Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN1- 22a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the nove...

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Datum:	2011
Hauptverfasser:	Dergai, M.V., Dergai, O.V., Tsyba, L.O., Novokhatska, O.V., Skrypkina, I.Ya., Rynditch, A.V.
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Sprache:	English
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2011
Schriftenreihe:	Вiopolymers and Cell
Schlagworte:	Short Communications
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spelling	irk-123456789-1560422019-06-18T01:25:15Z Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus Dergai, M.V. Dergai, O.V. Tsyba, L.O. Novokhatska, O.V. Skrypkina, I.Ya. Rynditch, A.V. Short Communications Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN1- 22a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules. Keywords: intersectin 1, isoform, homodimer. Мета. Раніше нами ідентифіковано нову ізоформу адаптерного білка ITSN1, названу ITSN1-22a. Вестерн-блот аналіз виявив наявність двох імунореактивних смуг 120 і 250 кДа, що відповідають ITSN1-22a. Мета цієї роботи полягала у вивченні здатності нової ізоформи формувати димери. Методи. Імунопреципітацію і Вестерн-блот аналіз використано для дослідження здатності ізоформи до димеризації. Ідентифікацію ділянки, що відповідає за формування димерів, здійснювали за допомогою сайтспрямованого мутагенезу та делеційного аналізу. Інгібування ендоцитозу індукували дефіцитом іонів калію. Для мітогенної стимуляції клітин HEK293 застосовано епідермальний фактор росту. Результати. Нами виявлено, що надекспресована в клітинах НЕК293 ізоформа ITSN1-22a формує димери. Димеризація обумовлена специфічним для цієї ізоформи С-кінцевим доменом. Нами показано, що цистеїни С1016 і С1019 залучені до процесу гомодимеризації. Інгібування клатрин-опосередкованого ендоцитозу та стимуляція клітин мітогеном не впливають на формування димерів ізоформою ITSN1-22a. Висновки. ITSN1-22a – це єдина відома на сьогодні ізоформа ITSN1, здатна утворювати гомодимери за допомогою дисульфідних зв’язків, що може бути важливим для формування ITSN1-вмісних білкових комплексів. Ключові слова: інтерсектин 1, ізоформа, гомодимер. Цель. Ранее мы идентифицировали новую изоформу адаптерного эндоцитозного белка ITSN1, названную ITSN1-22a. Вестерн-блот анализ выявил наличие двух иммунореактивных полос 120 и 250 кДа, соответствующих ITSN1-22a. Цель этой работы состояла в изучении возможности формирования димеров изоформой ITSN1-22a. Методы. Иммунопреципитация и Вестерн-блот анализ использованы для исследования возможности димеризации изоформы. Идентификацию участка, отвечающего за димеризацию, осуществляли с помощью сайт-направленного мутагенеза и делеционного анализа. Ингибирование эндоцитоза индуцировали дефицитом ионов калия. Для митогенной стимуляции клеток применен эпидермальный фактор роста. Результаты. Нами обнаружено, что сверхэкспрессированная в клетках НЕК293 изоформа ITSN1-22a формирует димеры. Димеризация изоформы обусловлена ее специфическим С-концевым доменом. Нами показано, что цистеины С1016 и С1019 вовлечены в процесс гомодимеризации. Ингибирование клатрин-опосредованного эндоцитоза и митогенная стимуляция клеток не влияют на формирование димеров изоформой ITSN1-22a. Выводы. ITSN1-22a – это единственная на сегодня изоформа ITSN1, способная образовывать гомодимеры посредством дисульфидных связей, что может быть важно для формирования ITSN1-содержащих белковых комплексов. Ключевые слова: интерсектин 1, изоформа, гомодимер. 2011 Article Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus / M.V. Dergai, O.V. Dergai, L.O. Tsyba, O.V. Novokhatska, I.Ya. Skrypkina, A.V. Rynditch // Вiopolymers and Cell. — 2011. — Т. 27, № 4. — С. 306-309. — Бібліогр.: 14 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000110 http://dspace.nbuv.gov.ua/handle/123456789/156042 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	Short Communications Short Communications
spellingShingle	Short Communications Short Communications Dergai, M.V. Dergai, O.V. Tsyba, L.O. Novokhatska, O.V. Skrypkina, I.Ya. Rynditch, A.V. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus Вiopolymers and Cell
description	Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN1- 22a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules. Keywords: intersectin 1, isoform, homodimer.
format	Article
author	Dergai, M.V. Dergai, O.V. Tsyba, L.O. Novokhatska, O.V. Skrypkina, I.Ya. Rynditch, A.V.
author_facet	Dergai, M.V. Dergai, O.V. Tsyba, L.O. Novokhatska, O.V. Skrypkina, I.Ya. Rynditch, A.V.
author_sort	Dergai, M.V.
title	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus
title_short	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus
title_full	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus
title_fullStr	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus
title_full_unstemmed	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus
title_sort	novel isoform of adaptor protein itsn1 forms homodimers via its c-terminus
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2011
topic_facet	Short Communications
url	http://dspace.nbuv.gov.ua/handle/123456789/156042
citation_txt	Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus / M.V. Dergai, O.V. Dergai, L.O. Tsyba, O.V. Novokhatska, I.Ya. Skrypkina, A.V. Rynditch // Вiopolymers and Cell. — 2011. — Т. 27, № 4. — С. 306-309. — Бібліогр.: 14 назв. — англ.
series	Вiopolymers and Cell
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fulltext	SHORT COMMUNICATIONS Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus M. V. Dergai, O. V. Dergai, L. O. Tsyba, O. V. Novokhatska, I. Ya. Skrypkina, A. V. Rynditch Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 m.dergai@gmail.com Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN1- 22a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dime- rization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclu- sions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules. Keywords: intersectin 1, isoform, homodimer. Introduction. Intersectin 1 (ITSN1) is a widely expres- sed cytosolic, multidomain scaffold protein, which is conserved from worms to humans. This protein coordi- nates several pathways controlling clathrin-mediated endocytosis as well as exocytosis. Initially the role of ITSN1 in endocytosis was de- monstrated at invagination and fission stages [1, 2]. Re- cently, ITSN1 has been shown to participate in other processes including the determination of cell polarity, cell cycle progression [3], mitogenic signalling [4] and cell survival [5] through the activation of the PI3K- AKT pathway [5]. Previously, two major ITSN1 isoforms were descri- bed: the ubiquitously expressed short isoform (ITSN1- s) and the long isoform (ITSN1-l), which are predomi- nantly expressed in brain [6]. ITSN1-s possesses two EH (Eps15 homology) domains at the N-terminus, fol- lowed by a coiled-coil region (CCR) and five SH3 do- mains (Src homology domains A-E). ITSN1-l additio- nally contains a C-terminal extension consisting of a conserved tandem DH (Dbl homology) and PH (Pleks- trin homology) domains, and a C2 domain [6]. Protein interactions of these ITSN1 isoforms were extensively studied during last decade (reviewed in [7]). Splicing affecting 5' UTR of ITSN1 transcripts was also shown [8]. Recently we have identified a novel isoform of ITSN1 that possess unique C-terminal domain instead of four last SH3 domains on its C-terminus [9]. This iso- form is produced by inclusion of the alternative exon 22a. ITSN1-22a migrated in SDS-PAGE as two immu- noreactive bands. Here we report that recently identifi- 306 ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 4. P. 306–309  Institute of Molecular Biology and Genetics, NAS of Ukraine, 2011 307 NOVEL ISOFORM OF ADAPTOR PROTEIN ITSN1 FORMS HOMODIMERS VIA ITS C-TERMINUS ed isoform ITSN1-22a forms homodimers via the C-ter- minal domain. Materials and methods. Antibodies. Polyclonal antibodies against the EH2 domain of ITSN1 [10] and anti-CTD antibodies [9] were described previously. Monoclonal anti-Omni (D-8, sc-7270) antibody was pur- chased from «Santa Cruz Biotechnology» (USA). DNA constructs. Constructs encoding Omni- ITSN1-22a, Omni-ITSN1-22a∆86, GST-SH3A-CTD and GST-SH3A-CTD∆39 were described previously [9]. Site-directed mutagenesis was carried out with reverse primer 5'-atgcggccgctcacaagtaatggggaagaGC gagataaaaaGCaccaa-3'. Protein expression, pull-down assays and Western blot analysis were conducted as described previously [9]. Cell culture and transfection. HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum, 50 U/ml penicillin and 100 µg/ml streptomycin. The cells were transiently transfected using polyethylenimine transfection reagent (JetPEI, Polyplus Transfection) and processed 24 to 36 h after transfection. Immunoprecipitation. For immunoprecipitation (IP) cells were lysed in IP buffer (20 mM Tris-HCl pH 7.5, 0.5 % NP40, 100 mM NaCl, 10 % glycerol, 1 mM PMSF and protease inhibitor cocktail). HEK293 cell lysate was mixed with antibodies and protein A/G. Af- ter overnight incubation at 4 °C the beads were washed three times with IP buffer. Bound proteins were eluted by boiling in Laemmli sample buffer and analysed by SDS-PAGE and Western blot. Inhibition of endocytosis. Endocytosis was inhi- bied by potassium depletion [10]. Cells were washed twice with depletion buffer (20 mM HEPES, pH 7.4, 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 g/l D- glucose). Subsequently, the cells were incubated for 5 min with a hypotonic buffer consisting of one part de- pletion buffer and one part H2O. Then, the cells were in- cubated for a further 30 min in depletion buffer at 37 °C. Control cells were incubated with the same buffer sup- plemented with 10 mM KCl. Results and discussion. To investigate functional features of ITSN1-22a isoform, a construct encoding Omni-tagged ITSN1-22a (Fig. 1, А) was expressed in HEK293 cells. Western blot analysis of ITSN1-22a im- munoprecipitates obtained with anti-Omni antibody re- vealed a major band of 116 kDa and an additional band of approximately doubled size (250 kDa). In similar ex- periments performed with ITSN1 short isoform, Omni- ITSN1-s appeared as a single band with the expected molecular weight of 136 kDa (Fig. 1, B). At the same time truncated Omni-ITSN1-22a∆86 mutant (110 kDa) lacking the last 86 amino acids did not produce band of higher molecular weight (Fig. 1, B). From this obser- vation we assumed that ITSN1-22a is able to form di- mers. However, it was not clear weather ITSN-22a forms homodimer or heterodimer with other protein of similar molecular weight. To exclude the presence of eukaryotic cell components we expressed the C-termi- nal region of ITSN1-22a in Escherichia coli. The GST- fusion protein containing SH3A domain and the C-ter- minal domain of ITSN1-22a (GST-SH3A-CTD) was purified from E. coli and visualized in Western blot as two bands with apparent molecular weight of 60 and 120 kDa (Fig. 1, C). Given that the bacterially expres- sed C-terminal part of ITSN1-22a was able to produce dimer we suggested that homodimerization occurs ra- ther than heterodimerization. To specify more precise- ly region engaged in dimerization we prepared trun- cated form of GST-SH3A-CTD lacking 39 residues at 170 130 100 IP: Omni-ITSN1-22a Omni-ITSN1-22a∆86 Omni-ITSN1-s + + + + + Omni-ITSN1-22a dimer Omni-ITSN1-s Omni-ITSN1-22a monomer Omni-ITSN1-22a∆86 B 120 56 Dimer Monomer WB: anti-CTD WB: anti-EH2 C Omni-ITSN1-22a Omni-ITSN1-22a)86 GST-SH3A-CTD GST-SH3A-CT∆D39 EH2 EH2 SH3ACCR CTD Omni EH2 EH2 CCR CTD Omni CTD GST CTD GST A 1 2 kDa kDa 1 2 SH3A SH3A SH3A Fig. 1. Novel isoform of ITSN1 adaptor forms dimer in vivo: A – schematic representation of recombinant proteins used in this study; B – HEK293 cells expressing Omni-ITSN1-s, Omni-ITSN1-22a and ITSN1-22a∆86 were lysed 24 h after transfection; cell lysates were subjected to immunoprecipitation with anti-Omni antibody or normal mouse serum (NMS) as a negative control followed by immunobloting with anti-EH2 antibodies; C – GST-SH3A-CTD and GST-SH3A- CTD∆39 were expressed in E. coli and affinity purified; recombinant proteins were immunoblotted with anti-CTD antibodies C-terminus (SH3A-CTD∆39). Western blot analysis demonstrated that SH3A-CTD∆39 did not form dimers (Fig. 1, C). Thus, the C-terminal residues of ITSN1-22a are supposed to be responsible for the formation of ho- modimers. We suggested that disulphide bonds could be en- gaged in homodimerization of ITSN1-22a. Non-cova- lent protein-protein interactions are unlikely given that ITSN1-22a dimer sustained prolonged boiling. Boiling of ITSN1-22a with mercaptoethanol during 30 min did not affected the amount of dimer significantly reducing it less than in 20 % of cases (data not shown). Homo- dimers formed by disulphide bonds could be extremely stable against mercaptoethanol [11–13]. Three cystei- nes were found within 39 C-terminal residues that con- tained clustering signal. The residue C1016 is conser- ved in all mammals whereas C990 and C1019 are spe- cific only for primates (data not shown). To assess a possible role of the cysteines C1016 and C1019 in ho- modimerization, we substituted alanines for these resi- dues (ITSN1-22aCA). Fig. 2 shows that the mutations significantly decreased the amount of dimerized form of ITSN1-22aCA in comparison with wild type ITSN1- 22a. However, these substitutions did not abolish dimerization completely (Fig. 2). Presumably, C990 is also able of forming disulphide bonds, however, its im- pact on dimerization is moderate in comparison with C1016 and C1019. We investigated cellular processes in which dime- rization could occur. As ITSN1 is a well-established endocytic protein we studied whether the formation of ITSN1-22a dimer depends on the endocytic activity. Our data (Fig. 3, A) indicated that dimerization of ITSN1-22a was not affected by inhibition of endocy- tosis and disruption of endocytic sites by potassium depletion. The dimerization of ITSN1-22a was also in- dependent on mitogenic stimulation of HEK293 cells with epidermal growth factor (EGF) (Fig. 3, B). Thus, the obtained data demonstrate that the novel isoform of ITSN1 adaptor ITSN1-22a is capable to form homodimers due to the C-terminal clustering sig- nal. Previously, Guipponi et al. proposed the coiled-co- il region of ITSN1 molecule as putative oligomeriza- tion signal [14], however, so far there are no published data confirming this interaction. The CTD of ITSN1- 22a is responsible for this isoform homodimerization signal. The dimerization of ITSN1-22a is not regulated during clathrin-mediated endocytosis or signaling from EGFR. The physiological significance of ITSN1-22a dimers remains a challenge for future investigations. М. В. Дер гай, О. В. Дер гай, Л. О. Циба, О. В. Но во хаць ка, І. Я. Скрипкіна, А. В. Рин дич Нова ізо фор ма адап тер но го білка ITSN1 фор мує го мо ди ме ри за по се ред ниц твом влас но го С-кінце во го до ме ну Ре зю ме Мета. Раніше нами іден тифіко ва но нову ізо фор му адап тер но го білка ITSN1, на зва ну ITSN1-22a. Вес терн-блот аналіз ви я вив на - явність двох іму но ре ак тив них смуг 120 і 250 кДа, що відповіда - 308 DERGAI M. V. et al. 170 130 100 A WB: anti-EH2 Anti-OmniIP: NMS Dimer Monomer B kDa 1 2 0.0 0.1 0.2 0.3 0.4 0.5 0.61 2 3 Fig. 2. Disulphide bonds within ITSN1-22a mediate homodimeriza- tion: A – HEK293 cells expressing Omni-ITSN1-22a (1, 2) or ITSN1-22aCA(3) were lysed 24 h after transfection; recombinant proteins were precipitated with anti-Omni antibody with subsequent Western blot with anti-EH2 antibodies; B – diagram demonstrates the amount of dimer of ITSN1-22a(1) and ITSN1-22aCA (2) normalized to corresponding monomer; densitometric analysis was performed with ImageJ software 170 130 100 WB: anti-EH2 IP: Anti-Omni Dimer Monomer A B 170 130 100 WB: anti-EH2 IP: Anti-Omni kDa kDa Dimer Monomer Fig. 3. Dimerization of ITSN1-22a does not depend on clathrin-media- ted endocytosis and mitogen stimulation: A – HEK293 cells expressing Omni-ITSN1-22a were treated to inhibit clathrin-mediated endocy- tosis (– KCl) or recover this process (+ KCl); precipitated with anti- Omni antibody recombinant ITSN1-22a was subjected to Western blot with anti-EH2 antibodies; B – immunoblotting of precipitated with anti-Omni antibody Omni-tagged ITSN1-22a from mitogen-starved (– EGF) and stimulated with 50 ng/ml EGF (+ EGF) HEK293 cells. Cells were mitogen-starved for 12 h and then stimulated by growth fac- tor for 10 min 309 NOVEL ISOFORM OF ADAPTOR PROTEIN ITSN1 FORMS HOMODIMERS VIA ITS C-TERMINUS ють ITSN1-22a. Мета цієї ро бо ти по ля га ла у вив ченні здат ності но вої ізо фор ми фор му ва ти ди ме ри. Ме то ди. Іму ноп ре ципіта- цію і Вес терн-блот аналіз ви ко рис та но для досліджен ня здат - ності ізо фор ми до ди ме ри зації. Іден тифікацію ділян ки, що відпо- відає за фор му ван ня ди мерів, здійсню ва ли за до по мо гою сайт- спря мо ва но го му та ге не зу та де леційно го аналізу. Інгібу вання ен - до ци то зу інду ку ва ли дефіци том іонів калію. Для міто ген ної сти - му ляції клітин HEK293 за сто со ва но епідер маль ний фак тор рос- ту. Ре зуль та ти. Нами ви яв ле но, що над е кспре со ва на в кліти нах НЕК293 ізо фор ма ITSN1-22a фор мує ди ме ри. Ди ме ри зація обу- мов ле на спе цифічним для цієї ізо фор ми С-кінце вим до ме ном. На- ми по ка за но, що цис теї ни С1016 і С1019 за лу чені до про це су го - мо ди ме ри зації. Інгібу ван ня клат рин-опо се ред ко ва но го ен до ци то - зу та сти му ляція клітин міто ге ном не впли ва ють на фор му ван ня ди мерів ізо фор мою ITSN1-22a. Вис нов ки. ITSN1-22a – це єдина відома на сьо годні ізо фор ма ITSN1, здат на утво рю ва ти го мо ди - ме ри за до по мо гою дис ульфідних зв’язків, що може бути важ ли - вим для фор му ван ня ITSN1-вмісних білко вих ком плексів. Клю чові сло ва: інтер сек тин 1, ізо фор ма, го мо ди мер. Н. В. Дер гай, А. В. Дер гай, Л. А. Цыба, О. В. Но во хац кая, И. Я. Скрип ки на, А. В. Рын дич Но вая изо фор ма адап тер но го бел ка ITSN1 фор ми ру ет го мо ди ме ры по сре дством сво е го С-кон це во го до ме на Ре зю ме Цель. Ра нее мы иден ти фи ци ро ва ли но вую изо фор му адап тер но го эн до ци тоз но го бел ка ITSN1, на зван ную ITSN1-22a. Вес терн-блот ана лиз вы я вил на ли чие двух им му но ре ак тив ных по лос 120 и 250 кДа, со от ве тству ю щих ITSN1-22a. Цель этой ра бо ты со сто я ла в из уче нии воз мож нос ти фор ми ро ва ния ди ме ров изо фор мой ITSN1-22a. Ме то ды. Имму ноп ре ци пи та ция и Вес терн-блот ана - лиз ис поль зо ва ны для ис сле до ва ния воз мож нос ти ди ме ри за ции изо фор мы. Иден ти фи ка цию учас тка, от ве ча ю ще го за ди ме ри за - цию, осу ще ствля ли с по мощью сайт-на прав лен но го му та ге не за и де ле ци он но го ана ли за. Инги би ро ва ние эн до ци то за ин ду ци ро ва ли де фи ци том ио нов ка лия. Для ми то ген ной сти му ля ции кле ток при- ме нен эпи дер маль ный фак тор рос та. Ре зуль та ты. Нами об на - ру же но, что сверх э кспрес си ро ван ная в клет ках НЕК293 изо фор - ма ITSN1-22a фор ми ру ет ди ме ры. Ди ме ри за ция изо фор мы обу- слов ле на ее спе ци фи чес ким С-кон це вым до ме ном. Нами по ка за но, что цис те и ны С1016 и С1019 вов ле че ны в про цесс го мо ди ме ри за - ции. Инги би ро ва ние клат рин-опос ре до ван но го эн до ци то за и ми - то ген ная сти му ля ция кле ток не вли я ют на фор ми ро ва ние диме- ров изо фор мой ITSN1-22a. Вы во ды. ITSN1-22a – это еди нствен - ная на се го дня изо фор ма ITSN1, спо соб ная об ра зо вы вать го мо ди - ме ры по сре дством дис ульфид ных свя зей, что мо жет быть важ- но для фор ми ро ва ния ITSN1-со дер жа щих бел ко вых ком плек сов. Клю че вые сло ва: ин тер сек тин 1, изо фор ма, го мо ди мер. REFERENCES 1. Sengar A. S., Wang W., Bishay J., Cohen S., Egan S. E. The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15 // EMBO J.–1999.–18, N 5.–P. 1159–1171. 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. Chabu C., Doe C. Q. Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression // Develop- ment.–2008.–135, N 16.–P. 2739–2746. 4. Mohney R. P., Das M., Bivona T. G., Hanes R., Adams A. G., Phi- lips M. R., O’Bryan J. P. 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Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

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