Intersectin (ITSN) regulation of Rab family GTPases

Abstract

ITSN is a multi‐domain scaffolding protein that regulates endocytosis and signal transduction. In addition, ITSN enhances receptor tyrosine kinase (RTK) trafficking in part through regulation of RTK ubiquitylation. We now report that ITSN regulates Rab‐mediated trafficking of RTKs. ITSN and Rab5 co‐localize on early endosomes. To examine the relationship of ITSN with various Rab family GTPases, we used bimolecular fluorescence complementation (BiFC) in which a YFP variant, Venus, is split into amino (VN)‐ and carboxyl (VC)‐terminal fragments, neither of which is fluorescent. When these fragments are fused to proteins that interact, fluorescence is reconstituted thus allowing for temporal and spatial monitoring of protein:protein interactions in vivo. Using BiFC, we shown that ITSN complexes with Rab5 and Rab7, but not with the dominant negative forms of these GTPases indicating that ITSN preferentially associates with active Rab5 and Rab7. In addition, we have identified a new component of the Rab pathway that forms a complex with ITSN and Rab5/Rab7 suggesting a role for this protein in Rab‐mediated trafficking. The localization of ITSN to vesicles as well as ITSN's interaction with the Rab pathway supports the model that ITSN regulates intracellular trafficking.K.A.W. supported in part by NIH PSTP Training Grant (5T32GM070388)