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

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

VI

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

VII

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