ACTIVATION INDEPENDENT FUNCTIONS OF COLLAGEN RECEPTOR INTEGRINS α1β1 AND α2β1. Maria Salmela - PDF

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ACTIVATION INDEPENDENT FUNCTIONS OF COLLAGEN RECEPTOR INTEGRINS α1β1 AND α2β1 Maria Salmela TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS Sarja - ser. A I osa - tom. 553 Astronomica - Chemica

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ACTIVATION INDEPENDENT FUNCTIONS OF COLLAGEN RECEPTOR INTEGRINS α1β1 AND α2β1 Maria Salmela TURUN YLIOPISTON JULKAISUJA ANNALES UNIVERSITATIS TURKUENSIS Sarja - ser. A I osa - tom. 553 Astronomica - Chemica - Physica - Mathematica Turku 2017 University of Turku Faculty of Mathematics and Natural Sciences Department of Biochemistry MediCity Research Laboratory National Doctoral Programme in Informational and Structural Biology (ISB) Doctoral Programme in Molecular Life Sciences (DPMLS) Supervised by Professor Jyrki Heino Department of Biochemistry University of Turku Turku, Finland Dr. Johanna Jokinen Department of Biochemistry University of Turku Turku, Finland Reviewed by Professor Kid Törnqvist Department of Cell Biology Åbo Akademi University Turku, Finland Professor Kari Airenne FinVector Vision Therapies Oy Kuopio, Finland Opponent Professor Pekka Lappalainen Institute of Biotechnology University of Helsinki Helsinki, Finland The originality of this thesis has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service. ISBN (PRINT) ISBN (PDF) ISSN (Print) ISSN (Online) Painosalama Oy - Turku, Finland 2017 To my Integrin family 4 Abstract Maria Lyydia Salmela Activation independent functions of collagen receptor integrins 1 1 and 2 1 Department of Biochemistry, MediCity Research Laboratory and Doctoral Programme in Molecular Life Sciences, University of Turku, Turku, Finland; National Doctoral Programme in Informational and Structural Biology (ISB), Finland ABSTRACT Cell adhesion to extracellular matrix (ECM) molecules, such as collagen, is mediated by the integrin family of cell surface receptors. Integrins are also exploited by several viruses during cell entry. Integrins literally integrate the ECM to the cytoskeleton by binding ECM ligands with their large head domain, and by connecting to the cytoskeleton through various focal adhesion proteins that bind integrin tails. Integrin tails recruit over 200 focal adhesion proteins to mediate cellular signaling events to control adhesion dependent cell growth and cell movement. Aberrant integrin signaling can lead to uncontrolled cell growth, which in turn can induce cancer and metastases. The affinity of integrins to their ligands, as well as interactions between integrin tails and focal adhesion proteins, are controlled by integrin activity and clustering. Integrins undergo conformational activation from a bent to primed/extended and finally to a fully active conformation. Integrin priming facilitates ligand binding, and ligand interactions further induce full activation through the separation of integrin legs, promoting protein recruitment to focal adhesions and integrin clustering. The bent integrin conformation has been considered nonfunctional, since the ligand binding domain faces the cell membrane and ligand interactions are thus mostly prevented. However, in this thesis work echovirus 1 was shown to interact specifically with the bent conformation of 2 1-integrins (I). Furthermore, it was shown that under flow conditions platelet 2 1-integrins can bind to collagen without receptor priming (III). The recruitment of focal adhesion proteins and the initiation of integrin signaling have been thought to require integrin conformational activation. Here, using echovirus 1 as a model system, I demonstrate that clustered 2 1-integrins can mediate Focal Adhesion Kinase signaling without conformational activation of the integrin (II). Integrins are controlled not only by regulating their conformational activity and clustering but also by influencing their expression levels and the pool on the cell surface. A fourth level of regulation occurs at focal adhesions where integrins are connected to the cytoskeleton. In my thesis I show that during cell spreading the reduced recruitment of the cytoskeleton protein vimentin to focal adhesions induces the formation of lamellipodia independently of 1 1-integrin conformational activation (IV). I also show that 1-integrin-mediated cell adhesion can be prevented without affecting the activity or surface expression of the integrin (V). In conclusion, this thesis emphasizes the importance of the nonactivated integrin conformation in regulating cell adhesion, and demonstrates that integrin mediated functions are regulated at several levels. Keywords: integrins, cell adhesion, cytoskeleton, focal adhesions Tiivistelmä 5 Maria Lyydia Salmela 1 1- ja 2 1-kollageenireseptori-integriinien aktivaatiosta riippumaton toiminta Biokemian laitos, MediCity-tutkimuslaboratorio ja Molekulaaristen biotieteiden tohtoriohjelma, Turun yliopisto, Turku; Bioinformatiikan ja biorakenteiden kansallinen tohtoriohjelma (ISB), Suomi TIIVISTELMÄ Integriinit ovat solukalvon reseptoreita, jotka säätelevät solujen tarttumista soluja ympäröivään soluväliaineeseen, kuten kollageeniin. Myös useat virukset käyttävät integriinejä sitoutuakseen solun pintaan. Integriinien kookas solun ulkopuolinen osa sitoutuu soluväliaineen molekyyleihin eli ligandeihin, ja integriinihäntien ympärille muodostuva adheesiokompleksi yhdistää integriinit solun tukirankaan. Adheesiokompleksin sadat signalointiproteiinit välittävät integriinien signalointia solukalvon puolin ja toisin ohjaten sekä solujen kasvua oikeanlaisessa ympäristössä että tarvittaessa solujen liikettä uuteen kasvuympäristöön. Integriinien muuttunut signalointi johtaa mm. solujen liikakasvuun ja syöpäkasvaimien muodostumiseen. Integriinien toimintaa säädellään integriinien aktivaatiolla ja klusteroimisella, eli muuttamalla integriinien muotoa taipuneesta ojentuneeksi ja tuomalla integriinit lähelle toisiaan. Suuret ligandit sitoutuvat helpommin integriinien ojentuneeseen muotoon. Integriini ligandi vuorovaikutuksen seurauksena tapahtuva integriinien klusteroituminen ja integriinihäntien erottuminen mahdollistavat adheesiokompleksien muodostumisen. Ligandien ei uskota sitoutuvan integriinien taipuneeseen muotoon, jossa sitoutumiskohta on painautuneena vasten solukalvoa ja siten useimpien suurten ligandien ulottumattomissa. Tästä syystä taipuneen muodon ei myöskään uskota osallistuvan solusignalointiin. Väitöstutkimukseni kuitenkin osoittaa ECHO-virus 1:n kykenevän sitoutumaan 2 1-integriinin taipuneeseen muotoon ja klusteroimaan integriinit (I). Lisäksi ECHO-virus 1:ä hyödyntäen havainnollistan, että myös taipuneessa muodossa olevat 2 1-integriinit voivat aktivoida solusignalointia aktivoimalla fokaaliadheesiokinaasia (II). Osoitamme myös verihiutaleiden sitoutuvan verisuonten kollageeniin ilman verihiutaleen pinnalla olevien 2 1-integriinien aktivaatiota (III). Integriinien ilmentyminen sekä pitoisuus solukalvolla vaikuttavat niiden aktivaation ja klusteroitumisen lisäksi integriinien toimintaan. Integriinien toimintaa voidaan säädellä myös muuttamalla niiden yhteyttä solun tukirankaan. Työssäni näytän, miten solujen leviämistä voidaan lisätä irrottamalla solutukirangan vimentiinisäikeiden yhteys adheesiokomplekseihin integriinien aktivaatiosta riippumattomasti (IV). Lisäksi osoitan solujen leviämisen voivan estyä muuttamatta integriinien aktiivisuutta tai pitoisuutta solukalvolla (V). Tutkimukseni painottaa integriinien taipuneen muodon merkitystä soluissa ja korostaa aktivaation ylitse ulottuvaa monipuolisuutta integriinien toiminnassa. Avainsanat: integriini, soluadheesio, solutukiranka, fokaaliadheesiot 6 Contents CONTENTS ABBREVIATIONS... 8 LIST OF ORIGINAL PUBLICATIONS INTRODUCTION REVIEW OF THE LITERATURE Integrins Integrins recognize several ligands Integrin structure Ligand interaction with the integrin I-domain induces integrin outside-in activation Protein interactions with integrin tails induce integrin inside-out activation Integrin clustering enhances ligand binding avidity Echovirus 1 uses 2 1-integrin in the cell entry process Integrin adhesions mediate integrin signaling and control cell attachment and movement Integrin mediated cell adhesions FAK orchestrates integrin adhesions and downstream signaling Integrin induced downstream signaling pathways Integrins connect the cytoskeleton to the extracellular matrix and act as mechanosensors Integrins mediate cell migration AIMS OF THE STUDY MATERIALS AND METHODS RESULTS Nonactivated integrins can bind to echovirus 1 (I) Integrin 2 can interact with EV1 in the nonactivated conformation EV1 induces 2-integrin clustering, but not conformational activation Nonactivated integrins can induce cell signaling (II) Cells can spread on an EV1 coated surface using nonactivated integrins Nonactivated integrins can induce the FAK pathway FAK activation by nonactivated integrins is dependent on PKC and independent of talin... 48 Contents Nonactivated 2-integrins can bind to collagen I under flow (III) Integrin small molecule inhibitors BTT-3033 and BTT-3034 bind to different conformations of 2-integrin BTT-3033 binds to the inactive integrin conformation and prevents platelet binding to collagen I under flow Tumor promoter TPA induces cell spreading and the formation of lamellipodia (IV) Integrin pre-activation (E317A) induces cell adhesion, but does not change the appearance of cell adhesions or increase cell spreading TPA treatment immediately modifies the cytoskeleton and induces cell spreading TPA affects the composition of 1-integrin mediated cell adhesions and significantly increases the amount of kindlin-2 and reduces the amount of vimentin Kindlin-2 is required for cell spreading, but kindlin-2 recruitment is not necessary for TPA effects TPA causes the retraction of vimentin filaments from cell adhesions PIM kinases regulate integrin mediated cell adhesion (V) Inhibition of PIM kinases prevents integrin mediated cell adhesion PIM kinases do not regulate the conformational activity of integrins DISCUSSION AND GENERAL PERSPECTIVES Nonactivated integrins can bind ligands and transmit cellular signaling Nonactivated integrins as virus receptors Currently known interactions mediated by the inactive integrin conformation The benefit of the nonactivated integrin conformation Integrin mediated cell adhesion and spreading can be controlled at several levels Do 1-integrins require conformational activation? Cell spreading and migration after established cell adhesion is controlled through the focal adhesion cell cytoskeleton axis Aspects of integrin based therapy CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES ORIGINAL PUBLICATIONS... 79 8 Abbreviations ABBREVIATIONS Amino acids Acidic D Asp Aspartate E Glu Glutamate Basic R Arg Arginine H His Histidine K Lys Lysine Polar S Ser Serine T Thr Threonine N Asn Asparagine Q Gln Glutamine Y Tyr Tyrosine C Cys Cysteine Nonpolar G Gly Glycine A Ala Alanine I Ile Isoleucine L Leu Leucine M Met Methionine F Phe Phenylalanine W Trp Tryptophan V Val Valine P Pro Proline Abbreviations 9 ADMIDAS adjacent to MIDAS I alpha inserted domain I beta inserted domain DHPCC-9 1,10-Dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde ECM extracellular matrix EDTA ethylenediaminetetraacetic acid EGF(R) epidermal growth factor (receptor) EV1 echovirus 1 FA focal adhesion FAK focal adhesion kinase FAT Focal adhesion targeting domain FERM band 4.1, ezrin, radixin, moiesin -domain ICAM intercellular adhesion molecule 1 ILK integrin linked kinase KD kinase domain LDV leucine-aspartate-valine LIMBS ligand-associated metal binding site MIDAS metal ion-dependent adhesion site PDGF(R) platelet derived growth factor (receptor) PI phosphatidylinositol PI3K phosphoinositide 3-kinase PIM proto-oncogene serine/threonine-protein kinase PIP 2 phosphatidylinositol (4,5)-bisphosphate PIP 3 phosphatidylinositol (3,4,5)-trisphosphate PKC protein kinase C PSI plexin-semaphorin-integrin PTEN phosphatase and tensin homolog RGD arginine-glycine-aspartate shrna short hairpin ribonucleic acid sirna small interfering ribonucleic acid TGF- transforming growth factor beta TPA 12-O-tetradecanoylphorbol-13-acetate (PMA) VCAM vascular cell adhesion protein 1 vwf von Willebrand factor 10 List of Original Publications LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following publications, which will be referred to according to their Roman numerals (I-V) I. Jokinen, J., White, D.J., Salmela, M., Huhtala, M., Käpylä, J., Sipilä, K., Puranen, J.S., Nissinen, L., Kankaanpää, P., Marjomäki, V., Hyypiä, T., Johnson, M., Heino J. Molecular mechanisms of 2 1 integrin interaction with human echovirus The EMBO Journal, vol 29, II. III. IV. Salmela, M., Jokinen, J., Tiitta, S., Rappu, P., Cheng, R.H., Heino, J. Integrin 2 1 in nonactivated conformation can induce focal adhesion kinase signaling. Manuscript. Nissinen, L., Koivunen, J., Käpylä, J., Salmela, M., Nieminen, J., Jokinen, J., Sipilä, K., Pihlavisto, M., Pentikäinen, O.T., Marjamäki, A., Heino, J. Novel 2 1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor pre-activation Journal of Biological Chemistry, vol 287, Salmela, M., Rappu, P., Lilja, J., Niskanen, H., Taipalus, E., Jokinen, J., Heino, J. Tumor Promoter PMA enhances kindlin-2 and decreases vimentin recruitment into cell adhesion sites International Journal of Biochemistry and Cell Biology, vol 78, V. Santio, N., Salmela, M., Arola, H., Eerola, S.K., Heino, J., Rainio, E-M., Koskinen, P. The PIM1 kinase promotes prostate cancer cell migration and adhesion via multiple signaling pathways Experimental Cell Research, vol 342, Introduction 11 1 INTRODUCTION In multicellular organisms, tissues are formed when different types of cells attach to the extracellular matrix (ECM) surrounding the cells. Cell adhesion to the ECM and other cells is strictly regulated by different cell adhesion receptors. Integrins are the most important ECM binding receptors in multicellular animals (Hynes, 2002). The binding of integrins to the ECM and counter receptors on adjacent cells establishes adhesion dependent cell signaling that drives essential cellular functions from differentiation to migration. Since integrins are key players in many normal cellular functions, abnormalities in their behavior can be connected to various diseases. Integrin mediated cell adhesion is regulated at several levels. The first level of regulation is the control of the transcription of the genes coding for integrin subunits. A common feature of cancer cells, which have escaped from the environmental control, is altered integrin expression on the cell surface. The second level of integrin regulation is the recycling of integrins to and from the cell membrane without changing their overall expression. This is important during cell migration, when cells constantly form and break adhesions, and can also be linked to the malignant and metastatic behavior of cancer cells (De Franceschi et al., 2015). In addition, several viruses use integrin trafficking mechanisms to invade cells (Hussein et al., 2015). The third way of influencing the interaction between integrins and ECM ligands is to regulate the conformation of integrins from a bent-inactive to an extended-active molecule (Hynes, 2002). The regulation of integrin activity is very important for directing the timing and place of platelet and immune cell binding, and aberrant regulation can lead to e.g. thrombosis. The final mode of regulating integrin adhesion occurs at cell adhesion complexes in the cytoplasm. These protein complexes transmit integrin signaling from the ECM to the nucleus, thereby changing gene expression and determining the long term response to environmental cues. Adhesion complexes also connect integrins to the cytoskeleton and control the movements of cells (Legate et al., 2009). This thesis focuses on the function and signaling of the collagen receptor integrins 2 1 and 1 1. The importance of the conformational activation of integrins for the binding of platelets to collagen and for the binding of echovirus 1 to its receptor, 2 1- integrin, are studied. In addition, cancer related cell spreading and migration are studied focusing on the regulation of interactions between integrins, cell adhesion complexes and the cytoskeleton. 12 Review of the Literature 2 REVIEW OF THE LITERATURE 2.1 Integrins Integrins are cell surface adhesion receptors that anchor cells to the surrounding extracellular matrix (ECM), as well as to other cells. Through integrins, cells can sense the properties of the pericellular environment, and respond to it (Hynes, 2002). By expressing 24 different integrin heterodimers with different ligand binding abilities, cells can respond to various stimuli coming from their surroundings, and adapt to different environments. While the composition of the ECM regulates cellular functions, the ECM is also constantly modified by the cells. Cell-ECM interactions create a unique environment in each tissue that rises from the diversity of the cells, integrins and ECM molecules involved (Frantz et al., 2010). The ECM is thus as important in regulating cellular functions as are soluble factors, such as growth factors and cytokines (Hynes, 2009). In addition to its composition, also the rigidity of the ECM plays a role in regulating cellular functions. Integrins bind their ligands with their large extracellular domains and integrate the extracellular matrix all the way to the cytoskeleton and the nucleus through their short intracellular tails. By forming a link from the ECM to the cytoskeleton, integrins participate in mechanotransduction and sense the rigidity of the matrix, too (DuFort et al., 2011). Through proteins that bind integrin tails, integrins mediate cellular signaling and control cell survival, proliferation and differentiation. Connections between integrins and the cytoskeleton dictate cell spreading and migration in response to mechanical forces. Due to their ability to regulate cell adhesion and behavior, integrins are involved in tissue morphogenesis, wound healing, and leukocyte and platelet binding (Figure 1) (Legate et al., 2009). PLATELET/LEUKOCYTE ADHESION WOUND HEALING TISSUE MORPHOGENESIS, CANCER ECM Integrins Cell adhesion complex Cell cytoskeleton Nucleus Cell adhesion Cell spreading and migration Cell survival, proliferation and differentiation Figure 1. Integrin adhesions control the very basic functions of cells. The interaction between an integrin and its ligand is strictly regulated and occurs through conformational changes in the integrin structure. Un-ligated integrins stay in their bent, inactive conformation until they are either primed for ligand binding from the inside of the cell, or interact with the ligand and the ligand induces their activation from the outside of the cell (Luo et al., 2007). Inside-out or outside-in activation leads to a firm integrin-ligand interaction and is followed by the recruitment of signaling proteins around the integrin tails. The recruited proteins form larger scaffolds called focal adhesions, where protein-protein interactions activate phosphorylation cascades that finally deliver the integrin dependent signals from the cell membrane to the nucleus (Legate et al., 2009). The details of integrin-ligand interactions, integrin conformational activation, protein binding to the tails of activated integrins, and the Review of the Literature 13 formation of integrin signaling complexes, the focal adhesions, will be discussed in detail in the following chapters. Furthermore, integrin connections both with the ECM and the cytoskeleton enable mechanical sensing and transmission of forces through the integrins. These forces are important for cells to be able to spread and migrate. The role of integrins in mechanotransduction and cell migration will also be discussed Integrins recognize several ligands The ECM is mainly composed of proteoglycans, which function as hydrating and buffering elements, and fibrous proteins such as collagen, fibronectin and laminin, which
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