Scheme of the domain organization of the NHERF family and various constructs used in this study. (a) NHERF1 and NHERF2 contain two PDZ domains and a C-terminal ERM-binding region. PDZ domains of NHERF2 share 60–70% identity with each other and with the equivalent domains of NHERF1. A19 is the partial clone of NHERF2 initially obtained from yeast two-hybrid screening using PC tail as bait. (b) PDZ domains of NHERF proteins prepared by in vitro translation or expressed as FLAG-tagged fusion proteins. N1P1, first PDZ domain of NHERF1; N2P1, first PDZ domain of NHERF2; N1P2, second PDZ domain of NHERF1; N2P2, second PDZ domain of NHERF2.

Distribution of PC, NHERF1, and NHERF2. PC and NHERF2 are expressed at high levels in the kidney and lung and are enriched in glomeruli. They are also detected in the brain, but not in heart, liver, or spleen. NHERF1 is present at highest levels in whole kidney and brain, but is not enriched in glomeruli. It is also present at very low levels in the liver and lung, but is not detected in heart and spleen. Ten micrograms of total protein from the indicated rat tissues was separated by 10% SDS-PAGE, transferred to PVDF membranes, and blotted with polyclonal anti-PC (0601), anti-NHERF1, or anti-NHERF2.

Immunogold localization of NHERF2 in the glomerulus. (a) Double labeling for PC and NHERF2 in the rat glomerulus. PC (10 nm gold) and NHERF2 (5 nm gold) colocalize along the apical PM of foot processes (fp) above the slit diaphragms and along the cell membrane of the cell bodies of GECs (arrowheads). PC is also localized along the luminal surface of the glomerular endothelium (En), but NHERF2 is absent. No labeling for NHERF2 is seen at the base of the foot processes facing the GBM (B). (b, c, and d) Double labeling for ezrin and NHERF2 in rat glomeruli. Ezrin (10 nm gold) and NHERF2 (5 nm gold) are colocalized mainly along the apical PM of foot processes (arrowheads) but are not found along the basal surface of the foot processes below the level of the slit diaphragms. Ultrathin cryosections were doubly incubated with polyclonal anti-NHERF2 and either anti-PC (5A) or anti-ezrin (3C12) IgG followed by 5- or 10-nm gold goat anti-rabbit or anti-mouse IgG conjugates as described in Methods. Bars, 0.1 μm.

Morphologic alterations seen in PAN-treated kidney. (a) Portion of a glomerular capillary from a normal, untreated rat showing the typical organization of the foot processes (fp) of the glomerular epithelium. (b) Portion of a capillary from a PAN nephrotic rat showing disruption of the foot process organization of GECs (Ep) and loss of the filtration slits between foot processes. BM, basement membrane; En, endothelium. Bar, 0.5 μm.

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.

PC associates with the second PDZ domain of NHERF proteins through its C-terminal PDZ-binding motif. (a) Interaction of GST-PCT with NHERF proteins. NHERF2 bind specifically to GST-PCT but not to GST alone (lanes 2 and 5), as well as NHERF1 (lane 3). Equivalent amounts of GST-PCT (7 μg) or GST alone (∼5 μg) bound to glutathione-agarose beads were incubated with in vitro–translated, radiolabeled NHERF1 or NHERF2. The precipitates were separated by 15% SDS-PAGE and detected by autoradiography. 5% input, 5% of the in vitro–translated products. (b) PC binds NHERF proteins, and the C-terminus of PC is essential for the interaction. PC coimmunoprecipitates with both NHERF proteins (lanes 4 and 5), but not with vector alone (lane 2) or with PSD95 (lane 3). PC, lacking the last four C-terminal residues [PC(ΔDTHL)], does not associate with either NHERF protein (lanes 6 and 7). Lane 1: 10 μg cell lysate. PC or PC(ΔDTHL) were transiently coexpressed in Cos 7 cells with either NHERF1, NHERF2, or PSD95 (control) followed by immunoprecipitation with anti-FLAG (M2). The precipitated proteins were immunoblotted with anti-PC (0601). The middle and lower panels demonstrate that the transfected cells express PC, PC mutant, and NHERF at comparable levels. (c) PDZ2 of both NHERF proteins specifically associates with GST-PCT (lanes 9 and 12). The A19 clone (see Figure 1a) is used as a positive control. Neither PDZ1 domain (lanes 3 and 6) associates with GST-PCT. Experiment performed as in a. Size markers indicate molecular mass in kilodaltons.

Localization of NHERF1, NHERF2, PC, and ezrin in the normal rat kidney. Strong staining for both NHERF2 (a and d) and PC (b and e) is seen in GECs in the glomerulus. PC is also present in the endothelium of peritubular capillaries (b, arrowhead) where NHERF2 is not seen. The yellow signal in the merged images (c and f) demonstrate overlap in the staining of GECs. In contrast, NHERF1 (g) is expressed mainly in the apical region of proximal tubules (arrowheads) and is not seen in glomeruli where PC is present (h and i). NHERF2 (j and m) also colocalizes with ezrin (k and n) in GECs where their staining overlaps (yellow staining in l and o). Ezrin is also expressed in proximal tubules (k, arrowheads) where NHERF1 (g) but not NHERF2 (l) is detected. Rat kidneys were processed for semithin (0.5 μm) cryosectioning, followed by double staining with mAbs anti-PC (5A) or anti-ezrin (3C12) and polyclonal anti-NHERF1 or anti-NHERF2, as described in Methods. Bars: (a–c and g–l) 20 μm; (d–f and m–o) 5 μm.

PC, NHERF2, and ezrin form a coprecipitable complex in rat glomeruli. (a) Immunoprecipitation was carried out on normal glomerular lysates with polyclonal anti-NHERF2 (lane 3) or control (lane 2) serum followed by immunoblotting with monoclonal anti-PC (5A). PC is seen in NHERF2 precipitates. Lane 1: 10 μg glomerular lysate. (b) A similar immunoprecipitation was carried out with anti-PC mAbs (1A + 5A) followed by blotting with polyclonal anti-ezrin, anti-NHERF1, or anti-NHERF2. Both NHERF2 and ezrin coprecipitate with PC, but NHERF1 is not detected (lane 3). Neither PC nor NHERF2 are detected in control serum precipitates (lane 2). Lane 1: 10 μg glomerular lysate. The experiment was performed twice with comparable results.

Disruption of PC/NHERF2/ezrin complexes in glomeruli from PAN-, SA-, and PS-treated rats. (a) Immunoprecipitation with anti-PC followed by immunoblotting for NHERF2 and ezrin as for Figure 6. The amount of ezrin and NHERF2 that coprecipitates with PC is reduced significantly in SA-treated (lane 5) and PS-treated (lane 6), but not in PAN-treated, glomeruli (lanes 3 and 4) compared with normal controls (lane 2). The amount of ezrin and NHERF2 that coprecipitates is restored to normal after heparin (Hep) perfusion of PS-treated rats (lane 7). Lane 1: 10 μg glomerular lysate. (b) When immunoprecipitation is carried out with anti-ezrin followed by immunoblotting for NHERF2 and PC, a similar reduction is seen in the amount of PC that coprecipitates with ezrin in SA (lane 5) and PS (lane 6) treated rats but not in PAN nephrotic rats (lanes 3 and 4). No change is seen in the amount of NHERF2 coprecipitated (bottom panel). Glomerular lysates from normal, PAN 3-d, and PAN10d, SA- and PS-treated rats were immunoprecipitated with anti-PC (1A + 5A) mAb (a) or polyclonal anti-ezrin (b) followed by immunoblotting as in Figure 6. Lane 1: 10 μg control lysate. Arrowheads indicate desialylated PC, which has a slower mobility than intact PC (lane 5). The arrow indicates IgG heavy chain. The experiment was performed twice with comparable results.

Models for the sites of disruption of the PC/NHERF2/ezrin complex with the actin cytoskeleton in pathological conditions associated with changes in foot process organization. (a) Normal glomerulus. PC binds to the second PDZ domain (PDZ2) of NHERF2 through its C-terminal PDZ-binding motif along the apical region of GECs. NHERF2 binds to the N-terminus of phosphorylated (unmasked) ezrin through its C-terminal ERM-binding region. Unmasked phosphorylated (P) ezrin links the complex to the actin cytoskeleton through its C-terminal actin-binding site. (b) Protamine sulfate (PS) treatment. Neutralization of the negative charge on the extracellular domain of PC by PS disrupts the binding of the cytoplasmic tail of PC to NHERF2. (c) PAN (10d) treatment. The PC/NHERF2/ezrin complex remains intact, but ezrin dissociates from the actin cytoskeleton with a concomitant decrease in the level of phosphorylated ezrin. (d) Sialidase (SA) treatment. When sialic acid is removed from PC by SA digestion, the complex is disrupted in two places: PC dissociates from NHERF2 and ezrin dissociates from the actin cytoskeleton with a concomitant reduction in the level of phosphorylated ezrin.