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Intersectin 1 Forms Complexes with SGIP1 and Reps1 in Clathrin-Coated

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Intersectin 1 Forms Complexes with SGIP1 and Reps1 in Clathrin-Coated Biochemical and Biophysical Research Communications 402 (2010) 408–413 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits ⇑ Oleksandr Dergai a, ,1, Olga Novokhatska a,1, Mykola Dergai a, Inessa Skrypkina a, Liudmyla Tsyba a, Jacques Moreau b, Alla Rynditch a a Department of Functional Genomics, Institute of Molecular Biology and Genetics, NASU, 150 Zabolotnogo Street, 03680 Kyiv, Ukraine b Molecular Mechanisms of Development, Jacques Monod Institute, Development and Neurobiology Program, UMR7592 CNRS – Paris Diderot University, 15 rue Hélène Brion, 75205 Paris Cedex 13, France article info abstract Article history: Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endo- Received 7 October 2010 cytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome Available online 12 October 2010 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome pheno- type. Here we report the identification of novel interconnections in the interaction network of this endo- Keywords: cytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth Endocytosis factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP Adaptor proteins associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated Intersectin 1 by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes compris- Protein interactions SGIP1 ing SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluores- cent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clath- rin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction cellular processes from cell survival [3,10], cell polarity and cell cycle progression [11] to dendritic spine development [12–14] Endocytosis is a fundamental process of internalization of and regulation of RTK (receptor tyrosine kinase) trafficking and sig- plasma membrane components, surface receptors, nutrients, naling [15]. Despite the fact that ITSN1 is implicated in a diverse pathogens and extracellular soluble molecules to regulate various set of cellular functions, its major role is thought to be regulation aspects of cell homeostasis and communication with the environ- of endocytic events by orchestrating recruitment of endocytic pro- ment [1]. Except for constituting the main pathway that allows teins such as adaptor Eps15 (epidermal growth factor receptor different substances to enter the cell, endocytosis is a starting point pathway substrate 15), GTPase dynamin, phosphoinositide phos- in a dramatically complicated network of intracellular trafficking. phatase synaptojanin and others [2,16–22]. The fidelity of this process relies on the precision and accuracy ITSN1 functions as a scaffolding protein regulating many of the endocytic components recruited. The endocytic scaffold aspects of membrane traffic. Disruption of ITSN1-containing endo- intersectin 1 (ITSN1) is one of the recruiters. It is an evolutionarily cytic complexes, caused by overexpression of the full-length conserved plasma membrane-associated adaptor protein involved protein, blocks receptor-mediated endocytosis [2]. Depletion of in clathrin-mediated endocytosis, apoptosis, signal transduction, ITSN1 leads to activation of the mitochondrial pathway of apopto- and regulation of cytoskeletal rearrangements [2–5]. ITSN1 is pres- sis in endothelial cells [3], whereas quadruple knockdown of ITSN1 ent in the cell as a set of alternatively spliced isoforms that are and ITSN2 together with Eps15 and Eps15R cause cytosolic locali- differentially expressed in various tissues [6–8]. The short form zation of the adaptor complex AP2 preventing clathrin-coated pit (ITSN1-s) consists of two N-terminal Eps15 homology domains (CCP) formation [23]. Furthermore through interactions with the (EH1 and EH2), a coiled-coil region and five Src homology domains regulators of the cytoskeleton N-WASP and Cdc42, ITSN1 is (SH3A-E) (Fig. 1). The long form (ITSN1-l) contains a C-terminal thought to link clathrin-mediated endocytosis to the actin nucle- extension with DH (a Dbl homology), PH (a pleckstrin homology), ation machinery and participate in actin assembly as a mechanism and a C2 domain [9]. ITSN1 was shown to regulate a plethora of of vesicle protrusion [5,24]. In the current report we attempted to extend our understanding ⇑ Corresponding author. Fax: +380 445260759. of the interaction network of ITSN1-s (further referred to as ITSN1). E-mail address: [email protected] (O. Dergai). An in silico prediction and interaction database search demon- 1 These authors contributed equally to this work. strated the association between the newly identified endocytic 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.045 O. Dergai et al. / Biochemical and Biophysical Research Communications 402 (2010) 408–413 409 Fig. 1. Schematic representation of the structure of ITSN1 and its binding partners. EH, Eps15 homology; DPF repeat, Asp-Pro-Phe motif of interaction with EH-domains; UIM, ubiquitin-interacting motif; PRD, proline-rich domain; MP, membrane phospholipid-binding; BAR, Bin/amphiphysin/Rvs; FCHo, Fer/Cip4 homology. adaptors Reps1 (RalBP associated Eps15-homology domain protein) Sepharose 4B beads (GE Healthcare) according to the manufac- and SGIP1 (Src homology 3-domain growth factor receptor-bound turer’s instructions. Lysates of transiently transfected HEK293T 2-like (endophilin) interacting protein 1) with ITSN1. cells were prepared in extraction buffer [20 mM Tris–HCl pH In current study we have identified interaction of ITSN1 with 7.4, 150 mM NaCl, 1% Triton X-100, and supplemented with SGIP1 and Reps1. Both Reps1 and SGIP1 colocalize with ITSN1 in complete EDTA-free protease inhibitor cocktail (Roche)] and clathrin-coated pits. Interaction data details evidence for triple centrifuged for 20 min at 12,000g at 4 °C. For pull-down assays complex formation implying scaffolding role for ITSN1. 2–5 lg of purified GST fusion proteins or GST were bound to 30 ll of 50% glutathione-Sepharose 4B beads and incubated with 2. Materials and methods cell lysates for 1 h at 4 °C. The beads were extensively washed and boiled in Laemmli sample buffer (150 mM Tris–HCl pH 6.8, 2.1. Expression constructs 2.5% glycerol, 10% SDS, 3% b-mercaptoethanol and 0.5% bromo- phenol blue). The eluted proteins were resolved by SDS–PAGE The following oligonucleotides were used to amplify the coding and transferred to nitrocellulose membranes. The blots were sequences of SGIP1 and Reps1 respectively: 50-TCTCTGCAGATG blocked for 1 h in 5% non-fat milk, 1Â PBS (phosphate-buffered ATGGAAGGATTG-30,50-CCGCGGCCG-CAAACCTTTTCTTGATG-30;50- saline) and 0.1% Tween 20, incubated with the corresponding pri- AGGAATTCATGGAAGGCTTAACGC-30,50-CTCTCGAGTCAAGCGTAAT mary antibodies for 1 h at room temperature and washed. Detec- CTGGAACATCGTATGGGTATTGTAGGTGAGAGAATGG-30 and PCR tion was performed by horseradish peroxidase-labeled secondary products were cloned into the pcDNA4Has/Max vector (Invitro- antibodies (Promega). gen). The glutathione S-transferase (GST) fusion SH3-domains and coiled-coil region of ITSN1 were described previously [25]. 2.4. Cell culture and transfection The region encoding the SH3 domain of amphiphysin 1 (amino acids 413–480) was amplified by PCR and subcloned into the Adherent cell lines HEK293T and MCF-7 cells were maintained pGEX-4T-3 vector (GE Healthcare); the full coding sequence of in Dulbecco’s modified Eagle’s medium (DMEM) supplemented ITSN1 was subcloned into the pEGFP-C1 (Clontech) and pmCher- with 10% fetal calf serum, 50 U/ml penicillin and 100 mkg/ml ry-C1 (Clontech) fluorescent vectors; the ITSN1-HA plasmid was streptomycin. The cells were transiently transfected using polyeth- kindly provided by Dr. Susana de la Luna (Barcelona, Spain); GFP- yleneimine transfection reagent (JetPEI, Polyplus Transfection) and clathrin was a kind gift of Dr. Serhiy Havrylov (Warsaw, Poland). processed 24–36 h after transfection. 2.2. Antibodies 2.5. Immunoprecipitation Polyclonal antibodies against the EH2 domain of ITSN1 were de- For immunoprecipitation (IP), HEK293 cells were lysed in IP scribed previously [25]. A monoclonal anti-omni (D-8): sc-7270 buffer (20 mM Tris–HCl pH 7.5, 0.5% NP40, 100 mM NaCl, 10% antibody was purchased from Santa Cruz Biotechnology. Anti-HA glycerol, 1 mM PMSF and protease inhibitor cocktail). The cell (hemagglutinin) antibody was purchased from Covance. lysate was mixed with antibodies and protein A/G Agarose beads (Santa Cruz Biotechnology) prewashed in IP buffer. After over- 2.3. Protein expression, pull-down assays and Western blot analysis night incubation at 4 °C the beads were washed three times with IP buffer. Bound proteins were eluted by boiling in 30 llof The recombinant GST fusion proteins were expressed in Laemmli sample buffer and analysed by SDS–PAGE and Western Escherichia coli BL21 (DE3) pLysE and purified on glutathione- blotting.
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