Biochemical and functional characterization of RhoSAP: a RhoGAP of the postsynaptic density

Abstract

Glutamatergic synapses in the central nervous system are characterized by an electron dense network of proteins underneath the postsynaptic membrane including cell adhesion molecules, cytoskeletal proteins, scaffolding and adaptor proteins, membrane bound receptors and channels, G-proteins and a wide range of different signalling modulators and effectors. This so called postsynaptic density (PSD) resembles a highly complex signaling machinery. We performed a yeast two-hybrid (YTH) screen with the PDZ domain of the PSD scaffolding molecule ProSAP2/Shank3 as bait and identified a novel interacting protein. This molecule was named after its Rho GAP domain: RhoSAP (Rho GTPase Synapse Associated Protein), which was shown to be active for Cdc42 and Rac1 by a GAP activity assay. Besides its Rho GAP domain, RhoSAP contains an N-terminal BAR domain that might facilitate membrane curvature in endocytic processes. At the C-terminus RhoSAP codes for several proline rich motifs that could possibly act as SH3 binding regions. Therefore, a second YTH screen was carried out with RhoSAP’s proline rich C-terminus as bait to discover putative interacting proteins. As an interacting partner syndapin I, a molecule involved in vesicle endocytosis via direct interaction with dynamin I was found. Furthermore, syndapin I contains a C-terminal SH3 domain that is most likely the binding motif for RhoSAP’s proline rich region. This novel interaction as well as the interaction with the ProSAP2 PDZ domain was verified by pull-down assays and coimmunoprecipitations. Coming from the finding that RhoSAP is associated with an endocytosis molecule, an FM4-64 endocytosis assay was performed in cell line to delineate the molecule’s significance in endocytic processes in general. Taken together, RhoSAP is a novel ProSAP2/Shank3 interacting PSD protein, which displays several protein/protein interaction domains. Due to the N-terminal BAR domain, and the interaction with syndapin I, RhoSAP might act within endocytic processes of the postsynaptic membrane.