Signaling in Endothelial Cells

Abstract

Objective-Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. Methods and Results-Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. Conclusions-Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking. Key Words: VEGFR2 Ⅲ Rab Ⅲ trafficking Ⅲ signaling Ⅲ migration V ascular endothelial growth factor A (VEGF-A) is an essential regulator of vascular physiology. 1 VEGF-A plays key roles in vasculogenesis and angiogenesis during development. 2 Angiogenesis is needed for embryogenesis and wound healing but is also associated with cancer, eye diseases, and other chronic disorders. The importance of VEGF-A is underlined by the fact that deletion of a single mouse VEGF-A allele causes embryonic lethality attributable to the impairment of vascular development. 3,4 The VEGF-A gene gives rise to at least 6 to 12 different variants, with VEGF-A 165 (termed VEGF-A) being the most abundant and proangiogenic isoform. 5,6 VEGF-A can bind to 2 different but closely related endothelial receptor tyrosine kinases (RTKs): fms-like tyrosine kinase (VEGFR1 or Flt-1) and kinase insert domain receptor (VEGFR2 or KDR). A major proangiogenic axis is based on VEGF-A binding to VEGFR2, stimulating tyrosine kinase activity, autophosphorylation, and signaling to downstream target proteins which regulate diverse processes including chemotaxis, migration, vascular permeability, cell survival, and proliferation. 7 Such physiological responses are regulated via multiple effectors including focal adhesion kinase (FAK), c-Akt/PKB, extracellular-signal related kinase (ERK) pathways, and stimulation of nitric oxide and prostaglandin synthesis. 8 Whereas VEGFR2 signaling pathways that regulate endothelial responses are well characterized, the spatial and temporal order underlying such events remains elusive. For example, VEGFR2 plasma membrane activation leads to trafficking to endosomes and degradation in lysosomes. 9 Simultaneously, VEGFR2 activation leads to tyrosine phosphorylation and intracellular signaling. 7,8 H...

Membrane Trafficking and Endothelial-Cell Dynamics During Angiogenesis

“…Rab7a GTPase regulates VEGFR2 trafficking from early to late endosomes. Rab7a depletion is inhibitory whereas Rab5a depletion is stimulatory for EC migration (Jopling et al, 2009). VEGF stimulation also can increase the rate of VEGFR2 recycling from sorting endosomes.…”

“…Thus, the intracellular distribution of VEGFR2 depends on VEGF stimulation. Endosomal trafficking and the signaling of VEGFR2 is dependent on the Rab5a protein, a Ras-related small GTPase associated with early endosomes (Jopling et al, 2009). Depletion of Rab5a enhances VEGFR2 tyrosine phosphorylation and MAPK signaling, whereas overexpression of a Rab5a GTPase-deficient (constitutively active mutant, Q79L) causes VEGFR2 accumulation within endosomes (Jopling et al, 2009).…”

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

“…Although the functions of Rab37 have not been elucidated in these cells types, it has been shown that several Rab isoforms are involved in tissue repair. In fact, Rab5A and Rab7A are involved in the trafficking of vascular endothelial growth factor receptor 2 during early/late endosome trafficking in endothelial cells [29]. Rab11 was also strongly expressed in differentiated epidermal keratinocytes [30].…”

Vascular Endothelial Growth Factor A-Stimulated Signaling from Endosomes in Primary Endothelial Cells

“…Rab GTPases are members of the Ras superfamily of GTPases and regulate many aspects of membrane trafficking and organelle fusion. Internalization of receptors from the plasma membrane into early endosomes requires Rab5a (Jopling et al, 2009). …”

“…Additionally, over-expressing the Rab5a GTPase deficient and constitutively active Q79L mutant causes accumulation of VEGFR2 in enlarged endosomes (Bruns et al, 2009;Jopling et al, 2009). Another member of the Rab family, Rab7a, is involved in the transport of VEGFR2 and other proteins from early to late endosomes (Bruns et al, 2009).…”