Gene 473 (2011) 67–75 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene 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 modifica- tions. 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 0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.11.016 68 L. Tsyba et al. / Gene 473 (2011) 67–75 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