Intersectin Multidomain Adaptor Proteins: Regulation of Functional Diversity

Gene 473 (2011) 67–75

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Review Intersectin multidomain adaptor proteins: Regulation of functional diversity

Liudmyla Tsyba, Oleksii Nikolaienko, Oleksandr Dergai, Mykola Dergai, Olga Novokhatska, Inessa Skrypkina, Alla Rynditch ⁎

Department of Functional Genomics, Institute of Molecular Biology and Genetics, NASU, 150 Zabolotnogo Street, 03680 Kyiv, Ukraine article info abstract

Article history: Adaptor/scaffold proteins serve as platforms for the assembly of multiprotein complexes and regulate the Accepted 30 November 2010 efficiency and specificity of signalling cascades. Intersectins (ITSNs) are an evolutionarily conserved adaptor Available online 9 December 2010 protein family engaged in endo- and exocytosis, actin cytoskeleton rearrangement and signal transduction. This review summarizes recent advances in the function of ITSNs in neuronal and non-neuronal cells, the role Received by A.J. van Wijnen of alternative splicing and alternative transcription in regulating the structural and functional diversity of ITSNs, their expression patterns in different tissues and during development, their interactions with proteins, Keywords: Intersectin family as well as the potential relevance of ITSNs for neurodegenerative diseases and cancer. The diversity of Adaptor/scaffold proteins mechanisms in the regulation of ITSN expression and specificity in different cells emphasizes the important Alternative splicing role of ITSN proteins in vesicle trafficking and signalling. Endocytosis © 2010 Elsevier B.V. All rights reserved.

1. Introduction shown to be essential components for initiation of clathrin-coated pit (CCP) formation (Henne et al., 2010). Adaptor/scaffold proteins have emerged as regulators of many In this review, we highlight recent advances in the study of the cellular processes, including proliferation, differentiation, cell-cycle functions of the ITSN family in neuronal and non-neuronal cells and control, cell survival and migration (Pawson and Scott, 1997; provide a picture of experimentally verified ITSN-specific interactions. Szymkiewicz et al., 2004; Zeke et al., 2009). Classical scaffolds usually We also describe the impact of alternative processing on the do not possess any type of enzymatic activity and are characterized by generation of diversity in the ITSN family and regulation of ITSN modular architecture with the presence of multiple protein/lipid genes expression. binding domains and sites for inducible posttranslational modifications. Scaffold molecules selectively control the spatial and temporal 2. Structure of ITSN family proteins assembly of multiprotein complexes. Specifically, they determine the formation and localization of protein complexes and may both ITSNs are evolutionarily conserved proteins present in diverse facilitate or inhibit signal transduction depending on their concen- metazoan organisms ranging from nematodes to mammals. There are tration in certain compartments, regulating the strength, specificity two ITSN genes in humans, ITSN1 and ITSN2 located on chromosomes and duration of signal propagation. 21 (q22.1–q22.2) and 2 (pter–p25.1), respectively (Guipponi et al., Intersectins (ITSNs) are adaptor/scaffold proteins with a unique 1998; Pucharcos et al., 2001). ITSN1 and ITSN2 share significant multidomain structure. By binding to numerous proteins, they sequence identity and a similar domain structure (Pucharcos et al., assemble multimeric complexes implicated in clathrin- and caveo- 2000). Moreover, they both have short and long isoforms produced by lin-mediated endocytosis, rearrangements of the actin cytoskeleton, alternative splicing (Guipponi et al., 1998; Pucharcos et al., 2001). The cell signalling and survival (Sengar et al., 1999; Predescu et al., 2003; short isoform (ITSN-S) consists of two Eps15 homology domains (EH1 Hussain et al., 2001; Mohney et al., 2003; Das et al., 2007; Predescu et and EH2), a coiled-coil region (CCR) and five Src homology 3 domains al., 2007). The roles of ITSN proteins as adaptors have been studied (SH3A–E) (Fig. 1). The EH domains which bind to Asn-Pro-Phe motifs intensively in different cell types and organisms. Recently, ITSNs were have been identified in several proteins implicated in endocytosis and vesicle transport. The SH3 domains bind to proline-rich sequences

Abbreviations: ITSNs, intersectins; ITSN1-S, short isoform of ITSN1; ITSN1-L, long and are commonly found in proteins implicated in cell signalling isoform of ITSN1; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; GEF, guanine pathways, cytoskeletal organization and membrane trafﬁc. They nucleotide exchange factor; CME, clathrin-mediated endocytosis; Dap160, Drosophila possess the most diverse speciﬁcity among interaction domains (Li, homolog of human ITSN; NMJ, neuromuscular junction; RTK, receptor tyrosine kinases; 2005). The long isoform (ITSN-L) contains three additional C-terminal EGFR, epidermal growth factor receptor; CCR, coiled-coil region; SV, synaptic vesicle; domains, a Dbl homology domain (DH), a Pleckstrin homology DS, Down syndrome; AD, Alzheimer's disease. ⁎ Corresponding author. Tel.: +380 44 526 9618; fax: +380 44 526 0759. domain (PH) and a C2 domain (Guipponi et al., 1998; Sengar et al., E-mail address: [email protected] (A. Rynditch). 1999). The DH and PH domains usually form a tandem in the Dbl

Fig. 1. Schematic representation of the structure of ITSN proteins in vertebrates, D. melanogaster and C. elegans. family of guanine nucleotide exchange factors (GEFs). The DH domain Pucharcos et al., 2001; Sengar et al., 1999). Numerous additional is sufficient to catalyse exchange of bound GDP for GTP and therefore splicing events affecting ITSN1 and ITSN2 transcripts will be briefly activate Rho GTPases. The DH domain of ITSN specifically activates the reviewed in this section. Cdc42 GTPase (Hussain et al., 2001). The PH domain of ITSN binds Four evolutionarily conserved in-frame alternative splicing events phosphoinositides (Snyder et al., 2001). C2 domains are usually affecting ITSN1 mRNAs were found in mice and humans (Okamoto et involved in Ca2+-dependent and Ca2+-independent phospholipid al., 1999; Pucharcos et al., 2001; Tsyba et al., 2004)(Fig. 2A). These binding (Rizo and Südhof, 1998). Multiple domains of ITSNs mediate events include: (1) the use of an alternative 3′-splice site internal to their association with a wide range of proteins. So far, we know of exon 6 that results in truncation of exon 6 and deletion of 37 amino more than 30 proteins that interact with ITSNs. However, the list of acids between the EH1 and EH2 domains, (2) splicing of the neuron- ITSN-binding proteins is not believed to be complete. The vast specific exon 20 that encodes 5 amino acids in the SH3A domain, (3) majority of the publications focuses on ITSN1. Despite limited deletion of exons 25 and 26 that encode the SH3C domain, (4) information on ITSN2, it is apparent that ITSN2 plays an important skipping of exon 35 encoding 31 amino acids of the DH domain and 25 role in clathrin- and caveola-mediated endocytosis (Pucharcos et al., residues of the DH–PH interdomain spacing. Inclusion of exon 22a 2000; Klein et al., 2009). results in the generation of the shortest ITSN1 transcript that encodes Proteins with similarity to the ITSN-S domain structure were found a protein containing two EH domains, the CCR, the SH3A domain and in mammals, frogs, flies and nematodes (Sengar et al., 1999; Okamoto the exon 22a-specific C-terminal sequence (Skrypkina et al., 2005). et al., 1999; Yamabhai et al., 1998; Roos and Kelly, 1998; Rose et al., Except for splicing of exon 30, three additional alternative splicing 2007), while ITSN-L isoforms have been identified only in vertebrates. events that do not introduce premature termination codons were The ITSN ortholog in Drosophila consists of two EH domains, a CCR and reported for ITSN2 (Fig. 2B). Truncation of exon 19 and brain-specific four SH3 domains (Roos and Kelly, 1998), while the Caenorhabditis inclusion of exon 17 change the structure of the CCR of ITSN2, while elegans ITSN contains five SH3 domains (Rose et al., 2007)(Fig. 1). skipping of exons 27 and 28 results in deletion of the SH3D domain Proteins homologous to ITSN were not found in S. cerevisiae. (exon numbering according to NM_006277) (Pucharcos et al., 2001; The organization of the ITSN1 and ITSN2 genes of vertebrates is Seifert et al., 2007). very similar (Pucharcos et al., 1999, 2001). They comprise more than Many splicing events cause frameshifts in ITSNs mRNA and 40 exons, while orthologous ITSN genes of nematodes (C. elegans) and introduce premature termination codons that lie more than 50 arthropods (D. melanogaster) contain 8 and 11 exons, respectively. nucleotides upstream of an exon–exon junction (Tsyba et al., 2004; Moreover, in vertebrates most of the exon boundaries are conserved Kropyvko et al., 2010a; Pucharcos et al., 2001; Seifert et al., 2007). between ITSN1 and ITSN2, and the mechanisms of generation of the Such mRNAs are expected to be degraded via the nonsense-mediated two major spliced variants are the same for these paralogous genes mRNA decay (NMD) pathway (McGlincy and Smith, 2008). The (Pucharcos et al., 2001). This suggests that, the long isoform produced relatively high abundance of mRNAs with premature termination by alternative splicing of exon 30 appeared earlier in evolution before codons suggests the participation of alternative splicing and NMD in gene duplication. Other known alternative splicing events are not regulation of the expression level of ITSNs. conserved between ITSN1 and ITSN2 (Pucharcos et al., 2001; Tsyba et ITSN1 and ITSN2 are widely expressed in mammal tissues al., 2004). An alternative promoter was identified in intron 5 of human (Guipponi et al., 1998; Sengar et al., 1999; Hussain et al., 1999; ITSN1 gene. This promoter generates the transcripts encoding ITSN1-S Okamoto et al., 1999; Pucharcos et al., 2000; Tsyba et al., 2004). isoform without the EH1 domain. However, it is not known, whether However, the tissue distribution of the ITSN proteins varies consid- these transcripts are translated into proteins (Kropyvko et al., 2010b). erably. The expression of some isoforms is tissue-dependent and Recently, the multi-modular protein, Cin1, whose domain struc- regulated during development. In contrast to the widely expressed ture is similar to that of ITSN, was found in the pathogenic fungus ITSN2-L isoform, ITSN1-L is expressed predominantly in neurons Cryptococcus neoformans (Shen et al., 2010). Cin1 contains an N- (Pucharcos et al., 2000, 2001; Guipponi et al., 1998). However, low terminal EH domain, a central CCR, a WASP-homology domain 2 levels of ITSN1-L expression were detected in adult kidney, liver and (WH2), two SH3 domains and C-terminal DH–PH domains. Interest- placenta (Pucharcos et al., 2001), as well as in fetal liver, lung, kidney ingly, alternative splicing resulted in two Cin1 isoforms, long and and muscle (Tsyba et al., 2004; Kropyvko et al., 2010a). Western blot short. The latter is similar to ITSN-S. analysis of neuron lysates and lysates of glial cells from rat brain did not reveal ITSN1-S isoforms in neurons (Yu et al., 2008). However, 3. Alternative splicing in the regulation of ITSN genes expression mRNAs of these isoforms were detected in mouse thalamic neurons by single-cell RT-PCR (Kropyvko et al., 2010a). Alternative splicing plays one of the major roles in the regulation ITSN1 transcripts containing exon 20 are neuron-specific and their of ITSN genes expression and functions. Two major ITSN isoforms are expression is developmentally regulated (Tsyba et al., 2004; Tsyba et produced by alternative splicing of exon 30 that provides the al., 2008). The frequency of the + exon 20 variant of ITSN1 increases termination codon for the short isoform (Guipponi et al., 1998; during fetal brain development whereas the level of the transcript L. Tsyba et al. / Gene 473 (2011) 67–75 69

Fig. 2. Alternative splicing of mammalian ITSNs. (A) Schematic representation of alternative splicing events affecting mouse and human ITSN1. Alternative splicing events are indicated above and below the ITSN1-L domain structure. Exon numbering is according to NM_003024. Asterisks indicate a stop codon. (B) Schematic representation of alternative splicing events affecting human ITSN2. Exon numbering is according to NM_006277. Inclusion of exons 30 and 31a results in the generation of two variants of ITSN2-S. lacking exon 20 decreases correspondingly. The ratio of ITSN1 neurons revealed its localization throughout the cytoplasm with isoforms with and without exons 25 and 26 (the SH3C domain) accumulation in the perinuclear region in Golgi-like structure varies in fetal and adult brain (Pucharcos et al., 2001; Tsyba et al., (Hussain et al., 1999; Mohney et al., 2003; Predescu et al., 2003; Ma 2004). In other tissues about 20% of all ITSN1 transcripts lack exon 25 et al., 2003; Pucharcos et al., 2000). ITSN1 was also observed in the cell and 26. Skipping of exon 35 occurs in approximately 10% of ITSN1-L periphery. Moreover, epidermal growth factor stimulation promoted isoforms, but the ratio of ITSN1-L transcripts with and without exon 35 relocalization of the ITSN–c-Cbl complex close to the plasma differs in brain and other tissues. Approximately 3% of ITSN1 membrane (Martin et al., 2006). At the plasma membrane ITSN1 is transcripts contain a truncated exon 6 (Kropyvko et al., 2010a). localized predominantly in CCPs (Hussain et al., 1999). Electron Taking into consideration that alternatively spliced exons could be microscopy immunogold labeling studies on endothelial cells indi- present in ITSN transcripts in different combinations, 16 variants of cated that ITSN1-S is associated with caveolae and clathrin-coated ITSN1-L, 8 variants of the short ITSN1 isoforms and 4 variants of the vesicles (CCVs), as well as with Golgi-derived vesicles and cytoskeletal ITSN1-22a isoforms could be generated in human tissues. Indeed, we elements (Predescu et al., 2003). recently cloned all eight variants of ITSN1-S (Kropyvko et al., 2010a). Transfection studies with ITSN1 deletion mutants suggest that the Although the function of most alternatively spliced isoforms of ITSNs EH domains and CCR are responsible for the localization of ITSNs in are poorly understood, it is evident that the generation of proteins with cells (Hussain et al., 1999; Scappini et al., 2007; Pucharcos et al., differences in functional domains or with a different domain composi- 2000). Moreover, it was shown that ITSN1 and Eps15 function tion could change the binding properties of ITSN proteins and may play a cooperatively to regulate subcellular localization of the ITSN1–Eps15 role in selecting specific interactions. Some isoforms of ITSNs have a complex (Sengar et al., 1999). ITSN2-S showed a subcellular brain-specific function. The brain-enriched ITSN1-L isoform that has GEF distribution similar to that of ITSN1-S (Pucharcos et al., 2000; Klein activity toward Cdc42 GTPase was shown to be involved in dendritic et al., 2009). The subcellular distribution of ITSN1 in neurons will be spine morphogenesis (Nishimura et al., 2006; Thomas et al., 2009). Our discussed later in this paper. Thus, the association of ITSNs with previous results demonstrated that neuron-specific inclusion of exon 20 different membranous organelles, caveolae, Golgi complex, CCPs and changes the binding properties of the SH3A domain, shifting its binding CCVs, suggests its involvement in different pathways of the cell specificity from the signalling proteins Sos1 and Cbl to the cytoskeleton membrane-trafficking system (Predescu et al., 2003). regulatory protein CdGAP and the endocytic proteins dynamin 1 and synaptojanin 1 (Tsyba et al., 2008). Skipping of the SH3C domain of 5. ITSN proteins are essential components of endo- and exocytosis ITSN1 or the SH3D domain of ITSN2 could change the interaction interfaces for certain ITSN protein partners or even abolish the Proteins of the ITSN family are clearly linked to endocytosis. interaction. For example, recently identified ITSN1-interacting protein Endocytosis is a complex and tightly controlled process that allows Reps1 binds only to the SH3C domain of ITSN1 (Dergai et al., 2010)and the delivery of proteins from the plasma membrane into the cell and is therefore could not interact with ITSN1ΔSH3C isoform. essential for maintaining cellular homoeostasis as well as for the interaction between the cell and its environment (Doherty and 4. Subcellular localization of ITSNs McMahon, 2009; Szymkiewicz et al., 2004). ITSNs have a modular structure composed of the EH and SH3 domains that are typical for Analysis of the subcellular distribution of endogenous and over- endocytic proteins. The subcellular localization of ITSNs also supports expressed ITSN1 in mammalian epithelial cells and rat hippocampal the involvement of these proteins in internalization events. 70 L. Tsyba et al. / Gene 473 (2011) 67–75

ITSN1 was shown to interact with a plethora of endocytic proteins ITSN1 by RNA interference attenuates internalization of epidermal (Fig. 3). The N-terminal tandem of the EH domains interacts with the growth factor receptor (EGFR) in HEK293T cells and reduces the rate endocytic accessory protein epsin (Yamabhai et al., 1998), which of transferrin endocytosis in neurons (Martin et al., 2006; Thomas et binds directly to inositol lipids, Eps15 and the α subunit of AP2, can al., 2009). Similarly, siRNA knockdown of ITSN1 decreases the rate of help drive membrane invagination as well as link ubiquitinated internalization of the renal outer medullar potassium 1 channel membrane proteins to clathrin. The EH domains of ITSN1 also interact (ROMK1) by a With-no-lysine (WNK) kinase-dependent mechanism with stonin 2, an endocytic sorting adaptor for synaptic vesicle cargo (He et al., 2007). It was shown that WNK1 and WNK4 kinases interact recognition (Martina et al., 2001; Kelly and Phillips, 2005) and the with the SH3 domains of ITSN1 and that the interactions are crucial for secretory carrier membrane protein SCAMP1 (Fernández-Chacón et stimulation of endocytosis of ROMK1 by WNKs (He et al., 2007). On al., 2000). The CCR of ITSN1 binds to synaptosome-associated proteins the other hand, overexpression of ITSN2-L enhanced the rate of T cell of 23 kDa and 25 kDa (SNAP-23 and SNAP-25) (Okamoto et al., 1999) antigen receptor (TCR) internalization, but this effect was dependent and forms heterodimers with the scaffolding adaptor Eps15 (Sengar et on the GEF activity of the DH domain of the long isoform (McGavin et al., 1999; Koh et al., 2007) that binds ubiquitinated cargo, AP2, epsin al., 2001). Similar effects of ITSN overexpression and silencing could and dynamin, and clusters AP2 appendages through its long C- be the result of disruption of the formation of higher order protein terminal tail (Doherty and McMahon, 2009). complexes between ITSN and its binding partners. The SH3 domains of ITSN1 bind the large regulatory GTPase Two laboratories reported the generation of loss-of-function dynamin involved in vesicle fission, a phosphoinositide phosphatase mutations that eliminate Dap160 (Drosophila homolog of human synaptojanin 1, a synaptic protein synapsin, a membrane-deforming ITSN). The studies demonstrated that the levels of dynamin, protein SGIP1, and the AP2 binding protein connecdenn (Roos and synaptojanin, endophilin and AP180 are severely reduced in Dap160 Kelly, 1998; Yamabhai et al., 1998; Okamoto et al., 1999; Evergren et mutant synapses, suggesting the role of Dap160 in recruitment of al., 2007; Dergai et al., 2010; Allaire et al., 2006). Recently the α- and endocytic proteins to the presynaptic terminals. Reduction of the β-appendage domains of AP2 were shown to directly interact with the number of synaptic vesicles, aberrant large vesicles, defects in vesicle linker between the SH3A and SH3B domains (Pechstein et al., 2010a). recycling and fission such as accumulation of collared pits and Ω-like Clathrin could be co-immunoprecipitated with anti-intersectin anti- structures were observed in Dap160 mutants (Koh et al., 2004; Marie bodies from rat brain lysates (Hussain et al., 1999) whereas direct et al., 2004). Similar defects in the development of neuromuscular interaction was not shown. It was reported that the SH3 domains of junction (NMJ) synapses and vesicle recycling were detected in ITSN1 also bind to the proline-rich region of inositol 5-phosphatase Drosophila Eps15-null mutants (Koh et al., 2007). Loss of Eps15 caused SHIP2, a negative regulator of CCP growth and insulin-mediated a severe reduction in dynamin and Dap160 protein levels at NMJs. signalling. Interaction between SHIP2 and ITSN1 leads to the Eps15 and Dap160 double mutants had a phenotype similar to that of recruitment of SHIP2 to CCPs (Xie et al., 2008; Nakatsu et al., 2010). the single Eps15 or Dap160 mutants, suggesting that Eps15 and More direct evidence for the role of ITSN in endocytosis was Dap160 act at the same step during endocytosis (Koh et al., 2007). A provided by data showing that overexpression or knockdown of ITSN reduced number of exocytosis events in chromaffin cells and a inhibits clathrin-mediated endocytosis (CME) (Sengar et al., 1999; slowing of endocytosis in neurons were also detected in ITSN1 null Pucharcos et al., 2000; Martin et al., 2006; Thomas et al., 2009). For mice (Yu et al., 2008). example, overexpression of ITSN1 blocked transferrin receptor ITSNs are also involved in caveolae endocytosis, an important step internalization in Cos-1 cells. However, this block could be rescued in mediating transcytosis of proteins in endothelial cells (Predescu et by overexpression of dynamin suggesting an important role of ITSN1 al., 2003; Klein et al., 2009). Overexpression of ITSN1-S inhibited in dynamin recruitment during CME (Sengar et al., 1999). Silencing caveolae-mediated uptake and affected caveolae morphology causing

Fig. 3. Schematic representation of the proteins that interact with ITSNs. Lines indicate the domains of ITSNs involved in these interactions. The interaction sites responsible for the binding of FCHo have not been determined. The K15 protein of Kaposi's sarcoma-associated herpesvirus was shown to interact with ITSN2 (Lim et al., 2007). The functional consequences of the interactions are shown in the shadowed areas. The numbers in circles indicate references: 1Yamabhai et al., 1998; 2Sengar et al., 1999; 3Koh et al., 2007; 4Dergai et al., 2010; 5Roos and Kelly, 1998; 6Okamoto et al., 1999; 7Pechstein et al., 2010a; 8Henne et al., 2010; 9Allaire et al., 2006; 10Das et al., 2007; 11Xie et al., 2008; 12Nakatsu et al., 2010; 13Lim et al., 2007; 14Nishimura et al., 2006; 15Martina et al., 2001; 16Kelly and Phillips, 2005; 17Fernández-Chacón et al., 2000; 18Evergren et al., 2007; 19Tong et al., 2000b; 20Nikolaienko et al., 2009a; 21Martin et al., 2006; 22Nikolaienko et al., 2009b; 23He et al., 2007; 24Jenna et al., 2002; 25Hussain et al., 2001; 26McGavin et al., 2001. L. Tsyba et al. / Gene 473 (2011) 67–75 71 formation of large clusters and “grape-like” structures. On the GDP from Rho GTPases and their subsequent activation by GTP contrary, siRNA-mediated ITSN2-L knockdown resulted in significant binding (Rossman et al., 2005). The DH domains of ITSN1 and ITSN2 increase of caveolae-mediated uptake that was accompanied by display high sequence homology (Pucharcos et al., 2000). Both decreased Cdc42 activation as well as by changes in cell shape and F- proteins are highly specific and promote exchange on Cdc42, but actin distribution probably due to decrease of GEF activity of the DH not on Rac1 or RhoA (Hussain et al., 2001; Klein et al., 2009). and PH domains of ITSN2-L. Indeed, overexpression of the DH–PH Overexpression of ITSN1-L causes actin rearrangements specific for domains causes a decrease of caveolae-mediated uptake (Klein et al., Cdc42 activation and stimulates filopodia formation in cultured 2009). fibroblasts (Hussain et al., 2001). ITSN is also a functional component of the exocytotic machinery. In the structure of most GEFs the DH domain is followed by the PH As mentioned previously ITSN1 binds to SNAP-25, a protein belonging domain that is responsible for phosphoinositide binding and is to the Q-SNARE family and involved in exocytosis of synaptic vesicles considered to positively or negatively modulate the efficiency of (Okamoto et al., 1999). Moreover, GEF activity of ITSN1-L is important GDP/GTP exchange (Pruitt et al., 2003). In vitro studies revealed that for actin cytoskeletal rearrangements required for exocytosis in exchange activity of the ITSN1 DH domain alone is reduced relative to neuroendocrine cells (Malacombe et al., 2006). Reducing endogenous that of the DH–PH domain (Zamanian and Kelly, 2003; Hussain et al., ITSN1 levels by siRNA prevented secretagogue-induced activation of 2001; Ahmad and Lim, 2010). At the same time, Pruitt et al. reported Cdc42 and resulted in inhibition of growth hormone secretion. that the presence of the PH domain does not alter GEF exchange Therefore, ITSN1-L was proposed to be an adaptor that coordinates activity of the ITSN1 DH domain in vitro but enhances it in vivo (Pruitt exo–endocytotic membrane trafficking in secretory cells. et al., 2003). Interactions of the PH domain with phosphoinositides Despite involvement of ITSN proteins in exo- and endocytosis is did not alter the ability of the DH domain of ITSN1 to activate Cdc42 in well established in vitro and in vivo, the precise mechanisms of its vitro (Snyder et al., 2001). Moreover, the crystal structure of the functioning remain unclear. ITSNs are considered as adaptors that ITSN1–Cdc42 complex shows no direct interactions between the PH organize multiprotein complexes at membrane compartments. ITSN1 domain and bound GTPase (Snyder et al., 2002). This contrasts with was shown to function as negative regulator of dynamin 1 the role of the PH domain in Dbs, which forms contacts with the recruitment in synaptic endocytic zones (Evergren et al., 2007). bound Cdc42 (Rossman et al., 2002). Deficiency of Dap160 causes temperature-sensitive paralytic pheno- Intact ITSN1-L shows lower Cdc42 exchange activity compared to type and endocytic abnormalities similar to dynamin (shibire) the isolated DH domain. Zamanian et al. demonstrated the existence mutants (Koh et al., 2004). Finally, analysis of loss-of-function of intramolecular interaction between the ITSN1-L SH3 region and the mutations in the Dap160 gene demonstrated that Dap160 is required DH domain that inhibits its exchange activity by reducing ITSN1-L for the proper localization of dynamin (Koh et al., 2004; Marie et al., binding to Cdc42 (Zamanian and Kelly, 2003). In line with this is the 2004). Taken together, these data suggest a role of ITSN in dynamin observation that binding of the N-WASP proline-rich region to the complex assembly and functioning. SH3 domains of ITSN1-L enhances the ability of the DH domain to The unique role of the SH3A domain of ITSN1 in the early stages of interact with GDP-bound Cdc42 and to catalyse its conversion to GTP- CME (Simpson et al., 1999) should be stressed. In contrast to other bound Cdc42 (Hussain et al., 2001). Similarly, interaction of the ITSN1 SH3 domains tested that selectively inhibit late events of CME SH3 domains with Numb, a protein implicated in cortical neurogen- involving membrane fission, the SH3A domain of ITSN1 inhibits esis during nervous system development, enhances the GEF activity of intermediate events leading to the formation of constricted coated ITSN1-L toward Cdc42 in vivo (Nishimura et al., 2006). Very recently, pits. the molecular mechanism of ITSN1-L autoinhibition was reported. Interestingly, using quantitative live-cell imaging by total internal Ahmad et al. showed that the GEF activity of ITSN1-L is controlled by reflection fluorescence microscopy (TIR-FM), ITSN2 was shown to be interaction of the SH3E domain with the C-terminal half of the DH important for maturation of CCPs at the later stages of CME together domain. The SH3E domain is both necessary and sufficient for with epsin and Eps15 (Mettlen et al., 2009). In view of controlling CCP repression of the DH domain (Ahmad and Lim, 2010). maturation, another recently established fact attracts attention: Another group of proteins possesses an effect opposite to that of interaction of ITSN1 with AP2 inhibits binding of synaptojanin 1 to GEF: these proteins enhance GTPase activity leading to rapid ITSN1 (Pechstein et al., 2010a). This could serve as a specific conversion to the inactive GDP-bound form. ITSN1 forms a complex checkpoint controlling CCP maturation. with CdGAP, a GTPase-activating protein with activity towards Cdc42 Recently, Henne et al. provided evidence that a family of and Rac1 (Jenna et al., 2002). In PDGF-stimulated fibroblasts, ITSN1 is membrane-sculpting proteins FCHo1/2 is responsible for marking colocalized with CdGAP and inhibits its GAP activity towards Rac1. the sites of CCV formation and act as CCP nucleators (Henne et al., These investigations expand the role of proteins of the ITSN family and 2010). FCHo1/2 proteins bind specifically to the plasma membrane suggest a function in Rac1 regulation. and recruit Eps15 and ITSNs, which in turn engage the adaptor The regulated assembly of actin filament networks is a crucial part complex AP2. ITSNs, Eps15 and Eps15R were shown to regulate FCHo of endocytosis (Galletta and Cooper, 2009). Actin assembly on clustering. Quadruple knockdown of ITSN1, ITSN2, Eps15 and Eps15R endocytic vesicles via N-WASP was reported to be a mechanism of abolished CCPs due to the inability of FCHo to initiate CCP formation, vesicle protrusion (Taunton et al., 2000). Regarding its domain suggesting that these proteins constitute an early module for nascent composition and interactions, the ITSN was considered to play a role CCP assembly (Henne et al., 2010). in coordinating endocytosis and actin cytoskeletal rearrangements. Indeed, McGavin et al. demonstrated that ITSN2-L is important for 6. ITSN proteins in actin cytoskeleton rearrangements recruiting Cdc42 and its effector WASP from perinuclear actin bundles to endocytic vesicles in T cells during T cell antigen receptor (TCR) It is noteworthy that endocytic events are tightly connected to endocytosis. Overexpression of ITSN2-L induces an increase of TCR actin cytoskeleton rearrangements and cargo income. Both members internalization, while both the DH domain-deleted form of ITSN2-L of ITSN family were shown to participate in actin cytoskeleton and latrunculin treatment severely reduce induction of TCR endocy- rearrangements by specific activation of the small GTPase Cdc42 and tosis (McGavin et al., 2001). direct interaction with Cdc42 effectors — the Wiskott–Aldrich Recently, interaction of cryptococcal proteins Cin1/ITSN, Cdc42 syndrome proteins, WASP and N-WASP (Hussain et al., 2001; and Wsp1/WASP was reported in the pathogenic fungus C. neofor- McGavin et al., 2001). GEF activity was shown only for ITSN-L mans, arguing for a conserved role in the regulation of the actin isoforms possessing a conserved DH domain catalysing the release of cytoskeleton (Shen et al., 2010). 72 L. Tsyba et al. / Gene 473 (2011) 67–75

7. ITSNs in RTK signalling As stated above, ITSN pre-mRNA undergoes complex splicing, that generate a number of isoforms. Some of them are neuron-specificor Activation of receptor tyrosine kinases (RTKs) by growth factors preferentially expressed in brain. Several studies argue that ITSN1-L initiates intracellular signalling pathways that control cell prolifera- isoforms completely replace the ITSN1-S isoforms in neurons of tion, differentiation, survival, metabolism and migration (Lemmon mammals (Hussain et al., 1999; Yu et al., 2008); however, ITSN1-S and Schlessinger, 2010). Several lines of evidence suggest that mRNA was detected in mouse neurons (Kropyvko et al., 2010a). endocytosis is a critical component in RTK signalling. Clathrin- The pattern of the subcellular localization of ITSN1 suggests its mediated endocytosis participates in downregulation of activated involvement in synaptic transmission. Roos and Kelly showed that RTKs via receptor internalization into endosomes with subsequent Dap160 is restricted to the synaptic nerve terminal of Drosophila NMJs targeting for lysosomal degradation or for recycling (Sorkin and Goh, (Roos and Kelly, 1998). Moreover, Dap160 localizes to a periactive 2009). However, endocytosis has also emerged as an essential step in zone that surrounds the active zone where much of the endocytic the successful activation of RTK-dependent signalling pathways, in machinery resides (Koh et al., 2007). Nematode ITSN was found to particular the extracellular signal-regulated kinase mitogen-activated also be enriched in presynaptic regions (Rose et al., 2007). Using cryo- protein kinase (ERK/MAPK) pathway (O'Bryan et al., 2001; McPher- electron microscopy, Evergren et al. demonstrated that lamprey ITSN son et al., 2001). is located at the presynaptic compartment of the lamprey giant Several experimental data suggest that ITSN1 provides a link synapse and that upon stimulation it undergoes dynamic redistribu- between endocytosis and signal transduction. ITSN1, through its SH3 tion from the synaptic vesicle cluster to the periactive zone (Evergren domains, forms complexes with Sos1, a guanine nucleotide exchange et al., 2007). factor for Ras, and stimulates Ras activation (Tong et al., 2000b; On the other hand, data on ITSN1 localization in mammalian Mohney et al., 2003). Furthermore, ITSN1 interacts with the E3- neurons is less consistent. Several studies provide evidence that ITSN1 ubiquitin ligase Cbl and enhances c-Cbl-mediated EGFR ubiquitylation is enriched in the somatodendritic regions of neurons, in particular in and degradation (Martin et al., 2006). Overexpression of the SH3 dendritic spines (Nishimura et al., 2006), and is absent from domains of ITSN1 inhibits EGF-induced activation of Ras indepen- presynaptic terminals (Thomas et al., 2009). It should be pointed dently of the ability of these domains to block endocytosis of the EGF out that ITSN homologues in worm and fly correspond to the short receptor (Tong et al., 2000a). Silencing ITSN1 by RNA interference mammalian isoform ITSN-S. They do not contain DH, PH and C2 attenuated EGFR internalization and activation of the ERK/MAPK domains, which could possibly lead to the observed difference in pathway (Martin et al., 2006). localization. As mentioned above, subcellular targeting of the ITSN1-S Our recent results demonstrate that ITSN1 forms a stable complex protein in fibroblasts is primarily determined by the interactions of with Ruk/CIN85, a scaffolding protein that regulates recruiting the the EH domains and CCR (Hussain et al., 1999; Scappini et al., 2007; Cbl–EGFR complex to endosomes for downregulation of RTK, as well Pucharcos et al., 2000). Therefore, localization of the ITSN1-L isoform as cytoskeletal rearrangements and apoptosis (Nikolaienko et al., in neurons of vertebrates could also be directed by the same 2009a). It should be noted that interactions of the SH3 domains of mechanism, or depend either on DH–PH–C2 extension or on the ITSN1 with Cbl and Ruk/CIN85 are not regulated by EGF neuron-specific interactions of other ITSN1 domains. It should be stimulations (Martin et al., 2006; Nikolaienko et al., 2009a,b). noted, that binding of the PH domain to phosphoinositides is Therefore, ITSN1 could be constitutively associated with Cbl as well relatively weak and appears to be insufficient to drive ITSN1 as with Ruk/CIN85. localization (Snyder et al., 2001). One of the major binding partners There is evidence that expression of either full-length ITSN1 or the of the ITSN1 EH domains, epsin 1, was detected in both the isolated EH domain region stimulate activation of the transcription presynaptic and postsynaptic sites (Yao et al., 2003), while dynamin factor Elk-1 independently of the MAPK pathway (Adams et al., 2000). 1, which strongly binds several ITSN1 SH3 domains, is concentrated It was also shown that overexpression of ITSN1 leads to oncogenic within the presynaptic compartment (Gray et al., 2003). Indeed, transformation of rodent fibroblasts (Adams et al., 2000). recently, Pechstein et al. demonstrated that ITSN1 accumulates Interestingly, siRNA knockdown of ITSN1-S reduced MEK and comparably at both pre- and postsynaptic membrane sites of mouse ERK1/2 phosphorylation and activated the mitochondrial pathway of hippocampal neurons (Pechstein et al., 2010a). Available biochemical apoptosis in endothelial cells (Predescu et al., 2007). ITSN1 was also data confirmed these findings: ITSN1 was detected as core component shown to regulate the survival of neuronal cells through the activation of presynaptic synaptotagmin-associated endocytic complex (Khanna of a PI3K–AKT pathway by interaction with PI3K–C2β. Decreasing et al., 2006) as well as in triton-insoluble postsynaptic density (PSD) ITSN expression by shRNA inhibited the PI3K–C2β–AKT signalling pellets from rat brain lysate (Nishimura et al., 2006). pathway and dramatically increased apoptosis in both neuroblastoma Evidence of ITSN1 functions in neurons is rather broad and cells and primary cortical neurons (Das et al., 2007). sometimes contradictory. The role of ITSN1 in the regulation of synaptic vesicle (SV) cycling has been extensively reviewed very 8. ITSN1 function in neurons recently (Pechstein et al., 2010b). The most comprehensive functional studies were performed on Drosophila larval NMJs. Loss-of-function Since ITSN1 was initially reported to be a major binding partner for Dap160 mutants exhibited a 10-fold increase in NMJ satellite bouton the neuronal GTPase dynamin 1, its high neuronal expression was formation probably due to altered signalling to the actin cytoskeleton established and confirmed by numerous studies. By immunofluores- via Nervous wreck (Nwk) and WASP (Koh et al., 2004; Marie et al., cence, ITSN1-specific staining was detected in the C. elegans nervous 2004). O'Connor-Giles et al. later confirmed this functional associa- system at all larval stages and in adult worms (Rose et al., 2007). tion. It was shown that Dap160 negatively regulates retrograde bone Dap160 is also expressed at high levels throughout Drosophila larval morphogenic protein (BMP) signalling pathway and Drosophila NMJ development in both central and peripheral neurons (Tomancak et al., growth through its direct association with Nwk (O'Connor-Giles et al., 2002; Marie et al., 2004). In situ hybridization revealed ITSN1 mRNA 2008). Partial loss-of-function Dap160 mutants display temperature- enrichment in developing nervous system of mouse embryos sensitive (ts) paralysis, whereas null mutants show ts defects in (Reymond et al., 2002). Further, Ma et al. reported the detailed endocytosis (Koh et al., 2004; Marie et al., 2004). Electron microscopy expression pattern of ITSN1 in the central nervous system of the adult revealed fewer vesicles, aberrant large vesicles, and accumulation of rat. The highest ITSN1 immunoreactivity was observed in layer III of endocytic intermediates at active and periactive zones in mutant the neocortex, hippocampus, globus pallidus, subthalamic nucleus, terminals, but evoked neurotransmission was normal. Thus, it was and substantia nigra (Ma et al., 2003). suggested that Dap160, like dynamin, is involved in synaptic vesicle L. Tsyba et al. / Gene 473 (2011) 67–75 73 retrieval at active and periactive zones. Finally, Dap160 mutants were sporadic AD (Cataldo et al., 2000). Similarly, early endosomes are shown to be lethal at midlarval stages, suggesting that Dap160 is an significantly enlarged in DS neurons and fibroblasts, as well as the essential gene (Marie et al., 2004). total number of endosomes of different sizes is increased (Cataldo et In contrast, Rose et al. demonstrated that the C. elegans ITSN gene is al., 2008). Surprisingly, DS fibroblasts possess a higher internalization nonessential for viability (Rose et al., 2007). In contrast to the Dap160 rate compared to normal cells (Cataldo et al., 2008); this contradicts mutant, no alterations in the levels of EHS-1 (epsin ortholog), AP180, previous observations indicating severe impairment of endocytosis or dynamin 1 were observed in ITSN-null worms. Further studies after overexpression of ITSN in cell lines and transgenic animals confirmed that C. elegans ITSN-null mutants are viable and display (Sengar et al., 1999; Pucharcos et al., 2000; Chang and Min, 2009). The grossly normal locomotion and development (Wang et al., 2008). DS brain is characterized by an altered shape, number, and density of However, motor neurons in these mutants show a dramatic increase synapses, that are thought to contribute to the pathological in large irregular vesicles and accumulate membrane-associated phenotype. Typically, spine density is decreased on the dendrites vesicles at putative endocytic hotspots. while presynaptic and postsynaptic elements are significantly Using lamprey giant synapse, Evergren et al. confirmed that ITSN1 enlarged (Belichenko et al., 2004). Similar defects were observed is essential for efficient SV recycling. They showed that ITSN and under conditions of overexpression of the ITSN1 SH3 domains and dynamin are redistributed after stimulation from the synaptic vesicle proline-rich region of Numb (Nishimura et al., 2006). cluster to the periactive zone where ITSN1 enhances dynamin- Using Drosophila as a model, Cheng and Min recently investigated mediated fission (Evergren et al., 2007). the effects of overexpression of three genes from human chromosome Numerous evidences of postsynaptic localization of ITSN1 were 21, ITSN/Dap160, DSCR1/nla and synaptojanin 1/synj. Overexpression also validated functionally. ITSN1 was shown to regulate AMPA-type of individual genes or of all three genes caused defects in synaptic glutamate receptors (GLR-1) trafficking in C. elegans interneurons terminal structure and synaptic activity (Chang and Min, 2009), but (Glodowski et al., 2007). Rat ITSN1-L was shown to bind the NMDA- all three genes were necessary to cause impaired vesicle recycling. type glutamate receptors NR1 and NR2B even in the presence of ionic Despite intense study of ITSN1 and its role in DS phenotype detergents, but not the AMPA-type glutamate receptor GluR1 formation, the precise mechanisms of ITSN1 overexpression resulting (Nishimura et al., 2006). However, the most thoroughly described in neuron dysfunction and other features of DS are still unknown. postsynaptic role of ITSN1-L lies in the regulation of dendritic spine Obviously, ITSN1 has a pleiotropic effect on DS phenotype formation. development. Spines are small actin-rich protrusions from dendrites Since ITSN1 is a scaffold protein, any changes in its expression could of neurons that form the postsynaptic component of most excitatory result in a wide spectrum of effects caused by unequal stoichiometry synapses in the brain. Spine growth and structural plasticity are of the components of multiprotein complexes. Thus, future investi- associated with synaptic plasticity in vitro and learning in vivo (Yang gation of ITSN1 function in the context of overexpression of human et al., 2009). It was demonstrated that ITSN1-L knock down alters chromosome 21 genes is very promising. dendritic spine development, but has little or no influence on SV Another neurodegenerative pathology with which ITSN1 was recycling (Thomas et al., 2009). Earlier studies provided evidence that shown to be associated is Huntington disease (HD). This genetic overexpression of ITSN1-L caused elongation of the spine neck, disorder arises from the expansion of a polyglutamine (polyQ) tract in whereas expression of the ITSN1 SH3 domains severely decreased the the huntingtin protein (Htt) resulting in aggregation of mutant Htt protrusion density and size of their heads, thus inhibiting spine into nuclear and/or cytosolic inclusions in neurons. ITSN1-S was formation (Irie and Yamaguchi, 2002; Nishimura et al., 2006). This shown to increase aggregate formation by mutant Htt through role of ITSN1-L appears to be linked to its GEF activity. Specifically, it activation of the JNK–MAPK pathway and to enhance polyQ-mediated was shown that the EphB2 receptor or Numb protein could physically neurotoxicity (Scappini et al., 2007). Moreover, overexpression of associate with ITSN1 and activate its GEF activity, which in turn ITSN1-S enhances aggregation of the androgen receptor, which activates the Cdc42 GTPase and spine morphogenesis (Irie and undergoes polyQ expansion in Kennedy's disease, suggesting a Yamaguchi, 2002; Nishimura et al., 2006). broader involvement of ITSN1 in neurodegenerative diseases through Recently, the generation of ITSN1 null mice was reported (Yu et al., destabilization of polyQ-containing proteins (Scappini et al., 2007). 2008). ITSN1 deficiency did not cause embryonic lethality. However, Given the role of ITSN in the activation of mitogenic pathways, its approximately every eighth homozygous null pup failed to thrive and involvement in cancer development was proposed. Indeed, it was often died early. Further analyses revealed significantly enlarged shown recently that a low level of ITSN2 expression is associated with endosomes in the brains of these pups compared with controls or poor prognosis of breast cancer patients after adjuvant chemotherapy their healthy littermates. The remaining homozygous null mice with cyclophosphamide, methotrexate and 5-fluorouracil (Specht et appeared to develop and reproduce normally and had no gross al., 2009). Moreover, the level of ITSN2 expression was considered to physiological abnormalities. However, the authors found that the rate be a predictive marker for breast cancer. Future studies are needed to of synaptic vesicle endocytosis in neurons of ITSN1 null mice was determine whether ITSN2 is involved in regulation of cell invasion and significantly reduced. Thus, disruption in ITSN1 expression causes a metastasis formation or if the ITSN2 gene is upregulated in tumors of disturbance in vesicle trafficking and endocytic function in the brain. patients with prolonged disease-free survival in parallel with some other currently unknown genes due to hyperactivation of the 9. ITSNs and diseases chromatin domain. Recently, new data of ITSN association with pathologies have Since the ITSN1 gene was mapped to chromosome 21 in the Down emerged. ITSN2 has been shown to interact with the K15 protein of syndrome (DS) critical region (Guipponi et al., 1998), its expression Kaposi's sarcoma-associated herpesvirus (KSHV) and regulate B-cell level and potential role in a specific Down syndrome phenotype were receptor internalization (Lim et al., 2007). This was the first finding of studied. Expression of the ITSN1 gene is upregulated in DS individuals hijacking of a protein of the ITSN family by a pathogen to manipulate (Pucharcos et al., 1999; Skrypkina et al., 2005) concurrently with cellular signalling. expression of synaptojanin 1 in DS brain (Arai et al., 2002) and decreasing of dynamin expression in the brain of DS and Alzheimer's 10. Concluding remarks disease (AD) patients (Greber et al., 1999). An association between the endocytic abnormalities and the pathological processes of DS and Numerous functional and genetic studies indicate that ITSN early AD was clearly stated (Keating et al., 2006). Enlarged early proteins are important scaffolds for protein assembly during endosomes are the earliest neuropathological alterations identified in endocytosis and synaptic transmission. In contrast to other scaffolds, 74 L. Tsyba et al. / Gene 473 (2011) 67–75 proteins of the ITSN family contain the catalytic DH domain that Arai, Y., et al., 2002. Excessive expression of synaptojanin in brains with Down syndrome. Brain Dev. 2, 67–72. allows assembly of endocytosis and exocytosis with dynamic Ballif, B., et al., 2004. Phosphoproteomic analysis of the developing mouse brain. Mol. regulation of the actin cytoskeleton. Recent advances in the Cell. Proteomics 11, 1093–1101. investigation of ITSN functions expanded its protein–protein interac- Belichenko, P., et al., 2004. Synaptic structural abnormalities in the Ts65Dn mouse model of Down syndrome. J. Comp. Neurol. 480, 281–298. tion network that engages ITSN proteins in many other processes Brill, L.M., et al., 2004. Robust phosphoproteomic profiling of tyrosine phosphorylation including cell survival and apoptosis, control of cell polarity (Chabu sites from human T cells using immobilized metal affinity chromatography and and Doe, 2008) and dendritic spine development. tandem mass spectrometry. Anal. Chem. 76, 2763–2772. Although significant progress has been made toward understand- Cataldo, A., et al., 2000. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects ing ITSN functions, the precise role of ITSN in dynamin regulation and of APOE genotype and presenilin mutations. Am. J. Pathol. 157, 277–286. control of CCP formation and maturation remains unclear. Another Cataldo, A., et al., 2008. Down syndrome fibroblast model of Alzheimer-related major unresolved question is stoichiometry of ITSN protein com- endosome pathology: accelerated endocytosis promotes late endocytic defects. Am. J. Pathol. 173, 370–384. plexes, competition between ITSN interactors and mechanisms of Chabu, C., Doe, C.Q., 2008. Dap160/intersectin binds and activates aPKC to regulate cell regulation of such complexes formation. The role of posttranslational polarity and cell cycle progression. Development 135, 2739–2746. modifications in regulation of the ITSN family of proteins is also Chang, K., Min, K., 2009. Upregulation of three Drosophila homologs of human chromosome 21 genes alters synaptic function: implications for Down syndrome. unresolved. Multiple serine/threonine phosphorylation sites for ITSN1 Proc. Natl Acad. Sci. USA 106, 17117–17122. were found in high throughput experiments, but none of them was Das, M., et al., 2007. Regulation of neuron survival through an intersectin investigated in detail (Tweedie-Cullen et al., 2009; Ballif et al., 2004; phosphoinositide 30-kinase C2b–AKT pathway. Mol. Cell. Biol. 27, 7906–7917. Dergai, O., et al., 2010. Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin- PHOSIDA database http://141.61.102.18/phosida/index.aspx). It coated pits. Biochem. Biophys. Res. Commun. 402, 408–413. should also be noted that several studies identified phosphorylated Doherty, G., McMahon, H., 2009. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, tyrosines in ITSN2, but not in ITSN1 (Zheng et al., 2005; Goss et al., 857–902. Evergren, E., et al., 2007. Intersectin is a negative regulator of dynamin recruitment to 2006; Rikova et al., 2007; Rush et al., 2005; Brill et al., 2004). the synaptic endocytic zone in the central synapse. J. Neurosci. 27, 379–390. Moreover, ITSN1, in contrast to ITSN2, practically does not contain Fernández-Chacón, R., et al., 2000. SCAMP1 function in endocytosis. J. Biol. Chem. 275, tyrosine residues in unstructured or surface exposed regions. 12752–12756. Whether these features are connected with functional divergency Galletta, B., Cooper, J., 2009. Actin and endocytosis: mechanisms and phylogeny. Curr. Opin. Cell Biol. 1, 20–27. between ITSN2 and ITSN1 remains a challenge for future research. Glodowski, D., et al., 2007. RAB-10 regulates glutamate receptor recycling in a The role of alternative splicing that diversifies module composi- cholesterol-dependent endocytosis pathway. Mol. Biol. Cell 11, 4387–4396. tions of the ITSN protein family and influences adaptor properties of Goss, V., et al., 2006. A common phosphotyrosine signature for the Bcr-Abl kinase. Blood 107, 4888–4897. ITSNs in a tissue- and developmentally-regulated manner should be Gray, N., et al., 2003. Dynamin 3 is a component of the postsynapse, where it interacts emphasized. Recently, we have shown that alternative splicing of with mGluR5 and Homer. Curr. Biol. 13, 510–515. microexons provides a mechanism for tissue-specific control of Greber, S., et al., 1999. Decreased levels of synaptosomal associated protein 25 in the – – brain of patients with Down syndrome and Alzheimer's disease. Electrophoresis 4 protein protein interactions. Insertion of a microexon in alternatively 5, 928–934. spliced ITSN1 leads to regulation of the SH3A domain specificity due Guipponi, M., et al., 1998. Two isoforms of a human intersectin (ITSN) protein are to the shifting of negatively charged amino acids towards the produced by brain-specific alternative splicing in a stop codon. Genomics 53, 369–376. interaction interface (Dergai et al., 2010). Multiple alternative spliced He, G., et al., 2007. Intersectin links WNK kinases to endocytosis of ROMK1. J. Clin. isoforms of ITSNs, some with already determined specificity, have Invest. 117, 1078–1087. been discovered. However, the individual ITSN isoform functions and Henne, W., et al., 2010. FCHo proteins are nucleators of clathrin-mediated endocytosis. fi Science 328, 1281–1284. the composition of the ITSN isoform speci c protein complexes Hussain, N., et al., 1999. Splice variants of intersectin are components of the endocytic remain to be elucidated. machinery in neurons and nonneuronal cells. J. Biol. Chem. 274, 15671–15677. Proteins of the ITSN family could be considered as a part of Hussain, N., et al., 2001. Endocytic protein intersectin-l regulates actin assembly via – molecular interfaces between different cellular processes such as Cdc42 and N-WASP. Nat. Cell Biol. 3, 927 932. Irie, F., Yamaguchi, Y., 2002. EphB receptors regulate dendritic spine development via endocytosis and signalling, vesicle traffic and signalling, endocytosis intersectin, Cdc42 and N-WASP. Nat. Neurosci. 11, 1117–1118. and cytoskeleton rearrangements, and mitogenic signalling and cell Jenna, S., et al., 2002. The activity of GTFase-activating protein CdGAP is regulated by – survival. Understanding the large molecular interfaces comprising endocytic protein intersectin. J. Biol. Chem. 277, 6366 6373. – – Keating, D., Chen, C., Pritchard, M., 2006. Alzheimer's disease and endocytic multiple adaptor proteins (e.g. ITSN Eps15 Ruk/CIN85) could shed dysfunction: clues from the Down syndrome-related proteins, DSCR1 and ITSN1. light on the mechanisms of cross-talks and integration of events in Ageing Res. Rev. 4, 388–401. cellular networks. These interfaces provide finely tuned interactions Kelly, L., Phillips, M., 2005. Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin). Biochem. J. of different cellular processes. 388, 195–204. Although ITSN proteins have been implicated in different diseases, Khanna, R., Li, Q., Stanley, E., 2006. ‘Fractional recovery’ analysis of a presynaptic no therapeutic approaches were developed to alter defects in their synaptotagmin 1-anchored endocytic protein complex. PLoS ONE 1, e67. Klein, I., et al., 2009. Intersectin-2L regulates caveola endocytosis secondary to Cdc42- functions. Hence, future efforts may have to be directed toward the mediated actin polymerization. J. Biol. Chem. 284, 25953–25961. development of such approaches to treat pathological conditions Koh, T., Verstreken, P., Bellen, H., 2004. 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