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Thesis Reference Thesis The interplay between endosomal sorting of the epidermal growth factor receptor and signaling BRANKATSCHK, Ben Abstract The epidermal growth factor (EGF)-induced removal of the EGF receptor (EGFR) from the plasma membrane and its endocytic downregulation is a major negative feedback mechanism controlling the intensity and duration of receptor signaling. Different mechanisms of ligand-accelerated endocytosis, rapid ubiquitination of activated EGFR, and sorting of the receptor into multivesicular bodies for lysosomal degradation, are the underlying principles of EGFR downregulation. The physical degradation of the EGFR is thought to protect cells from excessive stimulation. In addition, sequestering the receptor into intralumenal vesicles of endosomes, thereby uncoupling the intracellular tyrosine kinase domain from its downstream effectors, is proposed to contribute to signal attenuation. On the other hand, endosomal EGFR can be active and is able to compensate for signaling initiated at the plasma membrane. How the pool of active endosomal receptor is regulated, and to what extend it contributes to the biological response, has not been investigated conclusively to date. In this study, we aimed to dissect the precise contribution of [...] Reference BRANKATSCHK, Ben. The interplay between endosomal sorting of the epidermal growth factor receptor and signaling. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4265 URN : urn:nbn:ch:unige-142755 DOI : 10.13097/archive-ouverte/unige:14275 Available at: http://archive-ouverte.unige.ch/unige:14275 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie Département de biochimie Professeur Jean Gruenberg The Interplay Between Endosomal Sorting of the Epidermal Growth Factor Receptor and Signaling THÈSE présentée à la Faculté des sciences de l´Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie par Ben BRANKATSCHK de Bautzen (Allemagne) Thèse N° 4265 GENÈVE Atelier ReproMail 2010 Acknowledgements Jean, I am deeply grateful for your guidance during my PhD in your lab. Already the first time when I visited Geneva I was very impressed about you taking so much of your time to discuss, get to know to each other, and to show me around. This good feeling never vanished, and I wish to express my gratitude for your ability to always find the time for helpful troubleshooting or fundamental discussions. Many ideas originated from those spontaneous debates that led me through my research and taught me plenty about cell biology. Thank you also for your patience, for creating a truly pleasant and productive atmosphere in the lab, and for constant support, allowing me to pursue my scientific interests in great depth. Merci! Best of thanks to everybody in the lab! It is quite outstanding to be working within such a relaxed, cheerful and cooperative crew. Vero, Pierre, Zeina, Etienne, Cameron, Stefania, Fabrizio, Thomas, Karine, Katarina, Alejandra, Christin, Cansel, Shem, you rock! I am also much obliged to Brigitte, Marie-Claire, and Marie-Helene, for taking care of business, making the lab run smooth and our work more efficient. Special thanks to my good friend and collaborator Sven the Wichert. For ten years now, we have been experiencing a lot together, and it was both a challenge and a pleasure to do the microarray project with you. I have also learned a great deal about how to extract and illustrate information from large amounts of data, and I hope we will carry on in combining friendship and work. Danke. Many thanks to Moritz Rossner from the Max Planck Institute of Experimental Medicine in Göttingen for supporting this project in the course of a very interesting collaboration – to be continued. I am indebted to Olivier Schaad for the tremendous help in the handling and analysis of the microarray and NanoString data. Cheers to the Genomics Platform team, particularly to Mylène Docquier, Didier Chollet, and Patrick Descombes, for aiding two rather large-scale NanoString measurements in the summer heat of Geneva. I would also like to thank Christoph Bauer, Jérôme Bosset, and Michal Parkan from the Imaging Platform, for assistance with microscopy, image analysis, and computer issues, and for always being good for a joke! Sincere thanks to Pierre Cosson, Marcos González-Gaitán, and Moritz Rossner, for agreeing to be in my thesis defense committee and for reading through this piece of work. At this point, I must express my deepest and warmest gratitude to my family and to friends who became family. Iris, Ńćěpan, Jürgen, Mat, Bož, a Marke, mejće dźak! Basti und Sven, ich bin froh dass ich in euch Freunde für´s Leben gefunden habe. Chris, verdammt schade dass du nicht mehr bei uns bist, in Gedanken wirst du es immer sein. Teemu, dude, what would Geneva have been like without you? Thanks for everything! Best of thanks also to Henry, Tom, Alba, Andrea, Fabiana, Carole, Shem, Gordon, Leo, and many others, for enjoying ourselves together, frequent visits to good old Ferbi included, and for painting the city red on many an occasion. Your friendship is invaluable. Wutrobity a wosebity runks hińće raz na starych přećelow z Łužicy. Mat, Bož, Marke, Merč, Flaki, Micha, boroggnbezacunka, a wjele druhich, bjez was bych zawěsće žno dawno wobłudnił. Rjesće nutř a dale tak! Najwutrobnińi dźak słuńa mojej swójbje, bjez kotrejež mój studij njeby móžny był. Nano, mać a Jürgen, sym móhł stajnje na wańu podpěru ličić, a to je mi njesměrnje wjele hódne. Dźakuju za wńitko! Ben Table of contents Table of contents Summaries III 1. English summary of the thesis III 2. Résumé de la thèse en français V Introduction 1 1. The ERBB family of receptor tyrosine kinases and their ligands 1 1.1. Members of the ERBB family of receptor tyrosine kinases 1 1.2. The structure of ERBB ligand-binding domains 3 1.3. Members of the EGF family of ligands 4 1.4. Structure and processing of EGF family ligands 5 1.5. Ligand binding and ERBB receptor activation 6 1.6. Intracellular activation of the EGFR kinase domain 8 1.7. Regulatory sequences in the ERBB intracellular regions 10 2. The network of ERBB signaling 11 2.1. Phosphosites in the cytoplasmic tail of ERBBs as docking platforms 12 2.2. The initiation of signaling networks downstream of ERBBs 14 2.3. The RAF-MEK-ERK mitogen-activated protein kinase cascade 17 2.4. The architecture of transcriptional responses induced by ERBB ligands 19 2.5. Biological outcomes of differential signaling in the ERBB system 21 2.5.1. Biological responses of different EGF family ligands 21 2.5.2. Signaling specificity of distinct ligand-induced heterodimers 22 2.6. Intracellular modulation of ERBB signaling 24 2.6.1. Negative feedback regulators of the ERBB-mediated signaling cascade 25 2.6.2. Coordination of the ERK MAPK cascade by scaffold proteins 33 3. Receptor trafficking in the endosomal system 40 3.1. Overview of the endosomal system 40 3.1.1. Determinants of organelle identity in the endosomal system 41 3.1.2. Pathways of entry into cells 43 3.1.3. Recycling from early endosomes as the first sorting station 44 3.1.4. Cargo sorting to late endosomes as the second sorting station 45 3.1.5. The role of cholesterol and LBPA in late endosomal dynamics 51 3.1.6. Cargo delivery to lysosomes as the terminal degradative compartment 54 3.2. The EGFR as a model to study the interplay of trafficking and signaling 55 3.2.1. Internalization routes of the EGFR 56 3.2.2. EGFR sorting at early endosomes 59 3.2.3. EGFR sorting at late endosomes and delivery to lysosomes 63 3.2.4. The interplay between EGFR trafficking and signaling 64 4. Project outline 70 I Table of contents Results 71 1. Establishing a live-cell signaling assay to measure ELK1-driven transcription 71 2. EGFR signaling in cells depleted of ESCRTs or ESCRT-associated proteins 72 3. EGFR degradation and signaling under different EGF stimulation conditions 78 4. EGF signaling upon interference with EGFR internalization and ubiquitination 80 5. Outlook: specific effects of HRS and TSG101 depletion, and late EGFR activity 84 Discussion 86 Materials and Methods 96 1. Reagents, antibodies, siRNAs, and constructs 96 2. Cell culture, transfection, EGF stimulation, and harvest of cells 97 3. Live-cell signaling assay and quantitative real-time RT-PCR 98 4. Sample preparation for microarray analysis 98 5. mRNA measurements using the NanoString technology 99 6. Software used for data analysis 100 Bibliography 101 Appendix 140 1. List of abbreviations 140 2. Target sequences of ON-TARGETplus SMARTpool siRNAs from Dharmacon 145 3. Target sequences of NanoString probes 146 4. Microarray analysis – EGF response genes 150 5. Microarray analysis – knockdown effects 154 6. Manuscript “The phosphoinositide-binding protein SNX16 controls the formation (158) of tubulo-cisternal membrane domains in late endosomes” - 1 - II Summaries Summaries 1. English summary of the thesis The epidermal growth factor (EGF)-induced removal of the EGF receptor (EGFR) from the plasma membrane and its endocytic downregulation is a major negative feedback mechanism controlling the intensity and duration of receptor signaling. Different mechanisms of ligand-accelerated endocytosis, rapid ubiquitination of activated EGFR, and sorting of the receptor into multivesicular bodies for lysosomal degradation, are the underlying principles of EGFR downregulation. The physical degradation of the EGFR is thought to protect cells from excessive stimulation. In addition, sequestering the receptor into intralumenal vesicles of endosomes, thereby uncoupling the intracellular tyrosine kinase domain from its downstream effectors, is proposed to contribute to signal attenuation. On the other hand, endosomal EGFR can be active and is able to compensate for signaling initiated at the plasma membrane. How the pool of active endosomal receptor is regulated, and to what extend it contributes to the biological response, has not been investigated conclusively to date.
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