From the
Department of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676,
¶ Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0676,
|| Institute for Clinical Pathology, University of Vienna, Allgemeines Krankenhaus, Wahringer Guntel 18-20, Vienna, Austria 1090,
** Department of Veterans Affairs, Ann Arbor, Michigan 48105
Received for publication, February 5, 2003
, and in revised form, March 10, 2003.
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ABSTRACT |
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INTRODUCTION |
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The molecular composition and function of the foot process intercellular junction remains incompletely characterized. Emerging evidence suggests that this specialized junction is composed of a large complex of proteins associated in part within a specialized lipid microdomain. Presently, at least three transmembrane proteins have been identified at this site, including Nephrin (2), FAT1 (3), and P-cadherin (4). Significant attention has focused on Nephrin, the protein product of NPHS1. In humans, mutation of NPHS1 results in congenital nephrotic syndrome of the Finnish-type that is manifest by foot process effacement and proteinuria (5). That Nephrin participates in a protein complex important for maintenance of glomerular filter integrity has been suggested by observations that deletion or mutation of proteins that are physically associated with Nephrin, such as Podocin and CD2ap, also result in foot process effacement and proteinuria (6, 7, 8). Nephrin is a member of the immunoglobulin (Ig) superfamily characterized by eight C2 type Ig-like domains and a fibronectin type III repeat in its extracellular region. For this reason, it has been proposed that Nephrin is a cell adhesion molecule (CAM)1 that participates in forming the glomerular filter via homophilic or heterophilic interactions involving its extracellular domain (9, 10). However, the Nephrin ligand has not been identified.
Recent progress in genome and expressed sequence tag sequencing and work by mouse and Drosophila geneticists has provided important new clues regarding the true Nephrin ligand. Search of the genetic data base demonstrated the existence of three mammalian Nephrin-like Ig superfamily CAMs including Neph1 and two additional structurally similar proteins (11). While little characterization of Neph1 has been published, deletion of mouse Neph1 results in a podocyte effacement phenotype (12). A family of four CAMs was also identified in Drosophila that are structurally related to Nephrin (13). These proteins form two subfamilies: Hibris and Sticks and Stones that are most closely related to Nephrin, and Dumbfounded and Irregular Chiasm that are structurally similar to mammalian Neph1, Neph2, and Neph3. Importantly, Dumbfounded (the Neph1 homologue) interacts with Hibris and Sticks and Stones, but Hibris (the Nephrin homologue) does not interact with itself (13). These results predict that Nephrin interacts with Neph1.
In the present study we sought to investigate whether Neph1 and Nephrin interact via heterophilic and/or homophilic interactions. It was observed that both Nephrin and Neph1 are expressed in a glomerular epithelial cell-specific fashion in kidney and co-localize at the insertion of the glomerular slit diaphragm. Nephrin and Neph1 co-fractionate with a detergent-resistant lipid micro-domain and participate in a direct cis-interaction involving their cytoplasmic domains. In addition, interactions between the extracellular domain of Nephrin and itself and between the extracellular domain of Nephrin and that of Neph1 were detected. Neph1 did not interact via a homophilic interaction.
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EXPERIMENTAL PROCEDURES |
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Bacterial Fusion ProteinsPlasmid encoding GST-Neph1-CD (amino acid residues 554789 (11)) fusion protein was prepared using standard techniques. Briefly, PCR was performed using 5' primer (GGA ATT CCG ACG TCG CAA AGG CAG T-3') and 3' primer (CCC TCG AGC ACA TGA GTC TGC ATG CG-5') and using the full-length mouse Neph1 cDNA as a template. The PCR fragment was then cloned into pGEX4T-1 (Amersham Biosciences). This plasmid was expressed in Escherichia coli, then purified by affinity chromatography on glutathione-Sepharose (Sigma, G-4510). The eluted GST fusion protein was covalently linked to cyanogen bromide activated Sepharose C-L-4B beads (Sigma, CL-4B-200) and used for pull-down experiments as well as affinity purification of Neph1 antiserum. A plasmid (pRSET-A, Invitrogen) encoding hexahistidine-tagged Neph1-CD was prepared as above. A hexahistidine-tagged Nephrin-CD construct was described previously (2). Both proteins were prepared from bacterial lysate and purified using a commercially prepared nickel-agarose column according to the directions of the manufacturer (Invitrogen).
AntibodiesRabbit anti-Nephrin cytoplasmic domain (CD) polyclonal antibody was prepared as described previously (2). Anti-FLAG (M2, Sigma), anti-Myc (9E10, Oncogene Science), anti-V5 (Invitrogen), and anti-human IgG (Fc specific) (Sigma, A-0170) antibodies were obtained commercially. Mouse anti-rat GLEPP1 monoclonal antibody was a gift of Dr. Roger Wiggins (University of Michigan). Neph1 antiserum was produced by immunization of rabbits with hexahistidine-tagged-Neph1 cytoplasmic domain antigen. For antibody preparation, hexahistidine-Neph1 CD was solubilized in 6 M urea, purified by nickel affinity chromatography, separated on SDS-PAGE, and gel slices were used for rabbit inoculation. Antiserum was affinity purified on a GST-Neph1-CD affinity column after ammonium sulfate precipitation as described previously (2).
Cell CultureCOS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) and 200 units/ml of penicillin and streptomycin (Roche Molecular Biochemicals). Transient transfections were performed with FuGENE 6 (Roche Molecular Biochemicals) as described previously (16). Cell lysates were prepared 24 h after transfection using RIPA buffer (0.1 M HEPES, 0.15 M sodium chloride, 1.5 mM magnesium chloride, 1 mM EGTA, 10% glycerol, 0.1 M sodium fluoride, 10 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 1% Nonidet P-40, and protease inhibitor mixture tablet (Roche Molecular Biochemicals)).
Immunoblotting and ImmunoprecipitationsImmunoblotting was performed as described previously (17, 18). Blots were developed using the ECL chemiluminescent reagent (Amersham Biosciences) and were autoradiographed. Immunoprecipitations were performed as described previously (16). For affinity chromatography experiments using Fc-chimeric proteins, lysates were incubated with protein A-Sepharose (Sigma, P-5906). Beads were washed six times with 1x phosphate-buffered saline containing 0.1% Tween 20 and protease inhibitor. Immune complexes were separated under reducing conditions by SDS-PAGE, transferred to nitrocellulose, then immunoblotted with indicated antibodies.
Glomerular Isolation and Flotation Gradient PreparationAdult rat kidney glomeruli were isolated by sieving as described elsewhere (2, 19). The average purity of glomerular preparations was 90%. Glomeruli were lysed using RIPA buffer containing protease inhibitors at a concentration of
10,000 glomeruli/ml of extraction buffer.
For preparation of the floatation gradient, glomeruli were isolated and homogenized from rat kidneys at 4 °C with 14 strokes with a Dounce homogenizer in a buffer containing 250 mM sodium chloride, 5 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, and protease inhibitor mixture tablet (Roche Molecular Biochemicals). Triton X-100 was added to the homogenate to give a final concentration of 1%. The homogenate was mixed with 60% Optiprep solution (Nycomed Pharma AS) to obtain a final density of 40%. Five ml of this mixture was added to precooled centrifuge tubes and layered with 3 ml of 30% Optiprep and 4 ml of 5% Optiprep solution. Optiprep dilutions were obtained by diluting 60% stock Optiprep with Tricine buffer containing 20 mM Tricine, 0.25 mM sucrose, and 1 mM EDTA. The gradient was centrifuged at 37,000 rpm using a Beckman SW 41Ti rotor (169,000 x g) for 3 h at 4 °C. The Triton X-100-insoluble lipid layer was identifiable at the interface between the 5 and 30% Optiprep solutions. Top to bottom 1-ml fractions were sequentially collected.
ImmunofluorescenceSix-µm adult rat kidney cryosections were fixed with cold acetone. Affinity-purified rabbit anti-mouse Neph1 polyclonal antibody (1:50) or mouse anti-rat GLEPP1 monoclonal antibody (1:200) were used as a primary antibody. Species-appropriate secondary antibodies conjugated to fluorescein isothyocyanate or Cy3 (Jackson Laboratories, Westgrove, PA) were employed as indicated.
Immunogold Electron MicroscopyKidneys from adult rats were fixed in 4% freshly prepared formaldehyde, 0.1% distilled glutaraldehyde in 100 mM phosphate buffer, pH 7.2, for 612 h at 4 °C, soaked in sucrose, and stored in liquid nitrogen. Ultra-thin frozen sections of rat kidney were processed for indirect immunogold labeling as described previously (20, 21). In all experiments, control sections were prepared with affinity-purified rabbit anti-podocalyxin IgG to assess background and specificity.
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RESULTS |
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Neph1 Is Expressed in a Podocyte-specific Fashion in KidneyIndirect immunofluorescence was used to examine the expression pattern of Neph1 in adult rat kidney. Neph1 immunoreactivity was identified in a glomerular-specific pattern (Fig. 2, AC). To determine whether the immunoreactivity observed was podocyte-specific, cryosections of adult rat kidney were double labeled with a mouse monoclonal antibody directed against GLEPP1 and with rabbit anti-Neph1 antibody (Fig. 2, DF). GLEPP1 is a transmembrane protein-tyrosine phosphatase expressed only on podocytes in the kidney (22). Merged photomicrographs demonstrated that immunoreactivity for GLEPP1 and Neph1 produce superimposable signals and suggested that Neph1 is expressed in a podocyte-specific pattern in kidney.
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Neph1 Fractionates with the Triton X-100-insoluble Lipid Microdomain of Isolated Rat GlomeruliNephrin was found previously to associate in part with a Triton X-100-insoluble membrane fraction obtained from isolated glomeruli (23, 24). For this reason, the possibility that Neph1 also fractionates with this membrane fraction was investigated. Rat glomeruli were isolated and lysed at 4 °C in buffer containing 1% Triton X-100. Lysate was fractionated by floatation gradient centrifugation. The lipid fraction was identified in fraction four at the interface between 5 and 30% Optiprep (Fig. 3). One-ml fractions were collected from top to bottom, and each fraction was analyzed by immunoblotting for Neph1, Nephrin, Podocin, and CD2ap (Fig. 3). Like Podocin, another podocyte-specific detergent-resistant membrane fraction associated-protein (27), Neph1 was isolated only in the Triton X-100-insoluble membrane fraction (fraction 4). As described previously, Nephrin, in part, fractionated in the same lipid membrane fraction.
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Neph1 Subcellular Localization in GlomeruliTo examine the distribution of Neph1 within glomeruli in more detail, immunoelectron microscopy was carried out using post-embedding colloidal gold immunolabeling of rat kidney sections (Fig. 4). Gold particles were identified exclusively on lateral pedicel surfaces of podocytes in a distribution consistent with that of the insertion of the slit diaphragm. As anticipated, because the polyclonal antibody utilized in these experiments was raised against the cytoplasmic domain of Neph1, gold particles were not observed within the slit pore itself. There was no labeling for Neph1 detected elsewhere within membrane or cell body of the podocyte or within glomerular basement membrane or endothelial cells (Fig. 4). Importantly, this subcellular distribution of Neph1 within the podocyte was similar to that previously reported for Nephrin (2, 9).
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Neph1 and Nephrin InteractionsGiven the co-localization and structural similarity of Neph1 and Nephrin, it was hypothesized that Neph1 and Nephrin might engage in heterophilic and/or homophilic interactions occurring either in cis or trans at the podocyte foot process intercellular junction. To investigate this hypothesis, an initial series of co-immunoprecipitation experiments were performed after co-expressing Nephrin and/or Neph1, or their truncated mutants, by transient transfection of COS7 cells.
In preliminary experiments, full-length Neph1 co-immunoprecipitated with full-length Nephrin (Fig. 5A). However, full-length FLAG-tagged Neph1 did not co-immunoprecipitate with full-length V5-tagged Neph1 (Fig. 5B). A potential Nephrin-Nephrin interaction was examined in experiments described below. To assess whether endogenous Nephrin interacts with endogenous Neph1, Nephrin was immunoprecipitated from isolated glomerular lysate prepared by extraction with RIPA buffer (that had been shown in preliminary experiments to extract both Nephrin and Neph1 from membrane fractions). In these experiments, Neph1 co-immunoprecipitated with Nephrin immune complexes (Fig. 5C).
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To define the domains that mediate the observed interaction between Neph1 and Nephrin, additional co-immunoprecipitation experiments were performed. ECD interactions were examined by preparing plasmids encoding chimeric proteins of the ECD of Nephrin (Fc-mNephrin-ECD) and the ECD of Neph1 (Fc-FLAG-Neph1-ECD) fused to the Fc domain of human Ig (Fig. 6). In these experiments, the ECD of Nephrin co-immunoprecipitated with full-length FLAG-tagged Neph1 (Fig. 6A). Reciprocally, the ECD of Neph1 co-immunoprecipitated with full-length Nephrin (Fig. 6B). In similar experiments, Fc-mNephrin-ECD co-immunoprecipitated with full-length Nephrin, while Fc-FLAG-Neph1-ECD did not interact with full-length Neph1 (Fig. 6, D and C, respectively). In a control experiment, Nephrin ECD did not co-immunoprecipitate with the Nephrin CD (Fig. 6E). In summary, the extracellular domain of Nephrin interacts both with itself and with that of Neph1, while the extracellular domain of Neph1 does not interact with itself.
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Intracellular domain interactions were examined by preparing plasmids encoding the cytoplasmic domain of Nephrin. COS7 cells were co-transfected with Nephrin-CD and full-length FLAG-tagged Neph1 or full-length Nephrin. In these experiments, the CD of Nephrin co-immunoprecipitated with full-length FLAG-tagged Neph1 (Fig. 7A). However, the CD of Nephrin did not co-immunoprecipitate with full-length Nephrin (Fig. 7B). To confirm the interaction between the cytoplasmic domains of Neph1 and Nephrin, pull-down experiments were performed (Fig. 7, C and D). Rat glomerular lysate was incubated with either GST alone or GST-Neph1-CD (Neph1 cytoplasmic domain) covalently linked to Sepharose beads. As anticipated, Nephrin was pulled down from glomerular lysate with the GST-Neph1-CD but not with GST alone. Neph1 was not pulled down from glomerular lysate with GST-Neph1-CD or with GST protein (Fig. 7C). To determine whether the interaction between the cytoplasmic domain of Neph1 and Nephrin occurred via a direct interaction, GST alone or GST-Neph1-CD covalently linked to Sepharose beads was incubated in vitro with hexahistidine-tagged purified recombinant Nephrin-CD (Fig. 7D). A direct interaction was detected between Neph1-CD and Nephrin-CD, but not between GST protein and Nephrin-CD. In summary, the cytoplasmic domain of Nephrin directly interacts with the cytoplasmic domain of Neph1, while neither Nephrin nor Neph1 engage in homophilic interactions via their respective cytoplasmic domains.
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DISCUSSION |
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Nephrin and Neph1 share a number of important similarities. Deletion of either Neph1 (12) or Nephrin (5) results in similar phenotypes of foot process effacement with proteinuria and early postnatal death in mice. Both proteins are expressed in a glomerular epithelial cell-specific fashion in kidney and are localized to the podocyte slit diaphragm as demonstrated by immunoelectron microscopy of glomerular sections (2). Neph1 and Nephrin also have been shown to interact with Podocin, a lipid raft-associated component of the glomerular slit diaphragm (11, 27, 28). As suggested by recent work in Drosophila, a new family of structurally related Nephrin-like molecules has been described (13). Within this family is Hibris, a protein member of the Ig superfamily. Although their location and activity differ, Hibris bears strong structural similarity to mammalian Nephrin. In a study by Dworak et al. (13), Hibris mediated cell aggregation via a heterophilic interaction with Dumbfounded, a protein that bears structural similarity to Neph1. Therefore, these results predict that Nephrin interacts with Neph1.
The present results identify Neph1 as a newly recognized component of the Nephrin-associated protein complex existent at the intercellular junction between foot processes. Importantly, Nephrin and Neph1 appear to form a cis-interacting hetero-oligomeric complex associated with a detergent resistant lipid membrane microdomain. Nephrin and Neph1 directly interact via their cytoplasmic domains. In addition, Podocin, a stomatin-like protein, binds both Nephrin and Neph1 via their cytoplasmic domains (11, 27), and all three fractionate with the detergent-resistant membrane fraction. While Nephrin and Neph1 interact directly via their cytoplasmic domains, Podocin interaction might serve to stabilize their interaction and target the entire complex to the detergent-resistant membrane at the intercellular junction.
In the present study both homophilic and heterophilic interactions were also identified between the ECD of Nephrin and itself and between Nephrin and Neph1. Because multiple members of the Ig superfamily mediate cell-cell interaction via either homophilic or heterophilic associations, it is tempting to speculate that the Nephrin-Neph1 complex functions in a similar fashion. Several models have been proposed for cell adhesion mediated by classical CAMs (29, 30, 31, 32, 33, 34). In general, these models suggest that CAM-mediated cell-cell adhesion may be initiated by parallel cis-dimerization followed by an anti-parallel adhesive trans-interaction of the cis-dimers on opposing cells (31, 33, 34). Therefore, it is possible that Neph1 and Nephrin participate in foot process-to-foot process trans-interactions only after formation of a cis-hetero-oligomeric complex. This model would be consistent with our previous failure to demonstrate Nephrin-only mediated homophilic cell-cell interactions2 and suggests a new experimental direction.
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FOOTNOTES |
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These authors contributed equally to this work.
To whom correspondence should be addressed: University of Michigan Medical School, 1560 MSRB II, Ann Arbor, MI 48109-0676. Tel.: 734-764-3157; Fax: 734-763-0982; E-mail: lholzman{at}umich.edu.
1 The abbreviations used are: CAM, cell adhesion molecule; ECD, extracellular domain; GST, glutathione S-transferase; CD, cytoplasmic domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Fc, Fc domain of human Ig.
2 I. A. Kovari and L. B. Holzman, unpublished observation.
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REFERENCES |
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