Vascular Endothelial Growth Factor Receptor 2 Direct Interaction with Nephrin Links VEGF-A Signals to Actin in Kidney Podocytes

Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2011; 286(46):39933-44. DOI: 10.1074/jbc.M111.241620
Source: PubMed


The transmembrane protein nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and nephrin signaling pathways. We previously showed that in vivo nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

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    • "Sison et al. demonstrated that VEGF regulates slit diaphragm signaling through VEGFR2-nephrin cross-talk [17]. VEGF gain of function downregulates nephrin expression and distorts podocyte morphology, which concurs with the foot process effacement and proteinuria [18–20]. The autocrine VEGF signaling system in podocytes also regulates SD proteins by inducing podocin upregulation and increasing its interaction with CD2AP in vitro [16]. "
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    • "Exposure of differentiated podocytes to doxycycline for 48 hours resulted in ∼50% decrease of VEGF-A cell content, and ∼30% decrease in secreted VEGF-A, as compared to control (Figure 2B). VEGFR2 and nephrin co-localized within podocytes (Figure 2C), as previously described [30]. Nephrin, podocin, WT1 and VEGFR2 protein levels in differentiated podocytes were not altered by VEGF downregulation (Figure 2D). "
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