Phosphorylation of nephrin triggers its internalization by raft-mediated endocytosis.
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.
Qin
Xiao-Song
XS
2nd Department of Internal Medicine, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka, Japan.
Tsukaguchi
Hiroyasu
H
Shono
Akemi
A
Yamamoto
Akitsugu
A
Kurihara
Hidetake
H
Doi
Toshio
T
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism* / Q000648:ultrastructure; D007739:L Cells; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C407223:NPHS2 protein; D011994:Recombinant Proteins; C113267:nephrin