Abstract
Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.
Highlights
The regulation of biochemical pathways is mediated in part through numerous protein-protein interactions that are facilitated by protein scaffolds
There are two ITSN genes in mammals, ITSN1 and ITSN2, each encoding a short and long isoform and sharing 59% identity and greater than 70% similarity. Both ITSN short (ITSN-S) isoforms possess two amino-terminal Eps15 homology (EH) domains followed by a coiled-coil (CC) domain and five Src homology 3 (SH3) domains
The pool of ITSN binding proteins highlights the pivotal role of ITSNs in endocytosis, GTPase regulation, and receptor tyrosine kinase (RTK) regulation (Figure 1B), and suggests that ITSNs may have novel roles in regulating additional pathways as detailed
Summary
The regulation of biochemical pathways is mediated in part through numerous protein-protein interactions that are facilitated by protein scaffolds. Since the discovery of SH2 domains, a plethora of domains have been defined, each with unique specificity for distinct types of ligands (reviewed in [1]). The arrangement of these protein interaction domains results in intricate interaction networks for a given protein. There are two ITSN genes in mammals, ITSN1 and ITSN2, each encoding a short and long isoform and sharing 59% identity and greater than 70% similarity Both ITSN short (ITSN-S) isoforms possess two amino-terminal Eps homology (EH) domains followed by a coiled-coil (CC) domain and five Src homology 3 (SH3) domains. The DH and PH domains function together as a guanine nucleotide exchange factor (GEF)
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