Abstract
Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.
Highlights
With increasing knowledge of podocyte biology, it has become clear that normal glomerular filtration relies heavily on properly functioning podocytes
By mixing recombinant purified proteins we demonstrated a direct interaction between the cytoplasmic domain of NEPH1 and SH2 domain–containing protein tyrosine phosphatase-2 (SHP-2) (Fig. 1B)
NEPHRIN and NEPH1 are slit diaphragm proteins that are critical for podocyte function, and genetic defects in these proteins lead to renal dysfunction in mice and humans [7, 14, 25]
Summary
With increasing knowledge of podocyte biology, it has become clear that normal glomerular filtration relies heavily on properly functioning podocytes. Beads were removed and the phosphorylated GST-NEPH1 cytoplasmic domain (GST-NEPH1 CD) and GST-NEPHRIN CD proteins were incubated with purified recombinant SHP-2 for the indicated times and Western blot was performed with the respective phosphoantibodies, which confirmed SHP-2mediated dephosphorylation. We coexpressed NEPH1 or NEPHRIN with FYN in HEK293 cells and we treated the NEPH1- and NEPHRINexpressing stable cultured podocytes [2] with pervanadate and immunoprecipitated the proteins with their respective antibodies.
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