UNDERSTADNING THE REGULATION OF ENDOGENOUS TRPV2 BY GROWTH FACTORS IN NEURONAL CELLS by MATTHEW RYAN COHEN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Vera Moiseenkova-Bell Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY January 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of MATTHEW RYAN COHEN candidate for the degree of Physiology and Biophysics*. Witold Surewicz (Committee Chair) Sudha Chakrapani Vera Moiseenkova-Bell Xin Qi William Schilling David Van Wagoner November 5, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of tables VI List of figures VII Acknowledgements X Abstract XI CHAPTER 1: INTRODUCTION 1 1.1 The Transient Receptor Potential family of ion channels 2 1.2 TRPV channels as thermosensors 2 1.3 Domain structure of thermoTRPV channels 3 1.4 Involvement of TRPV2 in sensory transduction 4 1.5 Pharmacological Activation and Inhibition of TRPV2 6 1.6 Subcellular trafficking of TRPV2 10 1.7 Involvement of TRPV2 in nervous system function and axon extension 12 1.8 Neurotrophins and their receptors 13 1.9 Retrograde transport of neurotrophin signals 17 1.10 Ca2+ as a second messenger in neurotrophin signaling 20 1.11 TRP channels in neurite outgrowth and neurotrophin signaling 21 1.12 Purpose of this study 23 1.13 Figures 25 1.14 Table 33 i CHAPTER 2: EFFECT OF GROWTH FACTORS, INCLUDING IGF-1, ON TRPV2 TRANSLOCATION TO THE PLASMA MEMBRANE 34 2.1 Introduction 35 2.2 Materials and Methods 38 2.2.1 Ethics Statement 38 2.2.2 Plasmids 38 2.2.3 Protein expression and purificiation 38 2.2.4 TRPV2 antibody generation 39 2.2.5 Commercially available antibodies 39 2.2.6 Cell culture and transfection 39 2.2.7 Mouse tissue lysate generation and immunoprecipitation 40 2.2.8 Western blot analysis 41 2.2.9 Immunocytochemistry 41 2.2.10 Biotinylation of cell surface proteins 42 2.3 Results 43 2.3.1 Translocation of overexpressed TRPV2 in response to growth factors 43 2.3.2 Detection of recombinant TRPV2 and determination of TRPV2 binding region 45 2.3.3 Recognition of endogenously expressed TRPV2 46 2.3.4 Immunoprecipitation of TRPV2 from mouse brain and heart 47 2.3.5 Detection of TRPV2 by immunocytochemistry 48 2.3.6 Effect of IGF-1 on cell surface expression of TRPV2 49 2.4 Discussion 50 ii 2.5 Figures 55 CHAPTER 3: MAPK/ERK REUGLATES TRPV2 DOWNSTREAM OF NGF TO ENHANCE NEURITE OUTGROWTH 65 3.1. Introduction 66 3.2. Materials and Methods 68 3.2.1 Chemicals and antibodies 68 3.2.2 Cell culture and transfection 69 3.2.3 Dissociation and culture of primary E18 DRG neurons 69 3.2.4 Plasmids 70 3.2.5 Site-directed mutagenesis 70 2+ 3.2.6 Cytosolic Ca measurements 71 3.2.7 Western blot analysis 72 3.2.8 Removal of N-linked glycans 72 3.2.9 RNA extraction and cDNA synthesis 72 3.2.10 Semiquantitative RT-PCR of TRPV2 and GAPDH 73 3.2.11 Immunofluorescence 74 3.2.12 Cell surface biotinylation 74 3.2.13 Morphology analysis of PC12 cells 74 3.2.14 In vitro kinase assay 75 3.2.15 Isolation of TRPV2 from HEK293T cells and mass spectrometry analysis 76 3.2.16 Statistical analyses 77 3.3. Results 77 iii 3.3.1 TRPV2 is expressed in developing neurons and regulated by NGF 77 3.3.2 TRPV2 enhances NGF-induced neurite outgrowth 80 3.3.3 NGF-induced increase in TRPV2 protein is mediated by MAPK signaling 83 3.3.4 NGF does not induce TRPV2 translocation to the plasma membrane in PC12 cells 85 3.3.5 ERK phosphorylates TRPV2 to enhance neurite outgrowth 86 3.4. Discussion 89 3.5. Figures 95 CHAPTER 4: DISCUSSION AND FUTURE DIRECTIONS 112 4.1. Summary 113 4.2. Generation of monoclonal TRPV2 antibodies suitable for detection of endogenously expressed TRPV2 113 4.3. Regulation of TRPV2 by NGF signaling in developing neurons 117 4.4. Remaining questions 122 4.4.1 Is TRPV2 regulated downstream of NGF/MAPK in vivo? 122 2+ 4.4.2 Does TRPV2 activity directly affect endosomal Ca levels? 122 4.4.3 How is TRPV2 targeted to endosomes in developing neurons? 124 4.5. Potential mechanisms by which TRPV2 promotes neurite extension 125 2+ 4.5.1 TRPV2-mediated Ca signals might affect cytoskeletal structure in growing neurites 125 2+ 4.5.2 Potential influence of TRPV2-mediated Ca signals on Rab7 activity and signaling endosome function 127 iv 4.5.3 Potential role of TRPV2 in neuronal regeneration 130 4.5. Concluding remarks 132 4.6. Figures 133 APPENDIX 137 REFERENCES 138 v List of Tables Table 1.1. Modulators of TRPV2 activity 33 vi List of Figures Figure 1.1. The mammalian Transient Receptor Potential (TRP) Family 25 Figure 1.2. Function of TRPV subfamily members 26 Figure 1.3. Domain structure of thermoTRPV channels 27 Figure 1.4. Neurotrophins and their receptors 28 Figure 1.5. Effect of NGF on PC12 cell morphology 29 Figure 1.6. Schematic representing the major signaling pathways activated by NGF 30 Figure 1.7. Cartoon depicting receptor-mediated endocytosis 31 Figure 1.8. Cartoon depicting long-distance retrograde transport of NGF/TrkA-containing signaling endosomes 32 Figure 2.1. Treatment of F11 cells with IGF-1 does not induce translocation of overexpressed TRPV2 to the plasma membrane 55 Figure 2.2. IGF-1 treatment does not induce translocation of overexpressed TRPV2 to the plasma membrane in HeLa cells 56 Figure 2.3. Immuno-detection of recombinant TRPV2 and mapping of the TRPV2 binding region 57 Figure 2.4. TRPV2 monoclonal antibody recognizes endogenous TRPV2 by western blot 58 Figure 2.5. Immunoprecipitation of endogenous TRPV2 59 Figure 2.6. Immunostaining with TRPV2 antibodies 60 Figure 2.7. Regulation of TRPV2 trafficking by insulin-like growth factor-1 in CHO-K1 cells 61 vii Figure 2.8. Effect of IGF-1 on endogenous TRPV2 trafficking in F11 cells 63 Figure 3.1. NGF upregulates TRPV2 in models of developing neurons 95 Figure 3.2. NGF treatment results in sustained upregulation of TRPV2 expression in PC12 cells 96 Figure 3.3. Dominant negative TRPV2 co-assembles with WT TRPV2 98 Figure 3.4. Expression of DN TRPV2 reduces the Ca2+ response of F11 cells to 2-APB 99 Figure 3.5. TRPV2 overexpression enhances neurite outgrowth in F11 cells 100 Figure 3.6. TRPV2 activity enhances NGF-induced neurite outgrowth in PC12 cells 101 Figure 3.7. Silencing TRPV2 expression impairs NGF-induced neurite outgrowth 102 Figure 3.8. MAPK signaling mediates upregulation of TRPV2 103 Figure 3.9. NGF does not induce translocation of TRPV2 to the plasma membrane in PC12 cells 105 Figure 3.10. TRPV2 is phosphorylated by ERK 106 Figure 3.11. Mass spectrometry analysis of heterologously expressed TRPV2 to determine phosphorylation sites 107 Figure 3.12. Phosphorylation of TRPV2 by ERK enhances NGF-induced neurite outgrowth in PC12 cells 109 Figure 3.13. Model depicting the proposed mechanism by which NGF- activated MAPK signaling affects TRPV2 to enhance neurite outgrowth 111 viii Figure 4.1. Summary of findings 133 Figure 4.2. Rab GTPase cycle 134 Figure 4.3. Possible mechanism proposing how increased TRPV2 expression might affect longevity of NGF signals 135 Figure 4.4. Mechanism predicting how TRPV2 activity might increase neurite outgrowth 136 ix ACKNOWLEDGEMENTS I am especially grateful to my PhD advisor, Dr. Vera Moiseenkova-Bell. I approached Vera in the summer of 2010 as a first year student with very limited experience in the lab. I am lucky that she took a risk in allowing me to rotate in her lab and also in eventually accepting me as her first graduate student. I think in the end it worked out for both of us. I would also like to thank the members of the Moiseenkova-Bell lab: Kevin Huynh, who taught me all of the basic molecular biology techniques that I used to complete this dissertation; Teresa Cvetkov, who was always present as another mentor in my earlier days in the lab; Amrita Samanta and Xu Han, who both brought a great deal of energy during the latter portion of my time in the lab. I was very lucky to work with four very talented undergraduate students: Christian Marks, Monica Kane, Jennifer Pilat and Connor Dawedeit. In addition, I would like to thank Will Johnson. Will not only provided technical assistance but he also served as an objective critic, an ally, a colleague and a friend. He was certainly essential for taking my project to the next level. Thank you to my dissertation committee (Dr. Witold Surewicz, Dr. William Schilling, Dr. Sudha Chakrapani, Dr. David Van Wagoner and Dr. Xin Qi) for constantly challenging me, forcing me to stay focused and supporting me in my completion of these studies. Additional thanks go to Dr. Richard Zigmond, Alicia Lizowicz and Dr. Jana Kiselar. Thank you to my family for support throughout the past 6 years; to Kathleen – sometimes I feel like I could not have done this without you. Finally, thank you to all of the friends and colleagues who essential for my success during graduate school whom I did not mention here. x Understanding the Regulation of Endogenous TRPV2 by Growth Factors in Neuronal Cells Abstract By MATTHEW RYAN COHEN Transient receptor potential vanilloid 2 (TRPV2) is a non-selective Ca2+- permeable cation channel that belongs to the vanilloid subfamily of the TRP superfamily.