The SD complex contains at least three transmembrane proteins, nephrin (13
), P-cadherin (24
), and FAT (31
), that may account for its zipperlike structure. At the cytoplasmic insertion site of the SD, ZO-1 (32
), α-, β-, and γ-catenins (24
), and CD2AP (12
) are present. The SD may represent a modified adherens junction (24
) and is primarily responsible for the size selectivity of the glomerular filter (2
). Previous genetic studies have revealed that nephrin, CD2AP, and podocin are indispensable for the normal filtration function of the SD. In this study, podocin, a novel integral membrane protein possessing a characteristic hairpin loop topology, has been identified as another component of the SD.
The assembly of the SD complex commences when podocyte precursors differentiate from typical polarized epithelial cells of the S-shaped body to more mature mesenchymal-like cells of the capillary loop stage (20
). Apical tight junctions migrate downward and convert into SDs (32
). Podocyte dedifferentiation is observed in pathological situations in humans and experimental models (35
), suggesting dynamic regulation of the SD complex under these conditions. The temporal expression pattern of podocin during kidney development is similar to that of nephrin as shown by in situ hybridization (14
). Podocin first appears in the S-shape stage in the future podocytes. The expression of podocin is maintained during later stages of podocyte development and in mature kidneys (14
). CD2AP first appears in podocytes during the capillary loop stage (38
). CD2AP mutant mice show no alteration of nephrin distribution during kidney development, but in 7-week-old mice, when most of the glomeruli are already severely damaged, nephrin is hardly detectable in the glomeruli (12
). Taken together, these data and the data of the present study suggest that all three proteins, nephrin, CD2AP, and podocin, are necessary to maintain the structural integrity of the SD. Furthermore, the normal distribution of nephrin in postnatal kidneys of CD2AP-deficient mice may be due to its interaction with podocin, since podocin and nephrin are the first proteins of this group to be expressed in podocytes during kidney development.
This study shows that podocin can form complexes and associates with lipid rafts. The observed oligomerization not only resembles the oligomerization found for nephrin (Figure b) (27
); it may also represent a typical feature observed for other hairpin loop membrane proteins like caveolins and stomatin (15
). The oligomerization of caveolin clusters rafts and triggers plasma membrane invaginations termed caveolae (41
). Here, we showed that podocin is associated with Triton X-100–insoluble lipid membranes and is likely to be raft-associated, forming oligomeric complexes in rafts (Figure b). Hence, podocin may act as a scaffolding protein in podocyte lipid rafts, recruiting nephrin and CD2AP to these microdomains. Like caveolin, the oligomerization of podocin could cluster nephrin-containing rafts (27
) and thereby trigger SD assembly. The assembled SD complex could then be held together by a network of nephrin and podocin oligomers in which protein-protein interactions apart from lipid-protein interactions come into play. This study suggests that podocin interacts with nephrin via the COOH-terminus.
Apart from an interaction between nephrin and podocin, CD2AP could serve as a linker protein at the cytoplasmic side of the plasma membrane. Our data show that CD2AP interacts directly with podocin and is in part found in the same DRM fraction as podocin and nephrin. Further detailed biochemical studies are needed to elucidate the protein relations and the stochiometry of the SD complex. Where the assembly of the SD complex occurs is an interesting question. Podocin may have a crucial role in the assembly of the complex. It is not known whether the complex assembles during its transport along the biosynthetic pathway to the plasma membrane or directly at the level of the SD. Furthermore, the presence of other proteins within the SD complex needs to be examined carefully. In particular, signaling proteins, like protein kinases and phosphatases, may be required to regulate the assembly of the complex and the integration of newly synthesized proteins. Having a high affinity for lipid rafts, kinases of the src family represent good candidates.
In summary, we have demonstrated that podocin, the target protein of autosomal recessive steroid-resistant nephrotic syndrome (14
), is a novel component of the glomerular SD complex. In particular, (a) podocin is expressed in the podocyte foot process cell membrane at the insertion site of SD as shown by immuno–electron microscopy; (b) podocin, nephrin, and CD2AP are associated with lipid rafts of the SD; (c) podocin not only colocalizes but interacts with CD2AP and nephrin in vivo as shown by Co-IP studies of glomerular extracts; (d) podocin binds to CD2AP and nephrin via its COOH-terminus and may thereby serve as scaffolding protein in the organization of the SD complex; (e) podocin can directly bind to CD2AP. Since CD2AP has recently been shown to directly interact with nephrin in vitro (12
) and in vivo (42
), it may serve as an adapter protein mediating the interaction of podocin and nephrin. On the other hand, podocin and nephrin could also directly interact with each other. An intact SD is essential for normal glomerular filtration, and alterations of only one of its key components, nephrin, CD2AP, or podocin, leads to massive proteinuria (5
). The results of the present study may open new avenues for understanding the underlying pathomechanisms in glomerular diseases with proteinuria.
Note added in proof.
The interaction of podocin with nephrin was also found by T.B. Huber et al. (Huber, T.B., et al. 2001. Interaction with podocin facilitates nephrin signaling. J. Biol. Chem. 276:41543–41546.)