Intersectin multidomain adaptor proteins: Regulation of functional diversity

Gene 473 (2011) 67–75

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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

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

a r t i c l e

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Article history: Accepted 30 November 2010 Available online 9 December 2010 Received by A.J. van Wijnen Keywords: Intersectin family Adaptor/scaffold proteins Alternative splicing Endocytosis

a b s t r a c t Adaptor/scaffold proteins serve as platforms for the assembly of multiprotein complexes and regulate the efficiency and specificity of signalling cascades. Intersectins (ITSNs) are an evolutionarily conserved adaptor 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 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, as well as the potential relevance of ITSNs for neurodegenerative diseases and cancer. The diversity of mechanisms in the regulation of ITSN expression and specificity in different cells emphasizes the important role of ITSN proteins in vesicle trafficking and signalling. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Adaptor/scaffold proteins have emerged as regulators of many cellular processes, including proliferation, differentiation, cell-cycle control, cell survival and migration (Pawson and Scott, 1997; Szymkiewicz et al., 2004; Zeke et al., 2009). Classical scaffolds usually do not possess any type of enzymatic activity and are characterized by modular architecture with the presence of multiple protein/lipid binding domains and sites for inducible posttranslational modifications. Scaffold molecules selectively control the spatial and temporal assembly of multiprotein complexes. Specifically, they determine the formation and localization of protein complexes and may both facilitate or inhibit signal transduction depending on their concentration in certain compartments, regulating the strength, specificity and duration of signal propagation. Intersectins (ITSNs) are adaptor/scaffold proteins with a unique multidomain structure. By binding to numerous proteins, they assemble multimeric complexes implicated in clathrin- and caveolin-mediated endocytosis, rearrangements of the actin cytoskeleton, cell signalling and survival (Sengar et al., 1999; Predescu et al., 2003; Hussain et al., 2001; Mohney et al., 2003; Das et al., 2007; Predescu et al., 2007). The roles of ITSN proteins as adaptors have been studied intensively in different cell types and organisms. Recently, ITSNs were Abbreviations: ITSNs, intersectins; ITSN1-S, short isoform of ITSN1; ITSN1-L, long isoform of ITSN1; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; GEF, guanine nucleotide exchange factor; CME, clathrin-mediated endocytosis; Dap160, Drosophila homolog of human ITSN; NMJ, neuromuscular junction; RTK, receptor tyrosine kinases; EGFR, epidermal growth factor receptor; CCR, coiled-coil region; SV, synaptic vesicle; DS, Down syndrome; AD, Alzheimer's disease. ⁎ Corresponding author. Tel.: +380 44 526 9618; fax: +380 44 526 0759. E-mail address: [email protected] (A. Rynditch). 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.11.016

shown to be essential components for initiation of clathrin-coated pit (CCP) formation (Henne et al., 2010). In this review, we highlight recent advances in the study of the functions of the ITSN family in neuronal and non-neuronal cells and provide a picture of experimentally verified ITSN-specific interactions. We also describe the impact of alternative processing on the generation of diversity in the ITSN family and regulation of ITSN genes expression. 2. Structure of ITSN family proteins ITSNs are evolutionarily conserved proteins present in diverse metazoan organisms ranging from nematodes to mammals. There are two ITSN genes in humans, ITSN1 and ITSN2 located on chromosomes 21 (q22.1–q22.2) and 2 (pter–p25.1), respectively (Guipponi et al., 1998; Pucharcos et al., 2001). ITSN1 and ITSN2 share significant sequence identity and a similar domain structure (Pucharcos et al., 2000). Moreover, they both have short and long isoforms produced by alternative splicing (Guipponi et al., 1998; Pucharcos et al., 2001). The short isoform (ITSN-S) consists of two Eps15 homology domains (EH1 and EH2), a coiled-coil region (CCR) and five Src homology 3 domains (SH3A–E) (Fig. 1). The EH domains which bind to Asn-Pro-Phe motifs have been identified in several proteins implicated in endocytosis and vesicle transport. The SH3 domains bind to proline-rich sequences and are commonly found in proteins implicated in cell signalling pathways, cytoskeletal organization and membrane traffic. They possess the most diverse specificity among interaction domains (Li, 2005). The long isoform (ITSN-L) contains three additional C-terminal domains, a Dbl homology domain (DH), a Pleckstrin homology domain (PH) and a C2 domain (Guipponi et al., 1998; Sengar et al., 1999). The DH and PH domains usually form a tandem in the Dbl

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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 is sufficient to catalyse exchange of bound GDP for GTP and therefore activate Rho GTPases. The DH domain of ITSN specifically activates the Cdc42 GTPase (Hussain et al., 2001). The PH domain of ITSN binds phosphoinositides (Snyder et al., 2001). C2 domains are usually involved in Ca2+-dependent and Ca2+-independent phospholipid binding (Rizo and Südhof, 1998). Multiple domains of ITSNs mediate their association with a wide range of proteins. So far, we know of more than 30 proteins that interact with ITSNs. However, the list of ITSN-binding proteins is not believed to be complete. The vast majority of the publications focuses on ITSN1. Despite limited information on ITSN2, it is apparent that ITSN2 plays an important role in clathrin- and caveola-mediated endocytosis (Pucharcos et al., 2000; Klein et al., 2009). Proteins with similarity to the ITSN-S domain structure were found in mammals, frogs, flies and nematodes (Sengar et al., 1999; Okamoto et al., 1999; Yamabhai et al., 1998; Roos and Kelly, 1998; Rose et al., 2007), while ITSN-L isoforms have been identified only in vertebrates. The ITSN ortholog in Drosophila consists of two EH domains, a CCR and four SH3 domains (Roos and Kelly, 1998), while the Caenorhabditis elegans ITSN contains five SH3 domains (Rose et al., 2007) (Fig. 1). Proteins homologous to ITSN were not found in S. cerevisiae. The organization of the ITSN1 and ITSN2 genes of vertebrates is very similar (Pucharcos et al., 1999, 2001). They comprise more than 40 exons, while orthologous ITSN genes of nematodes (C. elegans) and arthropods (D. melanogaster) contain 8 and 11 exons, respectively. Moreover, in vertebrates most of the exon boundaries are conserved between ITSN1 and ITSN2, and the mechanisms of generation of the two major spliced variants are the same for these paralogous genes (Pucharcos et al., 2001). This suggests that, the long isoform produced by alternative splicing of exon 30 appeared earlier in evolution before gene duplication. Other known alternative splicing events are not conserved between ITSN1 and ITSN2 (Pucharcos et al., 2001; Tsyba et al., 2004). An alternative promoter was identified in intron 5 of human ITSN1 gene. This promoter generates the transcripts encoding ITSN1-S isoform without the EH1 domain. However, it is not known, whether these transcripts are translated into proteins (Kropyvko et al., 2010b). Recently, the multi-modular protein, Cin1, whose domain structure is similar to that of ITSN, was found in the pathogenic fungus Cryptococcus neoformans (Shen et al., 2010). Cin1 contains an Nterminal EH domain, a central CCR, a WASP-homology domain 2 (WH2), two SH3 domains and C-terminal DH–PH domains. Interestingly, alternative splicing resulted in two Cin1 isoforms, long and short. The latter is similar to ITSN-S. 3. Alternative splicing in the regulation of ITSN genes expression Alternative splicing plays one of the major roles in the regulation of ITSN genes expression and functions. Two major ITSN isoforms are produced by alternative splicing of exon 30 that provides the termination codon for the short isoform (Guipponi et al., 1998;

Pucharcos et al., 2001; Sengar et al., 1999). Numerous additional splicing events affecting ITSN1 and ITSN2 transcripts will be briefly reviewed in this section. Four evolutionarily conserved in-frame alternative splicing events affecting ITSN1 mRNAs were found in mice and humans (Okamoto et al., 1999; Pucharcos et al., 2001; Tsyba et al., 2004) (Fig. 2A). These events include: (1) the use of an alternative 3′-splice site internal to exon 6 that results in truncation of exon 6 and deletion of 37 amino acids between the EH1 and EH2 domains, (2) splicing of the neuronspecific exon 20 that encodes 5 amino acids in the SH3A domain, (3) deletion of exons 25 and 26 that encode the SH3C domain, (4) skipping of exon 35 encoding 31 amino acids of the DH domain and 25 residues of the DH–PH interdomain spacing. Inclusion of exon 22a results in the generation of the shortest ITSN1 transcript that encodes a protein containing two EH domains, the CCR, the SH3A domain and the exon 22a-specific C-terminal sequence (Skrypkina et al., 2005). Except for splicing of exon 30, three additional alternative splicing events that do not introduce premature termination codons were reported for ITSN2 (Fig. 2B). Truncation of exon 19 and brain-specific inclusion of exon 17 change the structure of the CCR of ITSN2, while skipping of exons 27 and 28 results in deletion of the SH3D domain (exon numbering according to NM_006277) (Pucharcos et al., 2001; Seifert et al., 2007). Many splicing events cause frameshifts in ITSNs mRNA and introduce premature termination codons that lie more than 50 nucleotides upstream of an exon–exon junction (Tsyba et al., 2004; Kropyvko et al., 2010a; Pucharcos et al., 2001; Seifert et al., 2007). Such mRNAs are expected to be degraded via the nonsense-mediated mRNA decay (NMD) pathway (McGlincy and Smith, 2008). The relatively high abundance of mRNAs with premature termination codons suggests the participation of alternative splicing and NMD in regulation of the expression level of ITSNs. ITSN1 and ITSN2 are widely expressed in mammal tissues (Guipponi et al., 1998; Sengar et al., 1999; Hussain et al., 1999; Okamoto et al., 1999; Pucharcos et al., 2000; Tsyba et al., 2004). However, the tissue distribution of the ITSN proteins varies considerably. The expression of some isoforms is tissue-dependent and regulated during development. In contrast to the widely expressed ITSN2-L isoform, ITSN1-L is expressed predominantly in neurons (Pucharcos et al., 2000, 2001; Guipponi et al., 1998). However, low levels of ITSN1-L expression were detected in adult kidney, liver and placenta (Pucharcos et al., 2001), as well as in fetal liver, lung, kidney and muscle (Tsyba et al., 2004; Kropyvko et al., 2010a). Western blot 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, mRNAs of these isoforms were detected in mouse thalamic neurons by single-cell RT-PCR (Kropyvko et al., 2010a). ITSN1 transcripts containing exon 20 are neuron-specific and their expression is developmentally regulated (Tsyba et al., 2004; Tsyba et al., 2008). The frequency of the + exon 20 variant of ITSN1 increases during fetal brain development whereas the level of the transcript

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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 isoforms with and without exons 25 and 26 (the SH3C domain) varies in fetal and adult brain (Pucharcos et al., 2001; Tsyba et al., 2004). In other tissues about 20% of all ITSN1 transcripts lack exon 25 and 26. Skipping of exon 35 occurs in approximately 10% of ITSN1-L isoforms, but the ratio of ITSN1-L transcripts with and without exon 35 differs in brain and other tissues. Approximately 3% of ITSN1 transcripts contain a truncated exon 6 (Kropyvko et al., 2010a). Taking into consideration that alternatively spliced exons could be present in ITSN transcripts in different combinations, 16 variants of ITSN1-L, 8 variants of the short ITSN1 isoforms and 4 variants of the ITSN1-22a isoforms could be generated in human tissues. Indeed, we recently cloned all eight variants of ITSN1-S (Kropyvko et al., 2010a). Although the function of most alternatively spliced isoforms of ITSNs are poorly understood, it is evident that the generation of proteins with differences in functional domains or with a different domain composition could change the binding properties of ITSN proteins and may play a role in selecting specific interactions. Some isoforms of ITSNs have a brain-specific function. The brain-enriched ITSN1-L isoform that has GEF activity toward Cdc42 GTPase was shown to be involved in dendritic spine morphogenesis (Nishimura et al., 2006; Thomas et al., 2009). Our previous results demonstrated that neuron-specific inclusion of exon 20 changes the binding properties of the SH3A domain, shifting its binding specificity from the signalling proteins Sos1 and Cbl to the cytoskeleton regulatory protein CdGAP and the endocytic proteins dynamin 1 and synaptojanin 1 (Tsyba et al., 2008). Skipping of the SH3C domain of ITSN1 or the SH3D domain of ITSN2 could change the interaction interfaces for certain ITSN protein partners or even abolish the interaction. For example, recently identified ITSN1-interacting protein Reps1 binds only to the SH3C domain of ITSN1 (Dergai et al., 2010) and therefore could not interact with ITSN1ΔSH3C isoform. 4. Subcellular localization of ITSNs Analysis of the subcellular distribution of endogenous and overexpressed ITSN1 in mammalian epithelial cells and rat hippocampal

neurons revealed its localization throughout the cytoplasm with accumulation in the perinuclear region in Golgi-like structure (Hussain et al., 1999; Mohney et al., 2003; Predescu et al., 2003; Ma et al., 2003; Pucharcos et al., 2000). ITSN1 was also observed in the cell periphery. Moreover, epidermal growth factor stimulation promoted relocalization of the ITSN–c-Cbl complex close to the plasma membrane (Martin et al., 2006). At the plasma membrane ITSN1 is localized predominantly in CCPs (Hussain et al., 1999). Electron microscopy immunogold labeling studies on endothelial cells indicated that ITSN1-S is associated with caveolae and clathrin-coated vesicles (CCVs), as well as with Golgi-derived vesicles and cytoskeletal elements (Predescu et al., 2003). Transfection studies with ITSN1 deletion mutants suggest that the EH domains and CCR are responsible for the localization of ITSNs in cells (Hussain et al., 1999; Scappini et al., 2007; Pucharcos et al., 2000). Moreover, it was shown that ITSN1 and Eps15 function cooperatively to regulate subcellular localization of the ITSN1–Eps15 complex (Sengar et al., 1999). ITSN2-S showed a subcellular distribution similar to that of ITSN1-S (Pucharcos et al., 2000; Klein et al., 2009). The subcellular distribution of ITSN1 in neurons will be discussed later in this paper. Thus, the association of ITSNs with different membranous organelles, caveolae, Golgi complex, CCPs and CCVs, suggests its involvement in different pathways of the cell membrane-trafficking system (Predescu et al., 2003). 5. ITSN proteins are essential components of endo- and exocytosis Proteins of the ITSN family are clearly linked to endocytosis. Endocytosis is a complex and tightly controlled process that allows the delivery of proteins from the plasma membrane into the cell and is essential for maintaining cellular homoeostasis as well as for the interaction between the cell and its environment (Doherty and McMahon, 2009; Szymkiewicz et al., 2004). ITSNs have a modular structure composed of the EH and SH3 domains that are typical for endocytic proteins. The subcellular localization of ITSNs also supports the involvement of these proteins in internalization events.

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ITSN1 was shown to interact with a plethora of endocytic proteins (Fig. 3). The N-terminal tandem of the EH domains interacts with the endocytic accessory protein epsin (Yamabhai et al., 1998), which binds directly to inositol lipids, Eps15 and the α subunit of AP2, can help drive membrane invagination as well as link ubiquitinated membrane proteins to clathrin. The EH domains of ITSN1 also interact with stonin 2, an endocytic sorting adaptor for synaptic vesicle cargo recognition (Martina et al., 2001; Kelly and Phillips, 2005) and the secretory carrier membrane protein SCAMP1 (Fernández-Chacón et al., 2000). The CCR of ITSN1 binds to synaptosome-associated proteins of 23 kDa and 25 kDa (SNAP-23 and SNAP-25) (Okamoto et al., 1999) and forms heterodimers with the scaffolding adaptor Eps15 (Sengar et al., 1999; Koh et al., 2007) that binds ubiquitinated cargo, AP2, epsin and dynamin, and clusters AP2 appendages through its long Cterminal tail (Doherty and McMahon, 2009). The SH3 domains of ITSN1 bind the large regulatory GTPase dynamin involved in vesicle fission, a phosphoinositide phosphatase synaptojanin 1, a synaptic protein synapsin, a membrane-deforming protein SGIP1, and the AP2 binding protein connecdenn (Roos and Kelly, 1998; Yamabhai et al., 1998; Okamoto et al., 1999; Evergren et al., 2007; Dergai et al., 2010; Allaire et al., 2006). Recently the α- and β-appendage domains of AP2 were shown to directly interact with the linker between the SH3A and SH3B domains (Pechstein et al., 2010a). Clathrin could be co-immunoprecipitated with anti-intersectin antibodies from rat brain lysates (Hussain et al., 1999) whereas direct interaction was not shown. It was reported that the SH3 domains of ITSN1 also bind to the proline-rich region of inositol 5-phosphatase SHIP2, a negative regulator of CCP growth and insulin-mediated signalling. Interaction between SHIP2 and ITSN1 leads to the recruitment of SHIP2 to CCPs (Xie et al., 2008; Nakatsu et al., 2010). More direct evidence for the role of ITSN in endocytosis was provided by data showing that overexpression or knockdown of ITSN inhibits clathrin-mediated endocytosis (CME) (Sengar et al., 1999; Pucharcos et al., 2000; Martin et al., 2006; Thomas et al., 2009). For example, overexpression of ITSN1 blocked transferrin receptor internalization in Cos-1 cells. However, this block could be rescued by overexpression of dynamin suggesting an important role of ITSN1 in dynamin recruitment during CME (Sengar et al., 1999). Silencing

ITSN1 by RNA interference attenuates internalization of epidermal growth factor receptor (EGFR) in HEK293T cells and reduces the rate of transferrin endocytosis in neurons (Martin et al., 2006; Thomas et al., 2009). Similarly, siRNA knockdown of ITSN1 decreases the rate of internalization of the renal outer medullar potassium 1 channel (ROMK1) by a With-no-lysine (WNK) kinase-dependent mechanism (He et al., 2007). It was shown that WNK1 and WNK4 kinases interact with the SH3 domains of ITSN1 and that the interactions are crucial for stimulation of endocytosis of ROMK1 by WNKs (He et al., 2007). On the other hand, overexpression of ITSN2-L enhanced the rate of T cell antigen receptor (TCR) internalization, but this effect was dependent on the GEF activity of the DH domain of the long isoform (McGavin et al., 2001). Similar effects of ITSN overexpression and silencing could be the result of disruption of the formation of higher order protein complexes between ITSN and its binding partners. Two laboratories reported the generation of loss-of-function mutations that eliminate Dap160 (Drosophila homolog of human ITSN). The studies demonstrated that the levels of dynamin, synaptojanin, endophilin and AP180 are severely reduced in Dap160 mutant synapses, suggesting the role of Dap160 in recruitment of endocytic proteins to the presynaptic terminals. Reduction of the number of synaptic vesicles, aberrant large vesicles, defects in vesicle recycling and fission such as accumulation of collared pits and Ω-like structures were observed in Dap160 mutants (Koh et al., 2004; Marie et al., 2004). Similar defects in the development of neuromuscular junction (NMJ) synapses and vesicle recycling were detected in Drosophila Eps15-null mutants (Koh et al., 2007). Loss of Eps15 caused a severe reduction in dynamin and Dap160 protein levels at NMJs. Eps15 and Dap160 double mutants had a phenotype similar to that of the single Eps15 or Dap160 mutants, suggesting that Eps15 and Dap160 act at the same step during endocytosis (Koh et al., 2007). A reduced number of exocytosis events in chromaffin cells and a slowing of endocytosis in neurons were also detected in ITSN1 null mice (Yu et al., 2008). ITSNs are also involved in caveolae endocytosis, an important step in mediating transcytosis of proteins in endothelial cells (Predescu et al., 2003; Klein et al., 2009). Overexpression of ITSN1-S inhibited 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; 13 Lim 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; 20 Nikolaienko 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.

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formation of large clusters and “grape-like” structures. On the contrary, siRNA-mediated ITSN2-L knockdown resulted in significant increase of caveolae-mediated uptake that was accompanied by decreased Cdc42 activation as well as by changes in cell shape and Factin distribution probably due to decrease of GEF activity of the DH and PH domains of ITSN2-L. Indeed, overexpression of the DH–PH domains causes a decrease of caveolae-mediated uptake (Klein et al., 2009). ITSN is also a functional component of the exocytotic machinery. As mentioned previously ITSN1 binds to SNAP-25, a protein belonging to the Q-SNARE family and involved in exocytosis of synaptic vesicles (Okamoto et al., 1999). Moreover, GEF activity of ITSN1-L is important for actin cytoskeletal rearrangements required for exocytosis in neuroendocrine cells (Malacombe et al., 2006). Reducing endogenous ITSN1 levels by siRNA prevented secretagogue-induced activation of Cdc42 and resulted in inhibition of growth hormone secretion. Therefore, ITSN1-L was proposed to be an adaptor that coordinates exo–endocytotic membrane trafficking in secretory cells. Despite involvement of ITSN proteins in exo- and endocytosis is well established in vitro and in vivo, the precise mechanisms of its functioning remain unclear. ITSNs are considered as adaptors that organize multiprotein complexes at membrane compartments. ITSN1 was shown to function as negative regulator of dynamin 1 recruitment in synaptic endocytic zones (Evergren et al., 2007). Deficiency of Dap160 causes temperature-sensitive paralytic phenotype and endocytic abnormalities similar to dynamin (shibire) mutants (Koh et al., 2004). Finally, analysis of loss-of-function mutations in the Dap160 gene demonstrated that Dap160 is required for the proper localization of dynamin (Koh et al., 2004; Marie et al., 2004). Taken together, these data suggest a role of ITSN in dynamin complex assembly and functioning. The unique role of the SH3A domain of ITSN1 in the early stages of CME (Simpson et al., 1999) should be stressed. In contrast to other SH3 domains tested that selectively inhibit late events of CME involving membrane fission, the SH3A domain of ITSN1 inhibits intermediate events leading to the formation of constricted coated pits. Interestingly, using quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), ITSN2 was shown to be important for maturation of CCPs at the later stages of CME together with epsin and Eps15 (Mettlen et al., 2009). In view of controlling CCP maturation, another recently established fact attracts attention: interaction of ITSN1 with AP2 inhibits binding of synaptojanin 1 to ITSN1 (Pechstein et al., 2010a). This could serve as a specific checkpoint controlling CCP maturation. Recently, Henne et al. provided evidence that a family of membrane-sculpting proteins FCHo1/2 is responsible for marking the sites of CCV formation and act as CCP nucleators (Henne et al., 2010). FCHo1/2 proteins bind specifically to the plasma membrane and recruit Eps15 and ITSNs, which in turn engage the adaptor complex AP2. ITSNs, Eps15 and Eps15R were shown to regulate FCHo clustering. Quadruple knockdown of ITSN1, ITSN2, Eps15 and Eps15R abolished CCPs due to the inability of FCHo to initiate CCP formation, suggesting that these proteins constitute an early module for nascent CCP assembly (Henne et al., 2010). 6. ITSN proteins in actin cytoskeleton rearrangements It is noteworthy that endocytic events are tightly connected to actin cytoskeleton rearrangements and cargo income. Both members of ITSN family were shown to participate in actin cytoskeleton rearrangements by specific activation of the small GTPase Cdc42 and direct interaction with Cdc42 effectors — the Wiskott–Aldrich syndrome proteins, WASP and N-WASP (Hussain et al., 2001; McGavin et al., 2001). GEF activity was shown only for ITSN-L isoforms possessing a conserved DH domain catalysing the release of

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GDP from Rho GTPases and their subsequent activation by GTP binding (Rossman et al., 2005). The DH domains of ITSN1 and ITSN2 display high sequence homology (Pucharcos et al., 2000). Both proteins are highly specific and promote exchange on Cdc42, but not on Rac1 or RhoA (Hussain et al., 2001; Klein et al., 2009). Overexpression of ITSN1-L causes actin rearrangements specific for Cdc42 activation and stimulates filopodia formation in cultured fibroblasts (Hussain et al., 2001). In the structure of most GEFs the DH domain is followed by the PH domain that is responsible for phosphoinositide binding and is considered to positively or negatively modulate the efficiency of GDP/GTP exchange (Pruitt et al., 2003). In vitro studies revealed that exchange activity of the ITSN1 DH domain alone is reduced relative to that of the DH–PH domain (Zamanian and Kelly, 2003; Hussain et al., 2001; Ahmad and Lim, 2010). At the same time, Pruitt et al. reported that the presence of the PH domain does not alter GEF exchange activity of the ITSN1 DH domain in vitro but enhances it in vivo (Pruitt et al., 2003). Interactions of the PH domain with phosphoinositides did not alter the ability of the DH domain of ITSN1 to activate Cdc42 in vitro (Snyder et al., 2001). Moreover, the crystal structure of the ITSN1–Cdc42 complex shows no direct interactions between the PH domain and bound GTPase (Snyder et al., 2002). This contrasts with the role of the PH domain in Dbs, which forms contacts with the bound Cdc42 (Rossman et al., 2002). Intact ITSN1-L shows lower Cdc42 exchange activity compared to the isolated DH domain. Zamanian et al. demonstrated the existence of intramolecular interaction between the ITSN1-L SH3 region and the DH domain that inhibits its exchange activity by reducing ITSN1-L binding to Cdc42 (Zamanian and Kelly, 2003). In line with this is the observation that binding of the N-WASP proline-rich region to the SH3 domains of ITSN1-L enhances the ability of the DH domain to interact with GDP-bound Cdc42 and to catalyse its conversion to GTPbound Cdc42 (Hussain et al., 2001). Similarly, interaction of the ITSN1 SH3 domains with Numb, a protein implicated in cortical neurogenesis during nervous system development, enhances the GEF activity of ITSN1-L toward Cdc42 in vivo (Nishimura et al., 2006). Very recently, the molecular mechanism of ITSN1-L autoinhibition was reported. Ahmad et al. showed that the GEF activity of ITSN1-L is controlled by interaction of the SH3E domain with the C-terminal half of the DH domain. The SH3E domain is both necessary and sufficient for repression of the DH domain (Ahmad and Lim, 2010). Another group of proteins possesses an effect opposite to that of GEF: these proteins enhance GTPase activity leading to rapid conversion to the inactive GDP-bound form. ITSN1 forms a complex with CdGAP, a GTPase-activating protein with activity towards Cdc42 and Rac1 (Jenna et al., 2002). In PDGF-stimulated fibroblasts, ITSN1 is colocalized with CdGAP and inhibits its GAP activity towards Rac1. These investigations expand the role of proteins of the ITSN family and suggest a function in Rac1 regulation. The regulated assembly of actin filament networks is a crucial part of endocytosis (Galletta and Cooper, 2009). Actin assembly on endocytic vesicles via N-WASP was reported to be a mechanism of vesicle protrusion (Taunton et al., 2000). Regarding its domain composition and interactions, the ITSN was considered to play a role in coordinating endocytosis and actin cytoskeletal rearrangements. Indeed, McGavin et al. demonstrated that ITSN2-L is important for recruiting Cdc42 and its effector WASP from perinuclear actin bundles to endocytic vesicles in T cells during T cell antigen receptor (TCR) endocytosis. Overexpression of ITSN2-L induces an increase of TCR internalization, while both the DH domain-deleted form of ITSN2-L and latrunculin treatment severely reduce induction of TCR endocytosis (McGavin et al., 2001). Recently, interaction of cryptococcal proteins Cin1/ITSN, Cdc42 and Wsp1/WASP was reported in the pathogenic fungus C. neoformans, arguing for a conserved role in the regulation of the actin cytoskeleton (Shen et al., 2010).

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7. ITSNs in RTK signalling Activation of receptor tyrosine kinases (RTKs) by growth factors initiates intracellular signalling pathways that control cell proliferation, differentiation, survival, metabolism and migration (Lemmon and Schlessinger, 2010). Several lines of evidence suggest that endocytosis is a critical component in RTK signalling. Clathrinmediated endocytosis participates in downregulation of activated RTKs via receptor internalization into endosomes with subsequent targeting for lysosomal degradation or for recycling (Sorkin and Goh, 2009). However, endocytosis has also emerged as an essential step in the successful activation of RTK-dependent signalling pathways, in particular the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK/MAPK) pathway (O'Bryan et al., 2001; McPherson et al., 2001). Several experimental data suggest that ITSN1 provides a link between endocytosis and signal transduction. ITSN1, through its SH3 domains, forms complexes with Sos1, a guanine nucleotide exchange factor for Ras, and stimulates Ras activation (Tong et al., 2000b; Mohney et al., 2003). Furthermore, ITSN1 interacts with the E3ubiquitin ligase Cbl and enhances c-Cbl-mediated EGFR ubiquitylation and degradation (Martin et al., 2006). Overexpression of the SH3 domains of ITSN1 inhibits EGF-induced activation of Ras independently of the ability of these domains to block endocytosis of the EGF receptor (Tong et al., 2000a). Silencing ITSN1 by RNA interference attenuated EGFR internalization and activation of the ERK/MAPK pathway (Martin et al., 2006). Our recent results demonstrate that ITSN1 forms a stable complex with Ruk/CIN85, a scaffolding protein that regulates recruiting the Cbl–EGFR complex to endosomes for downregulation of RTK, as well as cytoskeletal rearrangements and apoptosis (Nikolaienko et al., 2009a). It should be noted that interactions of the SH3 domains of the ITSN1 with Cbl and Ruk/CIN85 are not regulated by EGF stimulations (Martin et al., 2006; Nikolaienko et al., 2009a,b). Therefore, ITSN1 could be constitutively associated with Cbl as well as with Ruk/CIN85. There is evidence that expression of either full-length ITSN1 or the isolated EH domain region stimulate activation of the transcription factor Elk-1 independently of the MAPK pathway (Adams et al., 2000). It was also shown that overexpression of ITSN1 leads to oncogenic transformation of rodent fibroblasts (Adams et al., 2000). Interestingly, siRNA knockdown of ITSN1-S reduced MEK and ERK1/2 phosphorylation and activated the mitochondrial pathway of apoptosis in endothelial cells (Predescu et al., 2007). ITSN1 was also shown to regulate the survival of neuronal cells through the activation of a PI3K–AKT pathway by interaction with PI3K–C2β. Decreasing ITSN expression by shRNA inhibited the PI3K–C2β–AKT signalling pathway and dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons (Das et al., 2007). 8. ITSN1 function in neurons Since ITSN1 was initially reported to be a major binding partner for the neuronal GTPase dynamin 1, its high neuronal expression was established and confirmed by numerous studies. By immunofluorescence, ITSN1-specific staining was detected in the C. elegans nervous system at all larval stages and in adult worms (Rose et al., 2007). Dap160 is also expressed at high levels throughout Drosophila larval development in both central and peripheral neurons (Tomancak et al., 2002; Marie et al., 2004). In situ hybridization revealed ITSN1 mRNA enrichment in developing nervous system of mouse embryos (Reymond et al., 2002). Further, Ma et al. reported the detailed expression pattern of ITSN1 in the central nervous system of the adult rat. The highest ITSN1 immunoreactivity was observed in layer III of the neocortex, hippocampus, globus pallidus, subthalamic nucleus, and substantia nigra (Ma et al., 2003).

As stated above, ITSN pre-mRNA undergoes complex splicing, that generate a number of isoforms. Some of them are neuron-specific or preferentially expressed in brain. Several studies argue that ITSN1-L isoforms completely replace the ITSN1-S isoforms in neurons of mammals (Hussain et al., 1999; Yu et al., 2008); however, ITSN1-S mRNA was detected in mouse neurons (Kropyvko et al., 2010a). The pattern of the subcellular localization of ITSN1 suggests its involvement in synaptic transmission. Roos and Kelly showed that Dap160 is restricted to the synaptic nerve terminal of Drosophila NMJs (Roos and Kelly, 1998). Moreover, Dap160 localizes to a periactive zone that surrounds the active zone where much of the endocytic machinery resides (Koh et al., 2007). Nematode ITSN was found to also be enriched in presynaptic regions (Rose et al., 2007). Using cryoelectron microscopy, Evergren et al. demonstrated that lamprey ITSN is located at the presynaptic compartment of the lamprey giant synapse and that upon stimulation it undergoes dynamic redistribution from the synaptic vesicle cluster to the periactive zone (Evergren et al., 2007). On the other hand, data on ITSN1 localization in mammalian neurons is less consistent. Several studies provide evidence that ITSN1 is enriched in the somatodendritic regions of neurons, in particular in dendritic spines (Nishimura et al., 2006), and is absent from presynaptic terminals (Thomas et al., 2009). It should be pointed out that ITSN homologues in worm and fly correspond to the short mammalian isoform ITSN-S. They do not contain DH, PH and C2 domains, which could possibly lead to the observed difference in localization. As mentioned above, subcellular targeting of the ITSN1-S protein in fibroblasts is primarily determined by the interactions of the EH domains and CCR (Hussain et al., 1999; Scappini et al., 2007; Pucharcos et al., 2000). Therefore, localization of the ITSN1-L isoform in neurons of vertebrates could also be directed by the same mechanism, or depend either on DH–PH–C2 extension or on neuron-specific interactions of other ITSN1 domains. It should be noted, that binding of the PH domain to phosphoinositides is relatively weak and appears to be insufficient to drive ITSN1 localization (Snyder et al., 2001). One of the major binding partners of the ITSN1 EH domains, epsin 1, was detected in both the presynaptic and postsynaptic sites (Yao et al., 2003), while dynamin 1, which strongly binds several ITSN1 SH3 domains, is concentrated within the presynaptic compartment (Gray et al., 2003). Indeed, recently, Pechstein et al. demonstrated that ITSN1 accumulates comparably at both pre- and postsynaptic membrane sites of mouse hippocampal neurons (Pechstein et al., 2010a). Available biochemical data confirmed these findings: ITSN1 was detected as core component of presynaptic synaptotagmin-associated endocytic complex (Khanna et al., 2006) as well as in triton-insoluble postsynaptic density (PSD) pellets from rat brain lysate (Nishimura et al., 2006). Evidence of ITSN1 functions in neurons is rather broad and sometimes contradictory. The role of ITSN1 in the regulation of synaptic vesicle (SV) cycling has been extensively reviewed very recently (Pechstein et al., 2010b). The most comprehensive functional studies were performed on Drosophila larval NMJs. Loss-of-function Dap160 mutants exhibited a 10-fold increase in NMJ satellite bouton formation probably due to altered signalling to the actin cytoskeleton via Nervous wreck (Nwk) and WASP (Koh et al., 2004; Marie et al., 2004). O'Connor-Giles et al. later confirmed this functional association. It was shown that Dap160 negatively regulates retrograde bone morphogenic protein (BMP) signalling pathway and Drosophila NMJ growth through its direct association with Nwk (O'Connor-Giles et al., 2008). Partial loss-of-function Dap160 mutants display temperaturesensitive (ts) paralysis, whereas null mutants show ts defects in endocytosis (Koh et al., 2004; Marie et al., 2004). Electron microscopy revealed fewer vesicles, aberrant large vesicles, and accumulation of endocytic intermediates at active and periactive zones in mutant terminals, but evoked neurotransmission was normal. Thus, it was suggested that Dap160, like dynamin, is involved in synaptic vesicle

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retrieval at active and periactive zones. Finally, Dap160 mutants were shown to be lethal at midlarval stages, suggesting that Dap160 is an essential gene (Marie et al., 2004). In contrast, Rose et al. demonstrated that the C. elegans ITSN gene is nonessential for viability (Rose et al., 2007). In contrast to the Dap160 mutant, no alterations in the levels of EHS-1 (epsin ortholog), AP180, or dynamin 1 were observed in ITSN-null worms. Further studies confirmed that C. elegans ITSN-null mutants are viable and display grossly normal locomotion and development (Wang et al., 2008). However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membrane-associated vesicles at putative endocytic hotspots. Using lamprey giant synapse, Evergren et al. confirmed that ITSN1 is essential for efficient SV recycling. They showed that ITSN and dynamin are redistributed after stimulation from the synaptic vesicle cluster to the periactive zone where ITSN1 enhances dynaminmediated fission (Evergren et al., 2007). Numerous evidences of postsynaptic localization of ITSN1 were also validated functionally. ITSN1 was shown to regulate AMPA-type glutamate receptors (GLR-1) trafficking in C. elegans interneurons (Glodowski et al., 2007). Rat ITSN1-L was shown to bind the NMDAtype glutamate receptors NR1 and NR2B even in the presence of ionic detergents, but not the AMPA-type glutamate receptor GluR1 (Nishimura et al., 2006). However, the most thoroughly described postsynaptic role of ITSN1-L lies in the regulation of dendritic spine development. Spines are small actin-rich protrusions from dendrites of neurons that form the postsynaptic component of most excitatory synapses in the brain. Spine growth and structural plasticity are associated with synaptic plasticity in vitro and learning in vivo (Yang et al., 2009). It was demonstrated that ITSN1-L knock down alters dendritic spine development, but has little or no influence on SV recycling (Thomas et al., 2009). Earlier studies provided evidence that overexpression of ITSN1-L caused elongation of the spine neck, whereas expression of the ITSN1 SH3 domains severely decreased the protrusion density and size of their heads, thus inhibiting spine formation (Irie and Yamaguchi, 2002; Nishimura et al., 2006). This role of ITSN1-L appears to be linked to its GEF activity. Specifically, it was shown that the EphB2 receptor or Numb protein could physically associate with ITSN1 and activate its GEF activity, which in turn activates the Cdc42 GTPase and spine morphogenesis (Irie and Yamaguchi, 2002; Nishimura et al., 2006). Recently, the generation of ITSN1 null mice was reported (Yu et al., 2008). ITSN1 deficiency did not cause embryonic lethality. However, approximately every eighth homozygous null pup failed to thrive and often died early. Further analyses revealed significantly enlarged endosomes in the brains of these pups compared with controls or their healthy littermates. The remaining homozygous null mice appeared to develop and reproduce normally and had no gross physiological abnormalities. However, the authors found that the rate of synaptic vesicle endocytosis in neurons of ITSN1 null mice was significantly reduced. Thus, disruption in ITSN1 expression causes a disturbance in vesicle trafficking and endocytic function in the brain. 9. ITSNs and diseases Since the ITSN1 gene was mapped to chromosome 21 in the Down syndrome (DS) critical region (Guipponi et al., 1998), its expression level and potential role in a specific Down syndrome phenotype were studied. Expression of the ITSN1 gene is upregulated in DS individuals (Pucharcos et al., 1999; Skrypkina et al., 2005) concurrently with expression of synaptojanin 1 in DS brain (Arai et al., 2002) and decreasing of dynamin expression in the brain of DS and Alzheimer's disease (AD) patients (Greber et al., 1999). An association between the endocytic abnormalities and the pathological processes of DS and early AD was clearly stated (Keating et al., 2006). Enlarged early endosomes are the earliest neuropathological alterations identified in

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sporadic AD (Cataldo et al., 2000). Similarly, early endosomes are significantly enlarged in DS neurons and fibroblasts, as well as the total number of endosomes of different sizes is increased (Cataldo et al., 2008). Surprisingly, DS fibroblasts possess a higher internalization rate compared to normal cells (Cataldo et al., 2008); this contradicts previous observations indicating severe impairment of endocytosis after overexpression of ITSN in cell lines and transgenic animals (Sengar et al., 1999; Pucharcos et al., 2000; Chang and Min, 2009). The DS brain is characterized by an altered shape, number, and density of synapses, that are thought to contribute to the pathological phenotype. Typically, spine density is decreased on the dendrites while presynaptic and postsynaptic elements are significantly enlarged (Belichenko et al., 2004). Similar defects were observed under conditions of overexpression of the ITSN1 SH3 domains and proline-rich region of Numb (Nishimura et al., 2006). Using Drosophila as a model, Cheng and Min recently investigated the effects of overexpression of three genes from human chromosome 21, ITSN/Dap160, DSCR1/nla and synaptojanin 1/synj. Overexpression of individual genes or of all three genes caused defects in synaptic terminal structure and synaptic activity (Chang and Min, 2009), but all three genes were necessary to cause impaired vesicle recycling. Despite intense study of ITSN1 and its role in DS phenotype formation, the precise mechanisms of ITSN1 overexpression resulting in neuron dysfunction and other features of DS are still unknown. Obviously, ITSN1 has a pleiotropic effect on DS phenotype formation. Since ITSN1 is a scaffold protein, any changes in its expression could result in a wide spectrum of effects caused by unequal stoichiometry of the components of multiprotein complexes. Thus, future investigation of ITSN1 function in the context of overexpression of human chromosome 21 genes is very promising. Another neurodegenerative pathology with which ITSN1 was shown to be associated is Huntington disease (HD). This genetic disorder arises from the expansion of a polyglutamine (polyQ) tract in the huntingtin protein (Htt) resulting in aggregation of mutant Htt into nuclear and/or cytosolic inclusions in neurons. ITSN1-S was shown to increase aggregate formation by mutant Htt through activation of the JNK–MAPK pathway and to enhance polyQ-mediated neurotoxicity (Scappini et al., 2007). Moreover, overexpression of ITSN1-S enhances aggregation of the androgen receptor, which undergoes polyQ expansion in Kennedy's disease, suggesting a broader involvement of ITSN1 in neurodegenerative diseases through destabilization of polyQ-containing proteins (Scappini et al., 2007). Given the role of ITSN in the activation of mitogenic pathways, its involvement in cancer development was proposed. Indeed, it was shown recently that a low level of ITSN2 expression is associated with poor prognosis of breast cancer patients after adjuvant chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (Specht et al., 2009). Moreover, the level of ITSN2 expression was considered to be a predictive marker for breast cancer. Future studies are needed to determine whether ITSN2 is involved in regulation of cell invasion and metastasis formation or if the ITSN2 gene is upregulated in tumors of patients with prolonged disease-free survival in parallel with some other currently unknown genes due to hyperactivation of the chromatin domain. Recently, new data of ITSN association with pathologies have emerged. ITSN2 has been shown to interact with the K15 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) and regulate B-cell receptor internalization (Lim et al., 2007). This was the first finding of hijacking of a protein of the ITSN family by a pathogen to manipulate cellular signalling. 10. Concluding remarks Numerous functional and genetic studies indicate that ITSN proteins are important scaffolds for protein assembly during endocytosis and synaptic transmission. In contrast to other scaffolds,

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proteins of the ITSN family contain the catalytic DH domain that allows assembly of endocytosis and exocytosis with dynamic regulation of the actin cytoskeleton. Recent advances in the investigation of ITSN functions expanded its protein–protein interaction network that engages ITSN proteins in many other processes including cell survival and apoptosis, control of cell polarity (Chabu and Doe, 2008) and dendritic spine development. Although significant progress has been made toward understanding ITSN functions, the precise role of ITSN in dynamin regulation and control of CCP formation and maturation remains unclear. Another major unresolved question is stoichiometry of ITSN protein complexes, competition between ITSN interactors and mechanisms of regulation of such complexes formation. The role of posttranslational modifications in regulation of the ITSN family of proteins is also unresolved. Multiple serine/threonine phosphorylation sites for ITSN1 were found in high throughput experiments, but none of them was investigated in detail (Tweedie-Cullen et al., 2009; Ballif et al., 2004; PHOSIDA database http://141.61.102.18/phosida/index.aspx). It should also be noted that several studies identified phosphorylated tyrosines in ITSN2, but not in ITSN1 (Zheng et al., 2005; Goss et al., 2006; Rikova et al., 2007; Rush et al., 2005; Brill et al., 2004). Moreover, ITSN1, in contrast to ITSN2, practically does not contain tyrosine residues in unstructured or surface exposed regions. Whether these features are connected with functional divergency between ITSN2 and ITSN1 remains a challenge for future research. The role of alternative splicing that diversifies module compositions of the ITSN protein family and influences adaptor properties of ITSNs in a tissue- and developmentally-regulated manner should be emphasized. Recently, we have shown that alternative splicing of microexons provides a mechanism for tissue-specific control of protein–protein interactions. Insertion of a microexon in alternatively spliced ITSN1 leads to regulation of the SH3A domain specificity due to the shifting of negatively charged amino acids towards the interaction interface (Dergai et al., 2010). Multiple alternative spliced isoforms of ITSNs, some with already determined specificity, have been discovered. However, the individual ITSN isoform functions and the composition of the ITSN isoform specific protein complexes remain to be elucidated. Proteins of the ITSN family could be considered as a part of molecular interfaces between different cellular processes such as endocytosis and signalling, vesicle traffic and signalling, endocytosis and cytoskeleton rearrangements, and mitogenic signalling and cell survival. Understanding the large molecular interfaces comprising multiple adaptor proteins (e.g. ITSN–Eps15–Ruk/CIN85) could shed light on the mechanisms of cross-talks and integration of events in cellular networks. These interfaces provide finely tuned interactions of different cellular processes. Although ITSN proteins have been implicated in different diseases, no therapeutic approaches were developed to alter defects in their functions. Hence, future efforts may have to be directed toward the development of such approaches to treat pathological conditions associated with aberrant expression of ITSN proteins. Acknowledgments We thank Prof. Anne-Lise Haenni for critical reading of this manuscript and Inna Foenko for help preparing the manuscript. References Adams, A., et al., 2000. Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways. J. Biol. Chem. 35, 27414–27420. Ahmad, K., Lim, W., 2010. The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin. PLoS ONE 6, e11291. Allaire, P., et al., 2006. Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis. J. Neurosci. 26, 13202–13212.

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