Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Metho...

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Дата:	2012
Автори:	Gubar, O.S., Kropyvko, S.V., Tsyba, L.O., Gasman, S., Rynditch, A.V.
Формат:	Стаття
Мова:	English
Опубліковано:	Інститут молекулярної біології і генетики НАН України 2012
Назва видання:	Вiopolymers and Cell
Теми:	Structure and Function of Biopolymers
Онлайн доступ:	http://dspace.nbuv.gov.ua/handle/123456789/156729
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Цитувати:	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells / O.S. Gubar, S.V. Kropyvko, L.O. Tsyba, S. Gasman, A.V. Rynditch // Вiopolymers and Cell. — 2012. — Т. 28, № 6. — С. 429-423. — Бібліогр.: 29 назв. — англ.

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spelling	irk-123456789-1567292019-06-19T01:30:03Z Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells Gubar, O.S. Kropyvko, S.V. Tsyba, L.O. Gasman, S. Rynditch, A.V. Structure and Function of Biopolymers Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S coprecipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also colocalize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells. Keywords: ITSN1, alternatively spliced isoforms, adaptor/scaffold proteins. Інтерсетин 1 (ITSN1) – адатоний білок, залучений до мембранного транспорту та передачі клітинних сигналів. Довга і коротка ізоформи ITSN1 (ITSN1-L і ITSN1-S) утворюються в результаті альтернативного сплайсингу. Метою роботи було встановити, чи можуть ITSN1-L і ITSN1-S взаємодіяти в клітинах ссавців. Методи. Використано методи імунопреципітації та конфокальної мікроскопії. Результати. Показано, що ендогенний ITSN1- S копреципітується з надекспресованим ITSN1-L у клітинах PC12, 293 і 293T. Довга і коротка ізоформи ITSN1 також колокалізуються у клітинах 293T. Висновки. ITSN1-L і ITSN1-S формують комплекси в клітинах ссавців. Ключові слова: ITSN1, альтернативно сплайсовані ізоформи, адапторні білки. Интерсектин 1 (ITSN1) – адапторный белок, участвующий в мембранном транспорте и передаче клеточных сигналов. Длинная и короткая изоформы ITSN1 (ITSN1-L и ITSN1-S) образуются вследствие альтернативного сплайсинга. Целью исследования было выяснить, могут ли ITSN1-L и ITSN1-S взаимодействовать в клетках млекопитающих. Методы. Использованы методы иммунопреципитации и конфокальной микроскопии. Результаты. Показано, что эндогенный ITSN1-S копреципитируется со сверхэкпрессированным ITSN1-L в клетках PC12, 293 и 293T. Длинная и короткая изоформы ITSN1 так же колокализуются в клетках 293T. Выводы. ITSN1-L и ITSN1-S формируют комплексы в клетках млекопитающих. Ключевые слова: ITSN1, альтернативно сплайсированные изоформы, адапторные белки. 2012 Article Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells / O.S. Gubar, S.V. Kropyvko, L.O. Tsyba, S. Gasman, A.V. Rynditch // Вiopolymers and Cell. — 2012. — Т. 28, № 6. — С. 429-423. — Бібліогр.: 29 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000132 http://dspace.nbuv.gov.ua/handle/123456789/156729 577.22 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Structure and Function of Biopolymers Structure and Function of Biopolymers
spellingShingle	Structure and Function of Biopolymers Structure and Function of Biopolymers Gubar, O.S. Kropyvko, S.V. Tsyba, L.O. Gasman, S. Rynditch, A.V. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells Вiopolymers and Cell
description	Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S coprecipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also colocalize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells. Keywords: ITSN1, alternatively spliced isoforms, adaptor/scaffold proteins.
format	Article
author	Gubar, O.S. Kropyvko, S.V. Tsyba, L.O. Gasman, S. Rynditch, A.V.
author_facet	Gubar, O.S. Kropyvko, S.V. Tsyba, L.O. Gasman, S. Rynditch, A.V.
author_sort	Gubar, O.S.
title	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
title_short	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
title_full	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
title_fullStr	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
title_full_unstemmed	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
title_sort	alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2012
topic_facet	Structure and Function of Biopolymers
url	http://dspace.nbuv.gov.ua/handle/123456789/156729
citation_txt	Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells / O.S. Gubar, S.V. Kropyvko, L.O. Tsyba, S. Gasman, A.V. Rynditch // Вiopolymers and Cell. — 2012. — Т. 28, № 6. — С. 429-423. — Бібліогр.: 29 назв. — англ.
series	Вiopolymers and Cell
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first_indexed	2025-07-14T09:05:01Z
last_indexed	2025-07-14T09:05:01Z
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fulltext	UDC 577.22 Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells O. S. Gubar1, 2, S. V. Kropyvko1, L. O. Tsyba1, S. Gasman2, A. V. Rynditch1 1State Key Laboratory on Molecular and Cellular Biology Institute of Molecular Biology and Genetics, NAS of Ukaine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2Institut des Neurosciences Cellulaires et Integratives (INCI) UPR 3212 CNRS -Universite de Strasbourg, Centre de Neurochimie 5, rue Blaise Pascal, Strasbourg, France, 67084 ogubar@mail.ru Intersectin 1 (ITSN1) is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S) are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co- precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co- localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells. Keywords: ITSN1, alternatively spliced isoforms, adaptor/scaffold proteins. Introduction. ITSN1 is a multidomain and multifunc- tional adaptor protein which is involved in clathrin- and caveolin-dependent endocytosis [1, 2], Ca2+-regulated exocytosis [3] and synaptic vesicles retrieval [4]. It is also implicated in cellular signaling [5–7] and neuron survival [8]. Abnormalities of ITSN1 expression are as- sociated with the endocytic anomalies reported in Down syndrome brains and early stages of Alzheimer’s disea- se as well as with neurodegeneration in Huntington’s disease [9, 10]. ITSN1 is also associated with glioma and neuroblastoma tumorigenesis [11, 12]. Two major ITSN1 isoforms are produced by alter- native splicing [13]. Ubiquitously expressed ITSN1-S consists of two N-terminal EH (Eps15 homology) do- mains, coiled-coil region (CCR) and five SH3 (Src ho- mology) domains. EH domains interact with NPF motifs and are highly involved in clathrin coated pits assemb- ling [14], and SH3 domains interact with proline-rich PXXP motifs and provide protein-protein interactions in many cellular processes, including membrane traffi- cking and signaling [15]. The long isoform of ITSN1 (ITSN1-L) is expressed predominantly in neurons and has three additional C-terminal domains: DH (dbl homo- logy), PH (pleckstrin homology) and C2. The tandem of DH-PH domains is a GEF (guanine nucleotide exchange factor) for the Rho-family small GTPase Cdc42 [16]. ITSN1 domain composition implies that this is an adaptor/scaffold protein. These proteins possess multi- ple modular interaction domains and play a crucial role in spatial and temporal organization of cellular proces- ses. Scaffolds regulate selectivity in signaling path- ways using tethering mechanism and physically assemb- ling chosen components of signaling pathway or net- work [17]. It is known that some scaffold proteins di- merize or even oligomerize to perform their function [18, 19]. As we have recently shown that ITSN1-S forms a heterodimer with its minor isoform 22a [20, 21], we presumed that probably other isoforms of ITSN1 are able to interact with each other thereby assembling protein supercomplexes. 429 ISSN 0233–7657. Biopolymers and Cell. 2012. Vol. 28. N 6. P. 429–433 doi 10.7124/bc.000132  Institute of Molecular Biology and Genetics, NAS of Ukraine, 2012 Materials and methods. Antibodies. A monoclonal anti-Omni (D-8) antibody was from «Santa Cruz Biotech- nology» (USA). Rabbit polyclonal antibodies against the EH2 domain of ITSN1 were described previously [22]. HRP-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from «Invitrogen» (USA). DNA plasmid constructs. The coding sequence of ITSN1-L was amplified from human embryonic brain cDNA and cloned into the pcDNA4His/Max C vector («Invitrogen»). Plasmids encoding GFP-ITSN1-L and mCherry- ITSN1-S were described previously [23, 24]. Cell culture and transfection. 293 and 293T cells were obtained from the American Type Culture Collec- tion and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf se- rum («Sigma», USA), 50 U/ml penicillin and 100 mg/ml streptomycin. The cells were transiently transfected using JetPEI transfection reagent (Polyplus Transfec- tion) according to manufacturer recommendations and further processed 24 h after transfection. PC12 cells were maintained in DMEM supplemen- ted with 5 % fetal calf serum («Sigma»), 10 % horse serum («Sigma»), 50 U/ml penicillin and 100 mg/ml streptomycin. The cells were transiently transfected using Lipofectamine 2000 transfection reagent («Invitrogen») according to manufacturer recommendations and fur- ther processed 48 h after transfection. Immunoprecipitation and Western blot analysis. The immunoprecipitation and Western blot were per- formed as described previously [25]. In brief, the cells were lysed in IP buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 10 % glycerol, 0.5 % NP40, protease inhibitors co- cktail («Sigma») and centrifuged for 15 min at 16,000 g. Supernatant (2 mg of proteins) was incubated with 2 µg an- ti-Omni antibodies and 20 µl Protein A/G Plus Ultralink Resin («Thermo Scientific», USA) for 4 h at 4 °C. Then beads were washed four times with IP buf- fer without inhibitors. Immunoprecipitated complexes were eluted with Laemmli buffer, resolved by SDS- PAGE and transferred to nitrocellulose membranes («Bio-Rad», USA). The membranes were blocked with 5 % non-fat milk in TBS-T (1 × TBS («Euromedex», France), 0,1 % Tween 20) for 1 h, incubated with anti- Omni or anti-EH2 antibodies and washed. Detection was performed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies. Confocal microscopy. 293T cells were transfected with fluorescent protein constructs, fixed in 4 % para- formaldehyde in PBS 24 h after transfection, washed two times with PBS and mounted in Mowiol medium («Sigma»). The slides were analyzed using Leica SP5 confocal microscope. Results and discussion. In order to prove the exis- tence of ITSN1 macromolecular complexes in cells, we have performed a co-immunoprecipitation assay in dif- ferent mammalian cell lines (Fig. 1). Omni-tagged ITSN1-L was overexpressed in rat pheochromocytoma PC12 cell line and in human 293 or 293T cell lines. The immunoprecipitation was carried out with anti-Omni antibodies and further immunoblot- ting was performed with anti-EH2 antibodies to detect precipitated ITSN1-S. As a control of non-specific bin- ding appropriate amount of mouse IgG was used. In all tested cell lines endogenous ITSN1-S readily co-pre- cipitated with recombinant ITSN1-L. 293T cell line was of particular interest as it expressed endogenous ITSN1-L at relatively high level in contrast to 293 cell line (data not shown). And in addition to full-size ITSN1-L protein in 293T cells a minor band with slight- ly lower molecular weight is present which is presumab- 430 GUBAR O. S. ET AL. A B IB: α-Omni IB: α-EH2 170 130 kDa Input Input IP IP α-Omni α-OmniIgG IgG PC12 293 ←Omni-ITSN1-L ←ITSN1-S 150 kDa IB: InputInput IP IP α-Omni α-Omni α-Omni α-EH2 IgG IgG 293T ←Omni-ITSN1-L ←ITSN1-S Fig. 1. ITSN1-L and ITSN1-S co-precipitate in mammalian cells. PC12 and 293 cells (A) or 293T cells (B) were transfected with Omni-ITSN1- L and subjected to immunoprecipitation (IP) with anti-Omni antibody or control IgG followed by immunoblotting (IB) with anti-EH2 (anti- ITSN1) antibody ly one of the possible alternatively spliced isoforms of ITSN1-L. The bands below the ITSN1-S can also refer to one of its minor isoforms, e. g. ITSN1-22a [20, 21]. And these minor isoforms also co-precipitate with over- expressed ITSN1-L suggesting that ITSN1 can form he- terodimers of different isoform composition (Fig. 1, B). To explore whether ITSN1-S and ITSN1-L have simi- lar subcellular localization, we overexpressed GFP-tag- ged ITSN1-L and mCherry-tagged ITSN1-S in 293T cells (Fig. 2, A). As a control we co-expressed each of the proteins with empty vector (Fig. 2, B, C). Obtained data clearly indicate that both isoforms perfectly colocalize and have similar subcellular distribution. So we have demonstrated that ITSN1 isoforms in- teract in mammalian cells, thereby forming large comp- lexes with varying protein composition that can func- tion in membrane trafficking as well as in cell signal- ing. However the molecular mechanism controlling this clusterization remains unclear. Such ITSN1-ITSN1 interaction possibly could be mediated by CCR as it is the case for Eps15, another CCR and EH domain containing protein [26]. This sup- position is also supported by the work of Wong et al. [27]. They have used a high throughput yeast two hyb- rid screening to define the possible partners of the ITSN scaffolds and have identified ITSN1 as a target of a prey containing its EH2 domain and a half of CCR 431 ALTERNATIVELY SPLICED SHORT AND LONG ISOFORMS OF ADAPTOR PROTEIN INTERSECTIN 1 FORM mCherry-ITSN1-S mCherry-ITSN1-S mCherryGFP-ITSN1-L GFP-ITSN1-L GFP Merge Merge Merge A B C Fig. 2. Overexpressed GFP- ITSN1-L and mCherry-ITSN1- S colocalize in 293T cells. 293T cells were transfected with both GFP- ITSN1-L and mCherry-ITSN1-S (A) or with GFP-ITSN1-L alone and empty pmCherry vector (B) or with mCherry-ITSN1-S alone and empty pGFP-C1 vector (C). Scale bar repre- sents 5 µm (204–522 aa). They have further shown the existence of ITSN1 homo- and heteromeric complexes by bimolecu- lar fluorescence complementation. But we assume that ITSN1 SH3 domains may also contribute to this interaction as it has already been shown that some other proteins dimerize via their SH3 domains [28, 29]. These assumptions will be tested in our further research. Conclusions. We have shown that ITSN1 long and short isoforms form complexes in different mammalian cell lines. О. С. Гу бар, С. В. Кро пив ко, Л. О. Циба, С. Гас ман, А. В. Рин дич Альтернативно сплай со вані ко рот ка і дов га ізо фор ми адап тор но го білка інтер сек ти ну 1 фор му ють ком плек си у кліти нах ссавців Ре зю ме Інтер сек тин 1 (ITSN1) – адап тор ний білок, за лу че ний до мем - бран но го транс пор ту та пе ре дачі клітин них сиг налів. Дов га і ко - рот ка ізо фор ми ITSN1 (ITSN1-L і ITSN1-S) утво рю ють ся в ре- зуль таті аль тер на тив но го сплай син гу. Ме тою ро бо ти було вста- новити, чи мо жуть ITSN1-L і ITSN1-S взаємодіяти в кліти нах ссав- ців. Ме то ди. Ви ко рис та но ме то ди іму ноп ре ципітації та кон фо - каль ної мікрос копії. Ре зуль та ти. По ка за но, що ен до ген ний ITSN1- S коп ре ципітується з над е кспре со ва ним ITSN1-L у кліти нах PC12, 293 і 293T. Дов га і ко рот ка ізо фор ми ITSN1 також ко ло калізу - ють ся у кліти нах 293T. Вис нов ки. ITSN1-L і ITSN1-S фор му ють комплек си в кліти нах ссавців. Клю чові сло ва: ITSN1, аль тер на тив но сплай со вані ізо фор ми, адап торні білки. О. С. Гу барь, С. В. Кро пив ко, Л. А. Цыба, С. Гас ман, А. В. Рын дич Альтернативно сплай си ро ван ные ко рот кая и длин ная изо фор мы адап тор но го бел ка ин тер сек ти на 1 фор ми ру ют ком плек сы в клет ках мле ко пи та ю щих Ре зю ме Интер сек тин 1 (ITSN1) – адап тор ный бе лок, учас тву ю щий в мем бран ном транс пор те и пе ре да че кле точ ных сиг на лов. Длин - ная и ко рот кая изо фор мы ITSN1 (ITSN1-L и ITSN1-S) об ра зу ют ся всле дствие аль тер на тив но го сплай син га. Целью ис сле до ва ния было вы яс нить, мо гут ли ITSN1-L и ITSN1-S вза и мо де йство вать в клет ках мле ко пи та ю щих. Ме то ды. Исполь зо ва ны методы им - му ноп ре ци пи та ции и кон фо каль ной мик рос ко пии. Ре зульта ты. По ка за но, что эн до ген ный ITSN1-S коп ре ци пи ти ру ет ся со сверх - экпре сси ро ван ным ITSN1-L в клет ках PC12, 293 и 293T. Длин ная и ко рот кая изо фор мы ITSN1 так же ко ло ка ли зу ют ся в клет ках 293T. Вы во ды. ITSN1-L и ITSN1-S фор ми ру ют ком плек сы в клет - ках мле ко пи та ю щих. Клю че вые сло ва: ITSN1, аль тер на тив но сплай си ро ван ные изо - фор мы, адап тор ные бел ки. REFERENCES 1. Hussain N. K., Yamabhai M., Ramjaun A. R., Guy A. M., Bara- nes D., O’Bryan J. P., Der C. J., Kay B. K., McPherson P. S. Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells // J. Biol. Chem.– 1999.–274, N 22.–P. 15671–15677. 2. Predescu S. A., Predescu D. N., Timblin B. K., Stan R. V., Malik A. B. Intersectin regulates fission and internalization of caveolae in endothelial cells // Mol. Biol. Cell.–2003.–14, N 12.– P. 4997– 5010. 3. Momboisse F., Ory S., Calco V., Malacombe M., Bader M. F., Gasman S. Calcium-regulated exocytosis in neuroendocrine cells: intersectin-1L stimulates actin polymerization and exocy- tosis by activating Cdc42 // Ann. NY Acad. Sci.–2009.–1152.– P. 209–214. 4. 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Received 30.08.12 433 ALTERNATIVELY SPLICED SHORT AND LONG ISOFORMS OF ADAPTOR PROTEIN INTERSECTIN 1 FORM

Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

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