Neph1 Cooperates with Nephrin To Transduce a Signal That Induces Actin Polymerization

Abstract

While the mechanisms that regulate actin dynamics in cellular motility are intensively studied, relatively little is known about signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at the cell membrane. The kidney podocyte provides a unique model for investigating these mechanisms since deletion of Nephrin or Neph1, two interacting components of the specialized podocyte intercellular junction, results in abnormal podocyte morphogenesis and junction formation. We provide evidence that extends the existing model by which the Nephrin-Neph1 complex transduces phosphorylation-mediated signals that assemble an actin polymerization complex at the podocyte intercellular junction. Upon engagement, Neph1 is phosphorylated on specific tyrosine residues by Fyn, which results in the recruitment of Grb2, an event that is necessary for Neph1-induced actin polymerization at the plasma membrane. Importantly, Neph1 and Nephrin directly interact and, by juxtaposing Grb2 and Nck1/2 at the membrane following complex activation, cooperate to augment the efficiency of actin polymerization. These data provide evidence for a mechanism reminiscent of that employed by vaccinia virus and other pathogens, by which a signaling complex transduces an outside-in signal that results in actin filament polymerization at the plasma membrane.Precisely regulated actin polymerization provides the motive force necessary for intercellular junction formation and contributes to defining the shape and polarity of the cell. Similarly, directed actin polymerization proximate to the leading edge of the plasma membrane drives cell motility and is required in the complicated dynamics of lamellipodia, filopodia, and other specialized membrane structures including invadopodia and podosomes (6). While substantial progress has been made in understanding the cellular and molecular mechanisms that determine actin dynamics in these model systems (32), less is known about membrane-based proximal signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at these sites.Kidney glomerular visceral epithelial cells or podocytes are necessary for maintaining the glomerular filtration barrier (reviewed in reference 20). When mature, these cells have a unique octopus-like structure comprised of a central cell body that gives off branching primary, secondary, and tertiary processes. The tertiary processes or "foot processes" attach the podocyte to the glomerular capillary basement membrane, where they surround the glomerular capillary and where they interdigitate to form a specialized intercellular junction called the "slit diaphragm." Here foot processes provide a necessary element of the permeability-selective glomerular filter, allowing passage of water, solutes, and other small macromolecules from the capillary lumen to the urinary space while restricting the flux of cells and larger macromolecules.The podocyte provides a unique model for investiga...

Solution Structure Analysis of Cytoplasmic Domain of Podocyte Protein Neph1 Using Small/Wide Angle X-ray Scattering (SWAXS)

“…Currently, ZO-1 and Par proteins have been shown to interact with this domain, and our recent study demonstrates that Neph1 interacts with ZO-1 in a dynamic fashion that is regulated by Neph1 tyrosine phosphorylation (15,16). Further, Fyn-mediated tyrosine phosphorylation of Neph1 has been shown as a key signaling event that induces actin polymerization through recruitment of the adapter protein Grb2 (10). These studies collectively highlight the dynamic nature of the cytoplasmic domain of Neph1, suggesting that the structural changes induced by binding to other proteins and/or phosphorylation play a role in Neph1 function.…”

“…The GST-Neph1-CD protein was prepared as described previously (10,16). Further purification of GST-Neph1-CD protein for structural studies was carried out by subjecting the glutathione-eluted GST-Neph1-CD protein preparation to FPLC, and the final product was analyzed by SDS-PAGE and Western blot using Neph1 antibody.…”

“…In addition to its interaction with nephrin, the cytoplasmic domain of Neph1 interacts with various actin-associated proteins, including podocin, CD2AP, ZO-1 (zonula occludens), CASK, IQGAP1, ␤-arrestin, Nck, Grb2, ␣-actinin 4, synaptopodin, and the motor protein Myo1c (1,2,13). Although the role of each interaction is not entirely understood, it has been suggested that these interactions participate in transducing signals involved in actin cytoskeleton organization (10,14). Recent studies indicate that the interactions mediated by the PDZbinding domain of Neph1 are critical for Neph1 function (12).…”

“…Unlike nephrin, which is primarily a podocyte protein, Neph1 is widely distributed in tissues, including brain, heart, and liver. In podocytes, Neph1 co-localizes with nephrin, and evidence suggests that it forms a complex with nephrin both extracellularly and intracellularly (10,11). Whereas the extracellular interaction of Neph1 and nephrin determines the structural framework of the slit diaphragm, their intracellular engagement through their cytoplasmic domains plays a key role in signaling events that regulate podocyte actin organization (5,10,12).…”

“…Because the cytoplasmic domain of Neph1 is unique and displays multiple dynamic properties, determining the structure of this domain in solution will significantly enhance our understanding of the biology of this protein. Moreover, this protein is devoid of any disulfide bonds and therefore is fully functional when expressed as a recombinant protein in bacteria (10,16). To visualize the global structure of Neph1 and its complex with ZO-1, protein samples of tagless, GST-tagged, and His-tagged Neph1, His-ZO-1-PDZ1, and their equimolar mixture(s) were analyzed by SWAXS.…”