ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 3. P. 244–251 doi: 10.7124/bc.00081E UDC 577.21 ITSN protein family: Regulation of diversity, role in signalling and pathology L. O. Tsyba, M. V. Dergai, I. Ya. Skrypkina, O. V. Nikolaienko, O. V. Dergai, S. V. Kropyvko, O. V. Novokhatska, D. Ye. Morderer, T. A. Gryaznova, O. S. Gubar, A. V. Rynditch Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 [email protected] Adaptor/scaffold proteins of the intersectin (ITSN) family are important components of endocytic and signalling complexes. They coordinate trafficking events with actin cytoskeleton rearrangements and modulate the activity of a variety of signalling pathways. In this review, we present our results as a part of recent findings on the func- tion of ITSNs, the role of alternative splicing in the generation of ITSN1 diversity and the potential relevance of ITSNs for neurodegenerative diseases and cancer. Keywords: adaptor/scaffold proteins, intersectin family, alternative splicing, endocytosis. Introduction. Adaptor/scaffold proteins are important on chromosome 21, were associated with the endocytic components of many cellular processes and signalling anomalies reported in patients with Down syndrome systems. Classical scaffolds typically do not posses any and Alzheimer’s disease [5–7]. enzymatic activity. They function as platforms for the In this review, we present our results and summa- assembly of multiprotein complexes and can help to lo- rize recent findings of other laboratories concerning the calize signalling molecules to a specific compartment role of ITSN family members in the formation of clath- of the cell or/and regulate the efficiency of a signalling rin-coated vesicles and regulation of signal transduc- pathway [1, 2]. tion and actin cytoskeleton rearrangements. We also Scaffold proteins may both facilitate or inhibit sig- describe the impact of alternative processing on the ge- nal transduction depending on their concentration in cer- neration of diversity of the ITSN family and regulation tain compartments, regulating the strength, specificity of the ITSN genes expression. and duration of signal propagation. Role of ITSN in endocytosis and signalling.ITSN Adaptor/scaffold proteins of intersectin (ITSN) fa- family consists of proteins encoded by two genes, ITSN1 mily are characterized by the presence of multiple do- and ITSN2, located on human chromosomes 21 and 2, mains that mediate protein-protein interactions. By bin- respectively [8, 9]. ITSN1 and ITSN2 are evolutiona- ding to numerous proteins, ITSNs assemble multimeric rily conserved proteins and have the same domain orga- complexes implicated in clathrin- and caveolin-media- nization. The short isoform (ITSN-S) consists of two ted endocytosis, actin cytoskeleton rearrangements, cell Eps15 homology domains (EH1 and EH2), a coiled- signalling and survival (reviewed in [3, 4]). Abnorma- coil region (CCR) and five Src homology 3 domains lities of expression of the ITSN1 gene, which is located (SH3A–E). The long isoform (ITSN-L) contains an ex- tended C-terminus consisting of a Dbl homology (DH), Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013 a Pleckstrin homology (PH) and a C2 domain. 244 ITSN PROTEIN FAMILY: REGULATION OF DIVERSITY, ROLE IN SIGNALLING AND PATHOLOGY Fig. 1. ITSNs are important components of clathrin-mediated endocytosis and signalling. Upon ligand binding, plasma membrane signalling re- ceptors such as receptor tyrosine kinases undergo dimerization, auto-phosphorylation and ubiquitylation. Ubiquitylated receptors are recognized by adaptors, such as Eps15 and epsin, and recruited to clathrin-coated pits. After vesicle scission, receptors undergo sorting and are either recycled back to the plasma membrane or directed to lysosomes for degradation The DH domain is the only catalytic domain in ITSNs. scaffolding adaptor Eps15 [15], large GTPase dynamin It possesses guanine nucleotide exchange activity spe- involved in vesicle fission, a phosphoinositide phos- cific for the small GTPase Cdc42 [10]. Our data indica- phatase synaptojanin 1 [16], endocytic accessory protein te that the DH domain of ITSN2 regulates the activity epsin [17] and others. Thea-andb-appendage domains of Cdc42 during embryo development affecting actin of clathrin adaptor complex AP2 were shown to direct- cytoskeleton in Xenopus embryos. Overexpression of ly interact with the linker between the SH3A and SH3B the DH and PH domains in Xenopus embryos resulted domains of ITSN1 [18]. Our results demonstrate the in- in aberrant phenotypes [11]. Embryos displayed hyper- teraction of ITSN1 with a membrane-deforming protein pigmentation and gastrulation failure that were incom- SGIP1 and their colocalization in clathrin-coated pits patible with survival. The effect of expression of the con- [19]. stitutively active Cdc42 strongly resembled that of the The role of ITSN1 as an important endocytic scaf- DH–PH tandem. Moreover, the dominant negative Cdc42 fold has been confirmed by studies on Drosophila Dap partially rescued the developmental defects induced by 160 (homolog of human ITSN)-null mutants, which dis- the expression of the DH–PH domains [11]. ITSN2 was play severely reduced levels of dynamin, synaptojanin, reported to regulate Cdc42 activation during epithelial endophilin and AP180 in Dap160 mutant synapses, sug- morphogenesis. Silencing of either Cdc42 or ITSN2 dis- gesting the role of Dap160 in recruitment of endocytic rupted the proper orientation of the mitotic spindle and proteins to the presynaptic terminals [20, 21]. A redu- normal lumen formation [12]. Guanine nucleotide ex- ced number of exocytosis events in chromaffin cells and change activity of the brain-enriched ITSN1-L isoform a slowing of endocytosis in neurons were also detected was shown to be important for dendritic spine morpho- in ITSN1 null mice [22]. genesis [13, 14]. Initially, the role of ITSN1 in clathrin-mediated en- ITSN1 was shown to act at different stages of clath- docytosis was thought to be restricted to the invagi- rin-mediated endocytosis (Fig. 1). It forms complexes nation and fission stages [15]. Recently, ITSNs and with a plethora of endocytic proteins including the Eps15wereshowntobenecessaryforinitiationofcla- 245 TSYBAL.O.ETAL. thrin-coated pit assembly. It was found that ITSNs and well as is involved in cytoskeletal rearrangements and Eps15 regulate clustering of membrane-sculting pro- apoptosis [35]. CIN85/ITSN1/Cbl-b complexes were teins FCHo, which mark the sites of clathrin-coated pits found to be localized in clathrin-coated pits [31]. formation and act as clathrin-coated pit nucleators [23] Recently, it was shown that interaction of ITSN1 (Fig. 1). with endocytic adaptor Dab2 was important for interna- ITSN1 is also an important component of the exocy- lization of integrin b1, a specific cargo for Dab2 adap- tic machinery. ITSN1 interacts with SNAP-25 [16], a tors [36]. It was demonstrated that downregulation of protein of the SNARE complex that is critical for the do- ITSN1 via siRNA inhibits Erk1/2-MAPK and its direct cking and fusion of secretory granules to the plasma activator MEK and leads to mitochondrial apoptosis of membrane. Moreover, the DH domain of ITSN1-L is im- cultured human lung microvascular endothelial cells [37]. portant for the actin cytoskeletal rearrangements requi- Downregulation of ITSN1 expression in mouse lungs red for exocytosis in neuroendocrine cells [24]. Silen- by siRNA in vivo treatment caused endothelial cells death cing of ITSN1 strongly inhibited exocytosis and simul- and lung injury, followed by endothelial phenotypic taneously prevented secretagogue-induced activation of changes regarding hyper-proliferation and apoptosis-re- Cdc42. ITSN1-L has been proposed to couple Cdc42- sistance [38]. mediated actin remodelling involved in the late stages ITSN1 was also shown to regulate the survival of of exocytosis with the recruitment of the endocytic ma- neuronal cells through the activation of the PI3K–AKT chinery required for the subsequent compensatory endo- pathway by interaction with PI3K–C2b. Decreasing cytosis in secretory cells [25]. ITSN expression by shRNA inhibited the PI3K–C2b– Endocytosis is a critical component in membrane re- AKT signalling pathway and dramatically increased ceptor signalling. Although endocytosis terminates re- apoptosis in both neuroblastoma cells and primary corti- ceptor signalling [26], it also has emerged as an essential cal neurons [39]. step in the successful activation of receptor-dependent Recently, the results of yeast two-hybrid screen for signalling pathways [27, 28]. Very recently, it has been proteins that bind ITSNs were published. It was found shown that inhibition of early stages of clathrin-me- 127 potential ITSN-binding proteins involved in the diated endocytosis, by knockdown of ITSN2 or clath- membrane curvature, several cellular pathways inclu- rin heavy chain, attenuated granulocyte-macrophage ding Rab and Arf GTPase regulation, receptor tyrosine colony stimulating factor receptor (GMR)-mediated sig- kinase regulation, and inositol phosphate/phosphati- nalling, whereas inhibition of the membrane constric- dylinositol metabolism [40]. Several nuclear proteins tion step during clathrin-coated vesicle formation enhan- including RNA binding proteins and transcription fac- ced GMR signalling. Moreover, an ITSN2-non-binding tors were found to be potential ITSN partners. Therefo- mutant of GMR