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EGFR Phosphorylation Determines Its Progression From Early ... · PDF file machinery, compartments and pathways that ensure the integrity, sorting and correct contents of the endomembrane

Mar 12, 2020

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  • EGFR Phosphorylation Determines Its Progression From Early To Late

    Endosomes

    An Analysis of Receptor-Mediated Endocytosis by Advanced Confocal Imaging

    Merete Storflor

    Thesis for the Master of Science degree in Molecular Biology

    UNIVERSITY OF OSLO

    2015

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    © Merete Storflor

    2015

    EGFR phosphorylation determines its progression from early to late endosomes - An analysis

    of receptor-mediated endocytosis by advanced confocal imaging

    Merete Storflor

    http://www.duo.uio.no/

    Press: Reprosentralen, University of Oslo

    http://www.duo.uio.no/

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    Table of Contents

    Abstract .................................................................................................................................... IX

    Acknowledgements .................................................................................................................. XI

    Abbreviations ........................................................................................................................ XIII

    1 Introduction ...................................................................................................................... 17

    1.1 Endocytosis ................................................................................................................ 18

    1.1.1 Receptor-Mediated Endocytosis ........................................................................ 19

    1.1.2 Coat Proteins ...................................................................................................... 20

    1.1.3 Clathrin ............................................................................................................... 20

    1.1.4 Clathrin-Mediated Endocytosis .......................................................................... 21

    1.1.5 Clathrin-Independent Endocytosis ..................................................................... 22

    1.2 Epidermal Growth Factor Receptor ........................................................................... 23

    1.2.1 Epidermal Growth Factor, EGF ......................................................................... 23

    1.2.2 ErbB Family ....................................................................................................... 24

    1.2.3 EGFR Structure .................................................................................................. 25

    1.2.4 Conformational Change & Dimerization ........................................................... 26

    1.2.5 Activation ........................................................................................................... 27

    1.2.6 EGFR Signaling ................................................................................................. 28

    1.3 The Endocytic Pathway ............................................................................................. 29

    1.3.1 Cytoskeleton ....................................................................................................... 29

    1.3.2 Endosomal Sorting ............................................................................................. 30

    1.3.3 Rab Proteins ....................................................................................................... 30

    1.3.4 Early Endosomes ................................................................................................ 31

    1.3.5 Recycling ............................................................................................................ 33

    1.3.6 Multivesicular Bodies, MVBs ............................................................................ 34

    1.3.7 Ubiquitination ..................................................................................................... 35

    1.3.8 Late Endosomes ................................................................................................. 36

    1.3.9 Lysosomes .......................................................................................................... 37

    1.4 EGFR and Cancer ...................................................................................................... 38

    1.4.1 Therapies ............................................................................................................ 38

    2 Aim of the Study .............................................................................................................. 39

    3 Materials and Methods ..................................................................................................... 41

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    3.1 Cell Culture: Maintenance ......................................................................................... 41

    3.2 Cell Treatment ........................................................................................................... 41

    3.3 Microbiological Techniques ...................................................................................... 42

    3.4 Imaging Techniques .................................................................................................. 43

    3.5 DNA Techniques ....................................................................................................... 47

    3.6 Protein Techniques .................................................................................................... 51

    4 Results .............................................................................................................................. 53

    4.1 EGFR Sorting and Trafficking Analysis ................................................................... 53

    4.2 Rab5 Colocalization: Receptor Phosphorylation Determines Rab5-mCherry

    Recruitment .......................................................................................................................... 55

    4.2.1 Establishing the Wt- EGFR Progression through Early Stage Endocytic

    Trafficking ........................................................................................................................ 56

    4.2.2 Y1-Mutant (Y1045F) Shows Delayed Progression Towards Early Endosomes 58

    4.2.1 Y2-Mutant (Y1068/1086F) Shows Similar Trafficking to Wt-EGFR ............... 61

    4.2.2 Y3-Mutant (Y1045/1068/1086F) Induces a Significantly Altered Receptor

    Trafficking ........................................................................................................................ 63

    4.2.3 EGFR Trafficking is Determined by Receptor Phosphorylation ....................... 67

    4.2.4 Colocalization Rates ........................................................................................... 69

    4.2.5 Internalization and Initial Colocalization ........................................................... 70

    4.3 Rab7 Colocalization: Y3-Mutant Evades Degradation ............................................. 72

    4.3.1 Wt-EGFR was Intraluminally Sorted in Late Endosomes ................................. 73

    4.3.2 Y3-EGFR did not internalize in Rab7-mApple Positive Endosomes. ............... 76

    4.3.3 EGFR Phosphorylation Affects Trafficking Towards Late Endosomes ............ 79

    4.4 Y3-EGFR Showed Impaired Sorting to Lysosomes .................................................. 81

    4.5 EGFR Degradation .................................................................................................... 83

    4.6 Inhibition of Clathrin-Mediated Endocytosis by Pharmacologic Inhibitor, Pitstop2 85

    5 Discussion ........................................................................................................................ 89

    5.1 Phosphorylation Pattern Determines Receptor Trafficking ....................................... 89

    5.2 Phosphorylation Pattern: Cbl & Grb2 Functions ....................................................... 90

    5.2.1 Grb2 Mediates Internalization ............................................................................ 91

    5.2.2 Cbl is Involved in Receptor Trafficking ............................................................ 91

    5.2.3 Induced Instability in Endosome Maturation ..................................................... 92

    5.2.4 Ubiquitin-Threshold-Dependent Trafficking ..................................................... 92

    5.3 Inhibition of CME by Pitstop2 .................................................................................. 93

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    5.4 Conclusion ................................................................................................................. 93

    6 Future Perspectives .......................................................................................................... 95

    7 References ........................................................................................................................ 97

    8 Appendix ........................................................................................................................ 107

    8.1 List of Materials ....................................................................................................... 107

    8.2 Buffers and Solutions .............................................................................................. 109

    8.3 Protocol

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