CD2AP Structure And Progression Of Renal Disease

Abstract

CD2AP is a scaffolding molecule that was originally cloned as an interaction partner of CD2 in T lymphocytes. In the kidney, CD2AP is strongly expressed in podocytes, a cell type that regulates the filtration barrier. The protein directly interacts with filamentous actin and a variety of cell membrane proteins including the kidney filter protein nephrin. In addition to discrete binding sites for actin and nephrin, CD2AP possesses three SH3 domains and a proline-rich region containing, in turn, binding sites for SH3 domains. CD2AP is implicated in dynamic actin remodeling and membrane trafficking that occurs during receptor endocytosis and cytokinesis. We have initiated structural studies of recombinant CD2AP protein using electron microscopy and single particle image analysis. Negative stain electron microscopy of revealed uniform particles with a size and morphology suggesting a tetrameric organization, subsequently verified with chemical crosslinking. Single particle image analysis was used to generate a three-dimensional map of the CD2AP tetramer at 21 Å resolution. The electron density map reveals an extended structure allowing the identification of specific subdomains. The tetramer is organized around a central core, including density assigned to the C-terminal coiled-coil domain, surrounded by four loosely attached arms radiating out from the center, which we have assigned to the N-terminal SH3 domains. We have further identified CD2AP as a substrate for cytoplasmic cathepsin L, a protease that is induced in early podocyte damage. Cleavage of CD2AP with cathepsin L results in a C-terminal core domain that is structurally competent but releases the CD2AP binding partner dendrin resulting in translocation of dendrin to the nucleus where it promotes apoptosis. Based on our analysis of the cathepsin L cleavage sites within CD2AP we conclude that cytosolic cathepsin L releases the N-terminal arms producing a structurally competent C-terminal core domain.