Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton
Tetsuro Takeda, … , Robert A. Orlando, Marilyn G. Farquhar
Tetsuro Takeda, … , Robert A. Orlando, Marilyn G. Farquhar
Published July 15, 2001
Citation Information: J Clin Invest. 2001;108(2):289-301. https://doi.org/10.1172/JCI12539.
View: Text | PDF
Article

Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton

Abstract

Podocalyxin (PC), the major sialoprotein of glomerular epithelial cells (GECs), helps maintain the characteristic architecture of the foot processes and the patency of the filtration slits. PC associates with actin via ezrin, a member of the ERM family of cytoskeletal linker proteins. Here we show that PC is linked to ezrin and the actin cytoskeleton via Na+/H+-exchanger regulatory factor 2 (NHERF2), a scaffold protein containing two PDZ (PSD-95/Dlg/ZO-1) domains and an ERM-binding region. The cytoplasmic tail of PC contains a C-terminal PDZ-binding motif (DTHL) that binds to the second PDZ domain of NHERF2 in yeast two-hybrid and in vitro pull-down assays. By immunocytochemistry NHERF2 colocalizes with PC and ezrin along the apical domain of the GEC plasma membrane. NHERF2 and ezrin form a multimeric complex with PC, as they coimmunoprecipitate with PC. The PC/NHERF2/ezrin complex interacts with the actin cytoskeleton, and this interaction is disrupted in GECs from puromycin aminonucleoside–, protamine sulfate–, or sialidase-treated rats, which show a dramatic loss of foot processes, comparable to that seen in the nephrotic syndrome. Thus NHERF2 appears to function as a scaffold protein linking PC to ezrin and the actin cytoskeleton. PC/NHERF2/ezrin/actin interactions are disrupted in pathologic conditions associated with changes in GEC foot processes, indicating their importance for maintaining the unique organization of this epithelium.

Authors

Tetsuro Takeda, Tammie McQuistan, Robert A. Orlando, Marilyn G. Farquhar

×

Figure 9

Options: View larger image (or click on image) Download as PowerPoint
Dissociation of PC/NHERF2/ezrin complexes from the cytoskeleton in glome...
Dissociation of PC/NHERF2/ezrin complexes from the cytoskeleton in glomeruli from SA- and PAN-treated rats. (a) Immunoblot of normal, PS-, SA-, or PAN-treated (10d) rats. In normal glomeruli, PC, NHERF2, and ezrin distribute in all three fractions — that is, Triton X-100 soluble (TXS), RIPA soluble (RIPA), and RIPA insoluble (Ppt). In SA- and PAN-, but not PS-treated glomeruli, there is a significant increase in the amount of PC, NHERF2, and ezrin found in the Triton X-100 soluble (TXS) fraction. Actin is also distributed in all three fractions, but its pattern of distribution is unchanged among these experimental groups. C-terminal threonine phosphorylated ezrin [ezrin, pTyr(567)] is present only in the insoluble precipitate (Ppt) (lanes 3, 6, 9, and 12) under all conditions. The level of phosphorylated ezrin is significantly reduced in SA- and PAN-treated glomeruli. Isolated glomeruli from normal, PS-, SA-, and PAN-treated (10d) rats were solubilized sequentially with 0.5% Triton X-100 and RIPA lysis buffer and separated into Triton X-100–soluble (TXS), RIPA-soluble (RIPA), and RIPA-insoluble fractions (Ppt). Equal volumes of these fractions were immunoblotted with appropriate Ab’s. The arrowhead indicates the position of desialylated PC (lane 7). The experiment was performed twice with comparable results. (b) Quantification of protein bands shown in a was done by densitometry as described in Methods.