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Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis

Xiao-Song Qin^*, Hiroyasu Tsukaguchi^*, Akemi Shono^*, Akitsugu Yamamoto, Hidetake Kurihara and Toshio Doi^*

*Department of Clinical Biology and Medicine, University of Tokushima Graduate School of Medicine, Tokushima, Japan;
Faculty of Bio-Science, Nagahama Institutes of Bio-Science and Technology, Shiga, Japan; and
Department of Anatomy, Juntendo University School of Medicine, Tokyo, Japan

Correspondence: Dr. Hiroyasu Tsukaguchi, 2nd Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka, 570-0074, Japan. Phone: +81-6-6992-1001; Fax: +81-6-6992-1409; E-mail: tsukaguh{at}otokoyam.kmu.ac.jp

Received for publication January 5, 2009. Accepted for publication September 15, 2009.

Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.

Copyright © 2009 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673