1. ¹Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan
2. ²Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan
3. ³Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Correspondence: Dr H Kurihara, PhD, Department of Anatomy, Juntendo University School of Medicine, Tokyo 113-8421, Japan. E-mail: hidetake@med.juntendo.ac.jp; Dr Y Hata, PhD, Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan. E-mail: yuhammch@med.tmd.ac.jp

Received 20 June 2005; Revised 4 August 2005; Accepted 8 August 2005; Published online 12 September 2005.

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.