MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin

Abstract

MAGUK with inverted domain structure-1 (MAGI-1) is a membrane-associated protein with one guanylate kinase, six PSD-95/Dlg-A/ZO-1 (PDZ), and two WW domains and is localized at tight junctions in epithelial cells. MAGI-1 interacts with various proteins and is proposed to function as a scaffold protein. In the previous study, we discovered a MAGI-1-interacting cell adhesion molecule junctional adhesion molecule 4 (JAM4). Both proteins are highly expressed in glomerular podocytes in the kidney and partially colocalized. In this study, we have further searched for a binding partner of MAGI-1 in the kidney through yeast two-hybrid screening and obtained nephrin. Nephrin is a cell adhesion molecule specifically localized at the slit diaphragm between neighboring foot processes of podocytes. Biochemical studies reveal that nephrin directly binds to the middle PDZ domains of MAGI-1 through its carboxyl terminus but does not bind to ZO-1. MAGI-1 forms a tripartite complex with nephrin and JAM4 in vitro. Immunoelectron microscopy shows that the localization of MAGI-1 is restricted to the slit diaphragm, whereas JAM4 is also distributed on apical membranes of podocytes. In puromycin aminonucleoside-induced nephrotic podocytes, MAGI-1 is localized with nephrin at the displaced slit diaphragm. These data indicate that MAGI-1 is a component of the slit diaphragm and tightly interacts with nephrin and JAM4 in vivo. MAGI-1 may play a role in determining the boundary between the apical and the bosolateral domain at the level of slit diaphragm. Laboratory Investigation (2005) 85, 1528-1543.

Neph1, a Component of the Kidney Slit Diaphragm, Is Tyrosine-phosphorylated by the Src Family Tyrosine Kinase and Modulates Intracellular Signaling by Binding to Grb2

“…Tyr 637 Is Phosphorylated in Injured Podocytes in Vivo-Several tyrosine-phosphorylated proteins in the PS-induced podocyte injury model have been described by Kurihara and others (20,32). Therefore, we expected that treatment with PS may induce the phosphorylation of Neph1 in vivo.…”

“…Perfusion of rat kidneys with PS was carried out essentially as previously described (32). Six-week-old male Wistar rats were purchased from Charles River Laboratories Japan, Inc. (Atsugi, Japan).…”

“…Immunohistochemistry-Immunofluorescence studies were performed as previously described (32). Briefly, rat kidneys were perfused with 2% paraformaldehyde fixative buffered with 0.1 M phosphate buffer (pH 7.4).…”

“…The cryostat sections for immunohistological study and glomerular lysates were prepared as previously described (32). Induction of puromycin aminonucleoside (PAN) nephrosis was carried out as described previously (32). Young male rats (Wistar) were injected intraperitoneally with 10 mg/100 g body weight of PAN (Sigma).…”

“…Kidneys were perfused through the aorta at 5 ml/min with Hanks' balanced salt solution for 20 min followed by PS solution (500 g/ml in Hanks' balanced salt solution) for 20 min. The cryostat sections for immunohistological study and glomerular lysates were prepared as previously described (32). Induction of puromycin aminonucleoside (PAN) nephrosis was carried out as described previously (32).…”