The nephrin protein complex appears to play an important role in specifying cell junction formation by promoting interactions between specific cell types and in doing so participates in defining tissue organization. In C. elegans
, nephrin and Neph1 homolog ligation is necessary for directing synapse formation between a specific neuron and its specific target epithelial tissue (9
). Similarly, in D. melanogaster
, nephrin-Neph1 homolog interaction influences compound eye morphogenesis and patterning by specifying junction formation between appropriate cell types (10
). In a potentially related fashion in the podocyte, nephrin is necessary for establishing normal podocyte foot process morphology and normal intercellular junction structure (3
In general, the dynamic process of epithelial cell junction formation is dependent on proper regulation and integration of junctional adhesion with actin cytoskeletal dynamics (35
). This is a complex process, the details of which likely vary according to cell type. Nevertheless, central to this process is precisely regulated actin polymerization and organization; actin polymerization appears to provide the motive force necessary for junction formation, and disruption of the molecular machinery necessary for regulating actin cytoskeletal dynamics prevents proper junction formation (36
). Therefore the nephrin family of transmembrane proteins might function in specifying junction formation in part by transducing signals that serve to regulate associated cytoskeletal dynamics proximal to the forming junction.
The observation that mutation or deletion of nephrin results in both failure of proper foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics (3
). The observations made herein provide the first direct evidence to our knowledge for a tyrosine phosphorylation–mediated signaling mechanism by which this integrative function is derived. These data indicate that engagement of the nephrin ectodomain by its ligand induces SFK activity resulting in tyrosine phosphorylation of the nephrinCD on specific tyrosine residues. Subsequently, the adapter protein Nck and presumably proteins associated with Nck are recruited to phosphorylated nephrin, resulting in Nck-dependent regulation of local actin dynamics.
The discovery that Nck associated with activated nephrin and was necessary for nephrin-directed actin polymerization meshes well with the hypothesis that nephrin is one element in a mechanism that serves to integrate podocyte intercellular junction structure with cytoskeletal dynamics. As demonstrated in several systems, Nck serves as an adaptor protein that recruits to a plasma membrane locus a critical concentration of N-WASp, components of the Arp2/3 complex, and other components of the actin polymerization machinery sufficient to induce and regulate actin polymerization (30
). Similar mechanisms have been identified in a number of mammalian receptor systems including the EGF, PDGF, and T cell receptor complexes where Nck has been implicated as necessary in actin dynamics–dependent processes (30
). A related mechanism is employed by several bacterial and viral pathogens (38
). For example, insertion of the enteropathogenic E. coli
proteins Tir and intimin into the plasma membrane of host epithelial cells triggers robust actin pedestal formation at the plasma membrane when Tir is tyrosine phosphorylated by Fyn, creating a docking site for host cell–derived Nck and associated N-WASp (38
). The nephrin receptor complex appears to employ a remarkably similar mechanism for coordinating junctional events with podocyte cytoskeletal dynamics. Like other systems, nephrin at the podocyte intercellular junction appears to recruit a high local concentration of Nck, which facilitates actin polymerization because it provides multiple docking sites for Nck and because nephrin itself is concentrated by clustering in a lipid microdomain (32
Nephrin protein is first expressed when podocyte process formation is initiated in the podocyte precursor (7
). During this event, processes emerge from the basolateral aspect of the precursor cell in a polarized fashion and appear to push into neighboring cells; here nephrin is targeted specifically to the newly forming intercellular junctions (7
). Phosphorylation on mouse nephrin Y1191 and/or Y1208 occurred transiently during glomerulogenesis during this process of podocyte intercellular junction formation. Considered in the context of results obtained in cell culture, it is reasonable to hypothesize that within the glomerulus, nephrin is tyrosine phosphorylated upon ectodomain engagement during this event. Subsequent Nck recruitment to phosphorylated nephrin likely initiates directed actin polymerization and/or elongation at this site and by participating in regulating actin dynamics provides one element in a mechanism that specifies the specialized structure of the podocyte foot process and intercellular junction. Consistent with a role of Fyn in initiating this process is the observation that genetic deletion of Fyn in mice results in attenuated nephrin phosphorylation and is associated with abnormal foot process morphology (7
). The observation that in situ deletion of Fyn in the podocyte precluded recruitment of Nck to nephrin strengthens the conclusion that this pathway is operative in nature.
Phosphorylation of nephrin Y1191/1208 during glomerulogenesis is transient since nephrin does not remain phosphorylated on these sites in the fully differentiated adult glomerulus. What does the transient nature of phosphorylation on these residues suggest about the function of Nck in this system? While investigation of this process is required, it is possible that tyrosine phosphorylation–dependent Nck recruitment might be necessary only during active actin polymerization or elongation that occurs during junction formation. Like similar junctional receptor complexes, it is clear that actin filaments can associate with the slit diaphragm junctional complex via multiple intermediaries (e.g., at the podocyte intercellular junction via CD2ap, ZO-1/Neph1, JAM, etc.) that might provide stable, phosphorylation-independent contacts necessary for maintaining the structural integrity of foot processes and their formed intercellular junctions. In recent work the classical dogma that actin binds stably to classical cadherins at mature epithelial adherens junctions was challenged; it was suggested that cadherin-catenin complexes play a dynamic regulatory role, participating only in weak interactions with the cytoskeleton (40
). In a similar fashion, nephrin might play a dynamic regulatory role without necessarily forming stable interactions with the actin cytoskeleton via Nck.
It has been well appreciated that in human glomerular disease proteinuria is invariably associated with foot process effacement. Presumably, in the setting of disease, these integrated processes occur following podocyte injury that results in an inside-out signaling event culminating in effacement and alteration of the intercellular junction. Nephrin phosphorylation also occurred rapidly following induction of foot process effacement in the protamine sulfate model. At first inspection, this observation appears to contradict the model proposed above that Y1191/1208 phosphorylation initiates actin dynamics necessary for junction formation. Indeed, one might argue that because phosphorylation on Y1191/1208 is temporally associated with effacement that this phosphorylation event results in disassembly of the podocyte intercellular junction. However, deletion of Fyn in mouse podocytes was not associated with stabilization of the podocyte intercellular junction; rather, it was associated with failure of appropriate junction formation. Moreover, it is difficult to overlook the established biology of Nck recruitment in other systems in which Nck participates in junction formation. Therefore, while additional investigation is necessary to confirm the hypothesis, it is possible that Y1191/1208 phosphorylation occurs after podocyte injury as the podocyte actin cytoskeleton reorganizes; this might require reinduction of actin polymerization as new, albeit different, podocyte intercellular junctions are created.
Using both biochemical and genetic approaches, previous work demonstrated that Fyn can directly bind and phosphorylate nephrin in the context of the podocyte lipid raft (22
). The precise mechanism by which nephrin phosphorylation is initiated remains unresolved. In similar lipid raft–based receptor systems, it has been suggested that ectodomain engagement results in either (a) recruitment of the membrane-associated receptor to the lipid raft, where it is brought into proximity of the resident Src kinase and is tyrosine phosphorylated; or (b) clustering of unique rafts such that raft components are combined, leading to initiation of cellular signaling (reviewed in ref. 24
). One or both of these general mechanisms may hold for nephrin since it has been shown that podocin serves as a slit diaphragm lipid raft scaffold that recruits nephrin to the lipid raft (20
). However, at least in the artificial HEK 293 cell system, physical clustering of nephrin in a lipid raft does not appear to be required for induction of SFK catalytic activity and nephrin phosphorylation, since addition of the 50A9 monoclonal antibody alone, without a secondary clustering antibody, induced both events. Therefore engagement of the nephrin ectodomain might induce a nephrin conformational change that by a yet-to-be-identified mechanism transduces an SFK-activating signal.
Nephrin is necessary for establishing selective glomerular filtration. In part, this function may be dependent on the 3D structure created by the cis
- and trans
-junctional interactions of nephrin with other transmembrane components at this site (44
). However, given the generally accepted function of coordinated cytoskeletal dynamics in establishing intercellular junctional architecture (36
), the role of nephrin in regulating cytoskeletal dynamics may be as important in establishing a filtration barrier as nephrin’s potential for directly creating a physical barrier to the passage of macromolecules at the junction.