Intersectin 1 Research Articles

Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus

Brain development and function depend on the directed and coordinated migration of neurons from proliferative zones to their final position. The secreted glycoprotein Reelin is an important factor directing neuronal migration. Loss of Reelin function results in the severe developmental disorder lissencephaly and is associated with neurological diseases in humans. Reelin signals via the lipoprotein receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact mechanism by which these receptors control cellular function is poorly understood. We report that loss of the signaling scaffold intersectin 1 (ITSN1) in mice leads to defective neuronal migration and ablates Reelin stimulation of hippocampal long-term potentiation (LTP). Knockout (KO) mice lacking ITSN1 suffer from dispersion of pyramidal neurons and malformation of the radial glial scaffold, akin to the hippocampal lamination defects observed in VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin receptors, as evidenced by the prominent neuronal migration and radial glial defects in hippocampus and cortex seen in double-KO mice lacking ITSN1 and ApoER2. These defects were similar to, albeit less severe than, those observed in Reelin-deficient or VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 to facilitate Reelin signaling. Collectively, these data identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the cortex and hippocampus and to augment synaptic plasticity.

Intersectin1-S, a multidomain adapter protein, is essential for malignant glioma proliferation.

Glioblastomas, the most aggressive form of primary brain tumors with a tendency to invade surrounding healthy brain tissues, remains an incurable disease. Intersectin (ITSN) is a multidomain adapter protein implicated in endocytosis, exocytosis, and multiple signaling pathways. Prior research of ours has shown intersectin1-S (ITSN1-S) is critical for the migration and invasion of glioma cells by regulating several key proteins. In this study, we established ITSN1-S expression patterns in human tumor tissues. We discovered that ITSN1-S expression was positively correlated with histological grade of gliomas and with poor patient prognosis. We also found that the expression of ITSN1-S protein was essential to glioblastoma cell proliferation. Furthermore, through a series of expression constructs encoding different ITSN1-S domains, we identified the critical roles of ITSN1-S SH3 domains in the regulation of cell proliferation. This study also demonstrates evidence suggesting that the regulation of ITSN1-S on glioblastoma cells proliferation is through the Raf/MEK/ERK pathway. In conclusion, this study suggests critical roles of ITSN1-S in malignant glioma proliferation, indicating a potential usage of ITSN1-S in the therapeutic intervention as a novel molecular target.

Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia

Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.

Phosphatidylinositol 3-kinase, class 2 beta (PI3KC2β) isoform contributes to neuroblastoma tumorigenesis

Phosphatidylinositol 3-kinases (PI3Ks) play important roles in human tumorigenesis. Activation of the PI3K target AKT is frequent in neuroblastoma (NB) and correlates with poor prognosis. PI3K pan-inhibitors reduce NB tumor formation but present severe toxicity, which limits their therapeutic potential. Therefore, defining the importance of specific PI3K isoforms may aid in developing more effective therapeutic strategies. We previously demonstrated that PI3K Class IIβ (PI3KC2β) and its regulator intersectin 1 (ITSN1) are highly expressed in primary NB tumors and cell lines. Silencing ITSN1 dramatically reduced the tumorigenic potential of NB cells. Interestingly, overexpression of PI3KC2β rescued the anchorage-independent growth of ITSN1-silenced cells suggesting that PI3KC2β mediates ITSN1's function in NB cells. To address the importance of PI3KC2β in NBs, we generated PI3KC2β-silenced lines and examined their biologic activity. Herein, we demonstrate that PI3KC2β-silencing inhibits early stages of NB tumorigenic growth. We also show that loss of endogenous PI3KC2β or ITSN1 reduces AKT activation but does not impact ERK–MAPK activation. These data reveal a novel role for PI3KC2β in human NB tumorigenesis.

Abstract A17: Differential role of Ras isoforms in regulating PI3KC2β: Novel role for nucleotide-free H-Ras in cell signaling

Abstract Ras proteins, like all GTPases, cycle through an active GTP-bound state and an inactive GDP-bound state that arises through the intrinsic GTPase activity of the protein. Activation of Ras requires the activity of GEFs which promote the release of GDP leading to the subsequent binding of GTP due to the relatively higher levels of GTP in cells. Given the high affinity of nucleotide-free Ras (nf-Ras) for nucleotides in vitro, it has been proposed that nf-Ras is a transient intermediate in vivo. However, we have discovered a potentially new role for nf-H-Ras in the regulation of PI3KC2β (1). Previously, we discovered that the scaffold protein intersectin 1 (ITSN1) interacts with and activates H-Ras on endocytic vesicles (2). In addition, ITSN1 binds and activates PI3KC2β on a similar population of vesicles (3). PI3KC2β, like Class I PI3Ks, contains a Ras binding domain (RBD) although PI3KC2β does not interact with RasGTP or RasGDP (4). Our recent studies, however, indicate that nf-H-Ras binds the PI3KC2β RBD and inhibits PI3KC2β activity in vitro (1). Furthermore, the complex of nf-H-Ras with PI3KC2β RBD was not disrupted by addition of 1mM GTP or GDP suggesting that this complex may exist in the cellular milieu of high guanine nucleotides (1). Consistent with this in vitro data, PI3KC2β preferentially interacts with H-Ras dominant-negative (Ras17N)>H-Ras WT>activated H-Ras (Ras61L or 12V) in cells (1). To determine whether these results can be extended to other GTPases, we have examined the interaction of PI3KC2β with K-Ras and various Ras-related GTPases (see poster by RS Smith et. al.). Interestingly, PI3KC2β preferentially interacts with K-Ras12V>WT> dominant-negative (K-Ras15A). Furthermore, K-Ras localizes PI3KC2β to the plasma membrane whereas H-Ras localizes PI3KC2β to the perinuclear region. Thus, different Ras GTPases interact with PI3KC2β in distinct manners. We will present data on the contrasting role of K-Ras and H-Ras on regulation of PI3KC2β. Our findings suggest that PI3KC2β stabilizes nf-H-Ras and that interaction of H-Ras and PI3KC2β mutually inhibit one another. However, the role of K-Ras in regulation of PI3KC2β appears distinct. These findings have important implications for Ras-dependent tumorigenesis.

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.

MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1

The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1-3'UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3'UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3'UTR but not with 3'UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.

Adaptor Proteins Intersectin 1 and 2 Bind Similar Proline-Rich Ligands but Are Differentially Recognized by SH2 Domain-Containing Proteins

BackgroundScaffolding proteins of the intersectin (ITSN) family, ITSN1 and ITSN2, are crucial for the initiation stage of clathrin-mediated endocytosis. These proteins are closely related but have implications in distinct pathologies. To determine how these proteins could be separated in certain cell pathways we performed a comparative study of ITSNs.Methodology/Principal FindingsWe have shown that endogenous ITSN1 and ITSN2 colocalize and form a complex in cells. A structural comparison of five SH3 domains, which mediated most ITSNs protein-protein interactions, demonstrated a similarity of their ligand-binding sites. We showed that the SH3 domains of ITSN2 bound well-established interactors of ITSN1 as well as newly identified ITSNs protein partners. A search for a novel interacting interface revealed multiple tyrosines that could be phosphorylated in ITSN2. Phosphorylation of ITSN2 isoforms but not ITSN1 short isoform was observed in various cell lines. EGF stimulation of HeLa cells enhanced tyrosine phosphorylation of ITSN2 isoforms and enabled their recognition by the SH2 domains of the Fyn, Fgr and Abl1 kinases, the regulatory subunit of PI3K, the adaptor proteins Grb2 and Crk, and phospholipase C gamma. The SH2 domains mentioned were unable to bind ITSN1 short isoform.Conclusions/SignificanceOur results indicate that during evolution of vertebrates ITSN2 acquired a novel protein-interaction interface that allows its specific recognition by the SH2 domains of signaling proteins. We propose that these data could be important to understand the functional diversity of paralogous ITSN proteins.

ITSN protein family: regulation of diversity, role in signalling and pathology

Adaptor/scaffold proteins of the intersectin (ITSN) family are important components of endocytic and signalling complexes. They coordinate trafficking events with actin cytoskeleton rearrangements and modulate the activity of a variety of signalling pathways. In this review, we present our results as a part of recent findings on the function of ITSNs, the role of alternative splicing in the generation of ITSN1 diversity and the potential relevance of ITSNs for neurodegenerative diseases and cancer.

Emerging Roles for Intersectin (ITSN) in Regulating Signaling and Disease Pathways

Intersectins (ITSNs) represent a family of multi-domain adaptor proteins that regulate endocytosis and cell signaling. ITSN genes are highly conserved and present in all metazoan genomes examined thus far. Lower eukaryotes have only one ITSN gene, whereas higher eukaryotes have two ITSN genes. ITSN was first identified as an endocytic scaffold protein, and numerous studies reveal a conserved role for ITSN in endocytosis. Subsequently, ITSNs were found to regulate multiple signaling pathways including receptor tyrosine kinases (RTKs), GTPases, and phosphatidylinositol 3-kinase Class 2beta (PI3KC2β). ITSN has also been implicated in diseases such as Down Syndrome (DS), Alzheimer Disease (AD), and other neurodegenerative disorders. This review summarizes the evolutionary conservation of ITSN, the latest research on the role of ITSN in endocytosis, the emerging roles of ITSN in regulating cell signaling pathways, and the involvement of ITSN in human diseases such as DS, AD, and cancer.

Intersectin enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2‐Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multi‐domain scaffolding protein intersectin (ITSN) as an important regulator of this process. ITSN stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN enhanced Cbl activity was unclear. In this study, we find that ITSN enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN binds Pro‐rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2‐Cbl interaction resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2, ITSN1 binding site in Spry2 results in enhanced Cbl‐Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN and Spry2.

Interaction of ubiquitin ligase CBL with LMP2A protein of Epstein-Barr virus occurs via PTB domain of CBL and does not depend on adaptor ITSN1

Previously Latent membrane protein 2A (LMP2A) of Epstein-Barr virus was found to be ubiquitylated by CBL ubiquitin ligase but no direct interaction of LMP2A with CBL was reported. We aimed to explore this interaction and study a possibility of adaptor protein involvement. Taking into consideration that both LMP2A and CBL were shown to interact with endocytic adaptor protein intersectin 1 (ITSN1), we assumed that the latter could serve as a scaffold for LMP2A/CBL complex. Methods. We used an immunofluorescence and coimmunoprecipitation approaches to test a mutual complex formation of ITSN1, CBL and LMP2A proteins. Results. LMP2A coimmunoprecipitated with CBL while LMP2A did not interact with CBL G306E mutant harboring inactive phosphotyrosine-binding domain. We observed a triple colocalization of ITSN1, CBL and LMP2A signals in MCF-7 cells as well as coprecipitation of all mentioned proteins. Overexpression of ITSN1 did not affect the efficiency of complex formation of LMP2A with CBL. Moreover, LMP2A mutant unable to interact with ITSN1 was readily precipitated with CBL. Conclusions. LMP2A can be engaged in the complex together with endocytic adaptor ITSN1 and ubiquitin ligase CBL. We show that PTB domain of CBL is responsible for interaction with LMP2A. ITSN1 is not required for LMP2A recruiting to CBL.

Small molecule targeting Cdc42–intersectin interaction disrupts Golgi organization and suppresses cell motility

Signaling through the Rho family of small GTPases has been intensely investigated for its crucial roles in a wide variety of human diseases. Although RhoA and Rac1 signaling pathways are frequently exploited with the aid of effective small molecule modulators, studies of the Cdc42 subclass have lagged because of a lack of such means. We have applied high-throughput in silico screening and identified compounds that are able to fit into the surface groove of Cdc42, which is critical for guanine nucleotide exchange factor binding. Based on the interaction between Cdc42 and intersectin (ITSN), a specific Cdc42 guanine nucleotide exchange factor, we discovered compounds that rendered ITSN-like interactions in the binding pocket. By using in vitro binding and imaging as well as biochemical and cell-based assays, we demonstrated that ZCL278 has emerged as a selective Cdc42 small molecule modulator that directly binds to Cdc42 and inhibits its functions. In Swiss 3T3 fibroblast cultures, ZCL278 abolished microspike formation and disrupted GM130-docked Golgi structures, two of the most prominent Cdc42-mediated subcellular events. ZCL278 reduces the perinuclear accumulation of active Cdc42 in contrast to NSC23766, a selective Rac inhibitor. ZCL278 suppresses Cdc42-mediated neuronal branching and growth cone dynamics as well as actin-based motility and migration in a metastatic prostate cancer cell line (i.e., PC-3) without disrupting cell viability. Thus, ZCL278 is a small molecule that specifically targets Cdc42-ITSN interaction and inhibits Cdc42-mediated cellular processes, thus providing a powerful tool for research of Cdc42 subclass of Rho GTPases in human pathogenesis, such as those of cancer and neurological disorders.

Cdc42 and the RhoGEF Intersectin-1 collaborate with Nck to promote N-WASP-dependent actin polymerisation

Vaccinia virus enhances its cell-to-cell spread by inducing Arp2/3-dependent actin polymerisation. This process is initiated by Src- and Abl-mediated phosphorylation of the viral transmembrane protein A36, leading to recruitment of a signalling network consisting of Grb2, Nck, WIP and N-WASP. Nck is a potent activator of N-WASP-Arp2/3-dependent actin polymerisation. However, recent observations demonstrate that an interaction between Nck and N-WASP is not required for vaccinia actin tail formation. We found that Cdc42 cooperates with Nck to promote actin tail formation by stabilising N-WASP beneath the virus. Cdc42 activation is mediated by the Rho guanine-nucleotide-exchange factor (GEF) intersectin-1 (ITSN1), which is recruited to the virus prior to its actin-based motility. Moreover, Cdc42, ITSN1 and N-WASP function collaboratively in a feed-forward loop to promote vaccinia-induced actin polymerisation. Outside the context of infection, we demonstrate that ITSN1 also functions together with Cdc42, Nck and N-WASP during phagocytosis mediated by the Fc gamma receptor. Our observations suggest that ITSN1 is an important general regulator of Cdc42-, Nck- and N-WASP-dependent actin polymerisation.

Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

ГТФазо-активуючий білок олігофренін 1 – новий партнер багатофункціонального адапторного білка інтерсектину 1

Intersectin 1 (ITSN1) is a multifunctional adaptor protein which is involved in endocytosis, exocytosis and cellular signaling and it is also associated with such pathologies as Down syndrome and Alzheimer’s disease. The aim of this study was to identify new ITSN1 protein partners which are implicated in membrane trafficking. Methods. In silico analysis by Scansite online resource had identified a GTPase activating protein oligophrenin 1 (OPHN1) as a potential partner of ITSN1 SH3A domain. GST pull-down and immunoprecipitation were used to prove complex formation between ITSN1 and OPHN1. Subcellular protein localization was determined by immunofluorescence and confocal microscopy. Results. We have shown that brain-specific and ubiquitously expressed SH3A domain isoforms of ITSN1 interact with OPHN1. ITSN1 and OPHN1 form complexes in both resting and stimulated to exocytosis PC12 cell line. Conclusions. GTPase activating protein OPHN1 and adaptor protein ITSN1 interact in PC12 cell line independently of exocytosis stimulation.

A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinositol 3-kinase beta (PI3KC2β) regulation.

The intersectin 1 (ITSN1) scaffold stimulates Ras activation on endocytic vesicles without activating classic Ras effectors. The identification of Class II phosphatidylinositol 3-kinase beta, PI3KC2β, as an ITSN1 target on vesicles and the presence of a Ras binding domain (RBD) in PI3KC2β suggests a role for Ras in PI3KC2β activation. Here, we demonstrate that nucleotide-free Ras negatively regulates PI3KC2β activity. PI3KC2β preferentially interacts in vivo with dominant-negative (DN) Ras, which possesses a low affinity for nucleotides. PI3KC2β interaction with DN Ras is disrupted by switch 1 domain mutations in Ras as well as RBD mutations in PI3KC2β. Using purified proteins, we demonstrate that the PI3KC2β-RBD directly binds nucleotide-free Ras in vitro and that this interaction is not disrupted by nucleotide addition. Finally, nucleotide-free Ras but not GTP-loaded Ras inhibits PI3KC2β lipid kinase activity in vitro. Our findings indicate that PI3KC2β interacts with and is regulated by nucleotide-free Ras. These data suggest a novel role for nucleotide-free Ras in cell signaling in which PI3KC2β stabilizes nucleotide-free Ras and that interaction of Ras and PI3KC2β mutually inhibit one another.

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.

Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimer's disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.

Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks.

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.