Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin
Karin Schwarz, … , Lawrence B. Holzman, Peter Mundel
Karin Schwarz, … , Lawrence B. Holzman, Peter Mundel
Published December 1, 2001
Citation Information: J Clin Invest. 2001;108(11):1621-1629. https://doi.org/10.1172/JCI12849.
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Article

Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin

Abstract

NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH2- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.

Authors

Karin Schwarz, Matias Simons, Jochen Reiser, Moin A. Saleem, Christian Faul, Wihelm Kriz, Andrey S. Shaw, Lawrence B. Holzman, Peter Mundel

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The COOH-terminus of podocin interacts with nephrin and CD2AP. (a) The C...
The COOH-terminus of podocin interacts with nephrin and CD2AP. (a) The COOH-terminus of podocin associates with nephrin and CD2AP as shown by GST pull-downs. Nephrin and CD2AP are detectable in the final eluate (E) of the GST-podocin-COOH column. GST alone and the podocin NH2-terminus do not interact with these two SD components, as indicated by detection of nephrin and CD2AP in the flow-through (FT) fraction but not in the eluate. (b) The GST pull-down data were confirmed by Co-IPs with cross-linked antibodies against podocin, nephrin, CD2AP, or lamin (negative control) from glomerular extracts. Bound proteins were eluated and analyzed by SDS-PAGE and Western blot under nonreducing conditions (left panel) or in the presence of β-ME (right panel). Blots were then incubated with an irrelevant antibody (SRIB1), or with anti-podocin, anti-CD2AP, or anti-nephrin antibodies. The right panel shows Co-IPs of CD2AP and nephrin with anti-podocin antibody or preimmune serum (pre) under reducing conditions. (c) The ability of podocin to directly interact with CD2AP was tested by coimmunoprecipitation of in vitro translated proteins and by GST pull-downs. The left panel shows the in vitro translated S35-labeled proteins. Co-IP of unlabeled CD2AP and S35-labeled podocin showed direct interaction of both proteins (middle panel). The specificity of the podocin-CD2AP interaction was further confirmed by pull-down experiments, using recombinant podocin-GST fragments and radioactive-labeled CD2AP (right panel). Only with the COOH-terminal part of podocin, CD2AP was coprecipitated. IVTR, in vitro translation reaction.