. The majority of CD2AP staining was on the lateral membranes of the podocyte foot processes, with some gold particles clearly labeling the vicinity of slit diaphragm (Figure 1, B–D) . Only occasional gold particles were detected in the cell body. We suspect that the absence of gold particles in the cell body and the abundance of gold particles near the slit diaphragm reflects the increased concentration of CD2AP in this location. The pattern of staining was specific as no staining was detected on the basal podocyte membrane (adjacent to the basement membrane) nor was any staining detected in CD2AP KO kidney (Figure 1, A) . Immuno-electron-microscopic studies using two different antibodies to nephrin showed a similar pattern of expression (data not shown). Thus, CD2AP appears to localize in podocytes to a location consistent with the slit diaphragm.
| Figure 1.Immunoelectron microscopic localization of CD2AP in mouse podocytes. Immunoelectron microsopic localization of CD2AP in a 6-week-old normal mouse was performed using affinity purified CD2AP and secondary antibody conjugated with 10-nm gold particles. (more ...) |
Co-Precipitation of CD2AP with Nephrin in a Podocyte Cell Line
In our previous work, we were unable to test whether nephrin and CD2AP associate in the kidney because neither protein was soluble in our lysis solutions using weak detergents like digitonin or CHAPS (Figure 2A) . We therefore tested whether CD2AP and nephrin could be detected in a podocyte cell line. These cells were derived from a transgenic mouse that expresses a temperature-sensitive form of SV40 large T antigen under the control of an interferon inducible promoter. 11 At the permissive temperature, in the presence of γ-interferon, cells grow continuously in culture. At the non-permissive temperature and after interferon removal, cells stop growing, differentiate, and acquire a podocyte-like morphology. | Figure 2.Association of CD2AP and nephrin in podocyte cell line. A: Insolubility of nephrin and CD2AP from purified glomeruli. Glomeruli were purified from mouse kidneys by sieving. Lysates were prepared with digitonin based lysis buffer. A portion of the soluble (more ...) |
Lysates were prepared from early passage podocyte cells. Immunoblotting of cell lysates demonstrated that both nephrin and CD2AP were expressed in these cells (Figure 2 , and data not shown). It should be noted that the expression level of nephrin was not high and immunofluorescence staining showed that the protein is localized mainly at intracellular membranes. However, three different nephrin antibodies immunoblotted a band of between 180–190 kd in size and this band was similar in molecular mobility to nephrin immunoblotted from kidney. Importantly, both proteins were solubilized in a lysis buffer containing 1% digitonin.
Immunoblotting of CD2AP immunoprecipitates with an anti-nephrin antibody showed association of CD2AP with nephrin (Figure 2B , lane 4). This was specific to CD2AP, as immunoprecipitates prepared with a control antibody showed no nephrin reactivity (Figure 2B , lane 3). Thus, endogenous CD2AP/nephrin complexes are present in a cultured podocyte cell line.
CD2AP Interacts with the C Terminus of the Nephrin Cytoplasmic Domain
To confirm the specificity of the interaction between nephrin and CD2AP, we next focused on identifying the domains of nephrin and CD2AP critical for their interaction. Previously we showed that co-expression of CD2AP with a chimeric protein containing the cytoplasmic domain of nephrin fused to the extracellular and transmembrane domains of the VSV G protein in HeLa cells resulted in the formation of nephrin/CD2AP complexes. 3 We used this system to identify the portion of the nephrin cytoplasmic domain that interacts with CD2AP. We initially generated three mutated nephrin constructs. CT1 lacks the first third, CT2 lacks the second third, and CT3 lacks the last third of the nephrin cytoplasmic domain (Figure 3A) . | Figure 3.Mapping the binding site for CD2AP in nephrin. A: Three cytoplasmic truncation mutants of nephrin, CT1 (lacking residues 1119–1151), CT2 (lacking residues 1152–1200), and CT3 (lacking residues 1201–1241), fused with VSV-G were (more ...) |
Each of the mutated VSV G/nephrin constructs was co-expressed with myc-tagged CD2AP in HeLa cells. After lysis, immunoprecipitates prepared with antibodies to VSV G were immunoblotted with antibodies to myc (Figure 3B) . While CT1 and CT2 were both able to bind to CD2AP (Figure 3B , lanes 3 and 4), there was no CD2AP detected in CT3 immunoprecipitates (Figure 3B , lane 5). The last 41 residues of the nephrin cytoplasmic domain are therefore required for CD2AP association.
To test whether the last 41 residues were sufficient by themselves to bind to CD2AP, a VSV G/nephrin construct lacking all but the last 41 residues of the nephrin cytoplasmic domain was generated (CT4, Figure 3A ). CT4 bound poorly to CD2AP suggesting that the last 41 residues, while required, are not sufficient to fully reconstitute binding (Figure 3C , lane 3). We therefore generated another construct to test whether a larger fragment (the last 65 residues of nephrin) was sufficient to bind to CD2AP (CT5, Figure 3A ). CT5 was easily co-precipitated with CD2AP from cells overexpressing both proteins (Figure 3C , lane 4). Thus, while the last 41 residues of nephrin are required for CD2AP binding, a larger fragment of nephrin is required to fully reconstitute nephrin/CD2AP complex formation.
Nephrin Binds to the C-Terminal Domain of CD2AP We next focused on identifying the domain of CD2AP that binds to nephrin. CD2AP contains multiple functional domains. Three mutated constructs were generated to determine which domain of CD2AP binds to nephrin. The ΔSH3 construct lacks the three SH3 domains, the ΔPro construct lacks the proline-rich domain in the center of the molecule, and the ΔCT construct lacks the C-terminal 213 residues (Figure 4A) . All of the constructs contain a myc-epitope at the N terminus to facilitate detection.
| Figure 4.Mapping the binding site for nephrin in CD2AP. A: Schematic diagram of CD2AP deletion mutants. ΔSH3 lacks residues 8–323 which contain the three SH3 domains. ΔPro lacks residues 324–427 containing the proline-rich domain. (more ...) |
Each of the CD2AP constructs was co-expressed with the VSV G/nephrin fusion protein in HeLa cells. However, the analysis of CD2AP binding to nephrin was complicated by the similar electrophoretic mobilities of two of the mutated CD2AP constructs to immunoglobulin heavy and light chains. We therefore tested whether CD2AP in a cell lysate could bind to a nephrin fusion protein composed of glutathione S-transferase fused to the cytoplasmic domain of nephrin. The use of GST as a fusion partner allowed us to isolate the nephrin fusion protein without using an antibody.
Cell lysates were prepared from HeLa cells overexpressing wild-type CD2AP. Purified GST-nephrin was added to the lysate and precipitated using glutathione agarose beads. GST-nephrin readily co-precipitated CD2AP demonstrating that CD2AP can bind to nephrin in vitro (Figure 4B , lane 2). This was specific to nephrin as GST by itself was unable to co-precipitate CD2AP (Figure 4B , lane 1).
The ability of each of the three mutated CD2AP constructs to bind nephrin was then tested. Cell lysates prepared from cells overexpressing each of the mutated constructs was incubated with purified GST-nephrin. Both the ΔSH3 and the ΔPro construct were easily co-precipitated by GST-nephrin (Figure 4B , lanes 3 and 4). However, no association was detected with the ΔCT construct. The C-terminal 213 residues of CD2AP were therefore required for nephrin binding.
A coiled-coiled domain is present within the C-terminal 213 residues of CD2AP at the extreme C terminus. To test whether the coiled-coiled domain is responsible for binding to nephrin, we generated two additional constructs. Δ600–641 truncates the portion of the cytoplasmic domain containing the coiled-coiled domain while Δ428–600 deletes sequences preceding the coiled-coiled domain (Figure 4A) . The Δ600–641 construct was still able to co-precipitate with VSV G-nephrin (Figure 4C , lane 4) demonstrating that the coil-coil domain of CD2AP is not involved in nephrin binding. As expected, the Δ428–600 construct was unable to bind to nephrin (Figure 4C , lane 3). This region has no recognizable structural domains, suggesting that a novel domain in CD2AP mediates its interaction with nephrin.
Discussion We have provided further evidence that CD2AP is involved in the podocyte slit diaphragm. First, by immunoelectron microscopy we showed that CD2AP localizes in podocytes in a region close to or at the slit diaphragm. Second, using an immortalized podocyte cell line, we co-precipitated nephrin, the major component of the slit diaphragm with CD2AP. Third, we mapped the interaction between the two proteins to validate the specificity of the interaction. Overall, these data establish for the first time that CD2AP is a component of the slit diaphragm. Discovered over 30 years ago, there is still little known about the slit diaphragm. To date, only four molecules have been identified to be components of the slit diaphragm: nephrin, 7-9 ZO-1, 5 P-cadherin, 14 and FAT. 15 CD2AP can now be added to this list. Consistent with the idea that the slit diaphragm is an adhesion structure, all of these molecules appear to be involved in cell-cell adhesions. Nephrin, a transmembrane protein, mediates the interaction between the adjacent podocyte foot processes. P-cadherin is found in adherens junctions, while ZO-1 is found in both adherens junctions and tight junctions. 16,17 FAT is a novel member of the cadherin family of receptors containing 34 cadherin repeats. 15 Nephrin, a member of the immunoglobulin superfamily, was originally identified because it is mutated in the disease congenital nephrotic syndrome of the Finnish type (CNF). 10 Nephrin is expressed almost exclusively in glomerular podocytes. Several studies using immunoelectron microscopy demonstrate that nephrin localizes to the slit diaphragm. 7-9 Because CD2AP knockout mice develop nephrotic syndrome shortly after birth, it was logical to test whether there might be a relationship between nephrin and CD2AP. We had shown previously that nephrin and CD2AP could interact when both proteins were overexpressed in HeLa cells or when both proteins were co-expressed using the yeast two-hybrid system. 3 But these data did not prove that nephrin and CD2AP interact in vivo. Here we have extended these previous findings and show now that these complexes exist when both proteins are expressed at endogenous levels in a podocyte cell line. In addition, we were able to demonstrate that CD2AP/nephrin complexes form in vitro. The CD2AP binding domain of nephrin we have identified suggests that CD2AP may play a role in a subset of patients with CNF. Most cases of congenital nephrotic syndrome are caused by a mutation in the nephrin gene which completely abolishes expression. 18 However, a smaller group of patients have a nephrin mutation (Finn minor), which generates a stop codon within the cytoplasmic domain of nephrin resulting in a protein lacking the C-terminal 132 residues of nephrin. 18 As this domain is required for binding to CD2AP, loss of this CD2AP binding may account for the phenotype of these patients. It will be interesting to determine whether a subset of patients with CNF express smaller truncations of the nephrin cytoplasmic domain. While the exact role of CD2AP in adhesive contacts is unknown, we favor the idea that CD2AP links surface receptors with the cytoskeleton. We originally identified CD2AP as a molecule involved in the specialized adhesive complex termed the immunological synapse. 19 Now we have shown that it localizes to another specialized contact known as the slit diaphragm. This suggests that CD2AP may play a role in providing structural integrity to these adhesion complexes. Understanding CD2AP function will hopefully lead to further insights into the structure of the slit diaphragm and to a better understanding of epithelial surfaces. |
Acknowledgments We thank Marilyn Levy for performing the immunoelectron microscopy, Amy Holdorf, Gopa Green, Coky Nguyen, and Emil Unanue for critical review of the manuscript, Hui Wu for excellent technical assistance and Karl Tryggvason, Larry Holtzman, and Amin Arnout for antibodies to nephrin. |
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