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

Claudia Bertuccio,Delma Veron,Pardeep K Aggarwal,Lawrence Holzman,Alda Tufro

doi:10.1074/jbc.m111.241620

Abstract

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.

Highlights

Excess Vascular endothelial growth factor A (VEGF-A) down-regulates nephrin causing glomerular disease
We propose that the nephrin-VEGF receptor-2 (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
To evaluate VEGFR2 interaction with nephrin, VEGFR2 was immunoprecipitated from cultured podocyte lysates, and the immunoprecipitates were probed by Western blot with the corresponding antibodies (Fig. 2A)

Summary

Background

Excess VEGF-A down-regulates nephrin causing glomerular disease. Nephrin interacts with VEGFR2 in vivo. Results: Nephrin-VEGFR2 interaction is direct, modulated by tyrosine phosphorylation, the VEGR2-nephrin complex involves Nck and actin, and VEGF-A signaling via this complex decreases cell size. 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. Our studies showed that podocyte-specific VEGF164 gain of function during development causes nephrotic syndrome in mice [31] and in adult mice, leads to proteinuria, podocyte effacement, and loss of slit diaphragms [25]. We propose that the VEGFR2-nephrin complex transduces local VEGF-A signals to the podocyte actin cytoskeleton, thereby regulating cell shape and function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION