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 expression and purificiation 38

2.2.4 TRPV2 generation 39

2.2.5 Commercially available 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 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. Despite its discovery as a thermoTRPV channel over 15 years ago,

TRPV2 remains an “orphan TRP” due to its still unclear physiological function.

Here we took advantage of our ability to generate stable, functional recombinant

TRPV2 to gain new biological insights into the cellular function of TRPV2. Using

tetrameric TRPV2 as an antigen, we generated TRPV2 monoclonal antibodies

and used these antibodies to test the controversial hypothesis that growth

factors, including insulin-like growth factor 1 (IGF-1), cause translocation of

TRPV2 to the plasma membrane. We found that TRPV2 localizes to intracellular

membranes in both the absence and presence of growth factors in multiple cell

types, including dorsal root ganglion (DRG)-derived F11 cells. Additionally we

used these newly generated antibodies to explore the role of TRPV2 in neuronal

cell development. A detailed analysis revealed that nerve growth factor (NGF)

increases TRPV2 expression through the MAPK signaling pathway. Furthermore,

we discovered TRPV2 acts as a novel ERK substrate, whereby phosphorylation

xi of TRPV2 by ERK enhances TRPV2-mediated Ca2+ signals and neurite

outgrowth downstream of NGF. TRPV2 localizes to endosomal pools within

neurites of developing neurons, where it may serve to alter local Ca2+ signals.

Based on these data, we propose a previously uncharacterized mechanism in which TRPV2 acts as an endosomal Ca2+ channel regulated by NGF via the

MAPK pathway to alter local Ca2+ signaling within neurites and enhance neurite

outgrowth. Overall, our comprehensive analysis of the cellular function of TRPV2

indicates that TRPV2 physiology tightly depends on regulation of its expression

and localization in the cell.

xii Chapter 1

Introduction

1 1.1 The Transient Receptor Potential family of ion channels

The Transient Receptor Potential (TRP) superfamily of ion channels

contains 28 mammalian members categorized into 6 subfamilies based on

: TRPC (canonical), TRPV (vanilloid), TRPA (), TRPP

(polycystin), TRPM (melastatin) and TRPML (mucolipin) (1) (Figure 1.1). TRP proteins generally function as tetrameric non-selective cation channels (1). Each

monomer contains 6 transmembrane (TM) helices and a pore-forming loop

between the 5th and 6th TM helices (see Figure 1.3). The TM domain is relatively

similar amongst the TRP channels and resembles the TM domains of K+

channels (1). Most structural and functional differences between the subfamilies

resides in the cytoplasmic N- and C-termini. Understanding the structure and

function of TRP channels has garnered a great deal of interest recently due to

the function of key family members in temperature and pain sensation (2). To

date, the TRPV subfamily has been the most extensively studied amongst the

TRP channels due to the putative role of its members in heat and pain sensation

(2).

1.2 TRPV channels as thermosensors

The TRPV subfamily consists of 6 members (TRPV1-6). TRPV1-4 are

known as thermoTRPs due to their nearly 40% sequence identity and their

putative role in temperature sensation (2). TRPV5 and TRPV6 are highly Ca2+

selective and are involved in epithelial Ca2+ handling (3) (Figure 1.2). The

thermoTRPV channels were originally thought to act as temperature sensors

based on their ability to respond to temperature changes in heterologous

2 expression systems and their expression in sensory neurons (Figure 1.2) (4-6).

TRPV1 was the first TRPV channel to be identified and is the prototypical

thermoTRPV member (7). TRPV1 is activated by noxious heat (>42°C), protons,

as well as , the active ingredient in chili peppers (7). TRPV1 is

important for temperature sensation in vivo, as TRPV1-null mice display reduced

sensitivity to noxious heat and lack inflammatory thermal hyperalgesia (8)

(Figure 1.2). Furthermore, TRPV1 shows an intrinsic ability to respond to heat,

as purified channel protein reconstituted into artificial liposomes retains its heat

sensitivity (9). Since heat sensitivity is not fully lost after TRPV1 knockout, it

follows that other molecular heat sensors must exist. TRPV1 homologs were

prime candidates since they appeared to be heat sensitive in heterologous

expression systems (2, 4-6). However, knockout of TRPV2, TRPV3 or TRPV4 in

mice resulted in no differences in sensory transduction, including temperature

sensation, compared to wild type (WT) controls (10, 11) (Figure 1.2). Therefore, while temperature sensitivity of thermoTRPV channels may occur in heterologous expression systems, only TRPV1 appears to function as a heat sensor in vivo.

1.3 Domain structure of thermoTRPV channels

ThermoTRPV channels (TRPV1-4) share 40-50% sequence identity, and as such they share several key structural features (Figure 1.3). The channels function predominantly as a homotetramers (12). Each monomer contains 6 TM helices (Figure 1.3). TM5-6 and the linker between them serves as the pore of the channel. Within the TM5-6 linker is a pore turret, which is important for

3 modular gating and ion selectivity (13-15). The soluble N-terminus contains an

ankyrin repeat domain. Ankyrin repeats are the most common protein domain

structures, consisting of 33 amino acids that form an anti-parallel helix--helix

motif followed by a β-hairpin loop (16). The N-terminus of thermoTRPV channels

contains 6 ankyrin repeats (Figure 1.3). This domain is thought to be important

for protein-protein interactions as well as modulating allosteric channel gating

(17, 18). The soluble C-terminus of thermoTRPV channels has a conserved TRP box sequence (Figure 1.3), which is important for gating modulation and interaction with phospholipids in the membrane (9, 19-21). The least conserved regions of the thermoTRPV channels are the very distal N- and C-termini.

Despite these highly conserved regions within the thermoTRPV channels, evidence shows that regulation of expression, trafficking and activity of the different thermoTRPV channels is distinct for each individual channel. Detailed analysis into the structures and cellular function of the different thermoTRPV channels are necessary to further delineate their true physiological functions.

1.4 Involvement of TRPV2 in sensory transduction

Amongst the thermoTRPVs proteins, TRPV2 is considered an “orphan

TRP” since its physiological function remains unclear (22). TRPV2 shares nearly

50% sequence identity with TRPV1, and therefore was originally thought to perform a similar function in vivo (4). When rat TRPV2 was originally cloned and heterologously expressed in oocytes, it was shown to activate with a temperature threshold of 52°C (4). Based on this, TRPV2 was proposed to act as a sensor of noxious heat (4). TRPV2 is expressed in TRPV1-null sensory neurons in the

4 adult dorsal root ganglion (DRG). While TRPV1 localizes to small diameter C- fibers, TRPV2 was detected in medium and large diameter Aδ and Aβ neurons

(4, 7, 23). It was proposed that TRPV1 and TRPV2 allow subpopulations of DRG neurons to detect and process a range of thermal stimuli (24). However, the expression of TRPV2 in a wide-range of non-sensory tissue suggested that the channel has some function outside of sensory transduction (4).

Temperature-induced activation of TRPV2 became controversial as subsequent groups were unable to replicate these results (25). Later it was shown that activation of TRPV2 by noxious heat might be a species-specific phenomenon. While mouse and rat TRPV2 could be activated by noxious heat, TRPV2 was unresponsive (26). Consistent with this, heat activated currents with similar biophysical properties to heterologously expressed TRPV2 were detected in rodent derived F11 cells and medium and large diameter sensory neurons from the DRG, suggesting that TRPV2-expressing cell types display heat responses similar to those observed for heterologously expressed

TRPV2 (27, 28).

In addition to temperature sensitivity, it has been proposed that TRPV2 acts as a mechanosensor. TRPV2 immunoreactivity was detected in medium and large diameter mechanosensitive sensory neurons (23). Furthermore, osmo- sensitive currents detected in smooth muscle cells were attributed to TRPV2

(29). It remains to be determined if TRPV channels are intrinsically mechanosensitive. It is possible that association of the channels with the

5 confers TRPV2 the ability to respond to mechanical changes in the

cell (30-32).

TRPV2 knockout mice show normal thermal and mechanical sensation,

suggesting that TRPV2 does not function in sensory transduction in vivo (33).

TRPV2 is no longer considered a major receptor for noxious heat (34). The role of TRPV2 in neuronal cell function remains unclear.

1.5 Pharmacological Activation and Inhibition of TRPV2

One of the main sources of controversy and difficulty in studying TRPV2 function is that TRPV2 pharmacology remains poorly characterized. Currently, several promiscuous compounds are used to activate or inhibit TRPV2 activity experimentally (see Table 1.1). 2-aminoethoxydiphenyl borate (2-APB) was the first chemical compound discovered to activate TRPV2 (it was concurrently shown to activate TRPV1 and TRPV3 as well), although its activation properties and role in TRPV2 modulation have been inconsistent across laboratories (35).

Activation of TRPV2 by 2-APB occurred at concentrations of 1 and 3 mM in

HEK293 cells, which was considerably higher than the concentration thresholds for TRPV1 and TRPV3 (35). 2-APB has little effect on TRPV4, 5 and 6 (35).

Another group found that 100 µM 2-APB activates robust currents in TRPV3- expressing cells, modest currents in TRPV1-expressing cells and had no effect on TRPV2-expressing cells (36). 2-APB concentrations above 100 µM may induce Ca2+ influx in the absence of TRPV channel expression, which may account for the conflicting results between these two studies (26).

6 A detailed characterization of the effects of 2-APB on TRPV2 revealed

that TRPV2 orthologs respond differently to 2-APB. Whereas 2-APB activates

mouse and rat TRPV2, it has no effect on human TRPV2 (37). Since most of the

deviation in sequence between human and rodent TRPV2 occurs in the

cytoplasmic N- and C-termini, it was predicted that 2-APB activates TRPV2 from

inside the cell (37). Using chimeras exchanging domains between human and rat

TRPV2 and by truncating rat TRPV2 at the N- and C-termini, it was determined

that two regions of the channel are required for 2-APB activation: the first 120 N-

terminal residues and the last 55 C-terminal residues (26). Mutagenesis studies

of TRPV3 revealed two residues important for 2-APB activation. Mutations

H426N in the first intracellular loop of the transmembrane domain and R696K in

the C-terminal TRP box domain ablated the response of TRPV3 to 2-APB (38).

Interestingly, the equivalent residues in TRPV1 and TRPV2 are Asn and Lys,

suggesting that the molecular mechanism of 2-APB activation differs between the

TRPV3 compared to TRPV1 and TRPV2.

Based on these studies, 2-APB is considered an activator of rodent

TRPV2. However, 2-APB is promiscuous in its actions. It has been shown to

modulate other Ca2+ channels and transporters, including TRPC channels,

TRPM8, IP3 receptor, SERCA2, and store-operated Ca2+ channels (39) (Table

1.1). Due to its non-specific effect on Ca2+ signaling proteins, including other

TRPV channels, and its inconsistent effects on TRPV2 activity, results obtained

using 2-APB as a TRPV2 activator should be interpreted with caution.

7 Cannabinoids also activate both rodent and human TRPV2 (26, 37, 40).

Cannabidiol was found to be most potent cannabinoid activator, followed by

(THC) and then (40) (Table 1.1). In DRG neurons, activation of TRPV2 by evoked calcitonin related peptide (CGRP) release, indicating a role for TRPV2 in inflammatory pain signaling (40). However, cannabinoids are not specific activators of TRPV2, as they also activate TRPV1, TRPA1, TRPM8 and cannabidiol receptors (41).

Probenecid may be the most specific known activator of TRPV2 (Table

1.1). Probenecid was originally used to decrease renal excretion of antibiotics and was shown to increase serum levels of penicillin (42). Later it was found to increase clearance of urate in the and therefore was used in treatment of gout. Its use in the clinical setting decreased once antibiotics were optimized and urate levels did not correlate with symptoms of gout (42). Probenecid was however used in the research setting to measure Ca2+ transients in cells by preventing fura-2 leak from cells (42).

Later, probenecid was shown to activate rat TRPV2 (43). Probenecid increased currents in HEK293 cells transiently expressing TRPV2 with an EC50 of

31.9 µM (43). Importantly, probenecid treatment did not activate currents or Ca2+

increases in HEK293 cells expressing the other thermoTRP channels, TRPV1, 3

and 4, TRPA1 and TRPM8, which are also expressed in sensory neurons (43).

Probenecid also caused Ca2+ increases in large diameter sensory neurons (43) and increased contractility in wild type, but not TRPV2-null cardiomyocytes (44), indicating that it serves as an activator of endogenously expressed TRPV2.

8 Interestingly however probenecid had similar effects on activity of TRPV2-null neurons compared to WT neurons, indicating that it may not be a direct activator of TRPV2 (10). Consistent with this, probenecid has been shown to affect Ca2+

handling of pannexin channels and purinergic signaling (45).

TRPV2 blockers are also relatively non-specific (Table 1.1). Ruthenium

red, a pan-TRP channel inhibitor blocks heat, 2-APB and probenecid activated

currents in endogenous and heterologous expression systems (4, 26, 43).

Trivalent captions such as La3+ and Gd3+ have also been identified as TRPV2

blockers, although they also block other Ca2+ channels (28). SKF96365, a Ca2+

uptake inhibitor, as well as K+ channel blockers tetraethyl ammonium (TEA) and

4-Aminopyridine (4-AP), inhibit 2-APB activated currents in HEK293 cells

expressing TRPV2 (37). Tranilast, an anti-allergic drug, also may act as a TRPV2

inhibitor, though the specificity and mechanism of channel inhibition are poorly

characterized (46). Overall there is no specific validated inhibitor of TRPV2

activity.

As of yet, no endogenous mediators of TRPV2 have been identified and

validated. It has been suggested that phosphatidylinositide 3-kinase (PI3K)

signaling augments and phosphoinositide depletion attenuates TRPV2-mediated

currents (47, 48); however the molecular mechanisms by which this occurs are

unknown. Furthermore, there is evidence that TRPV2 displays constitutive basal

activity in the absence of applied activators (47, 49). Therefore, in addition to

modulators of channel activity, regulators of channel expression and localization

are likely important for the function of TRPV2.

9 1.6 Subcellular trafficking of TRPV2

Since TRPV2 displays constitutive basal activity (33, 47), it follows that the

subcellular distribution of the channel might have a major influence on channel

function in the cell. Therefore, understanding the mechanisms that dictate the

subcellular distribution of TRPV2 is essential for understanding its function. It

was originally observed that treatment of Chinese hamster ovary (CHO) cells

transiently expressing TRPV2 with insulin-like growth factor 1 (IGF-1) led to an increase in plasma membrane levels of the channel (50). IGF-1 caused Ca2+

increases that depended on the presence of extracellular Ca2+ and that could be

blocked by application (50). An increase in Cs+ conductance was

also apparent after treatment with IGF-1 that was inhibited by wortmannin,

suggesting that PI3K signaling was involved in potentiating the plasma

membrane current. Immunocytochemistry revealed that more TRPV2 was

present at the plasma membrane after IGF-1 treatment; plasma membrane

translocation of TRPV2 was also inhibited by wortmannin and by coexpression of

TRPV2 with a dominant negative PI3K subunit (50). Together, this study

suggested that increased plasma membrane expression of TRPV2 is responsible

for IGF-1 induced increases in Ca2+ influx, and translocation of the channel is

mediated by PI3K signaling downstream of IGF-1.

IGF-1 was later shown to affect translocation of TRPV2 to the sarcolemma

in skeletal and cardiac myocytes (51). Stretch also induced plasma membrane

translocation of the channel (51). It was observed that myocytes derived from

rodent models of muscular dystrophy displayed increased sarcolemmal TRPV2,

10 suggesting that structural disturbances in myocytes due to muscular dystrophy

may lead to stretch-induced increase in TRPV2 expression at the sarcolemma

(51). Translocation did not occur in the presence of Gd3+, a TRPV2 blocker (51).

Treatment of myocytes isolated from rodent models of muscular dystrophy with

Gd3+ decreased sarcolemmal TRPV2 (51). This indicates that TRPV2-mediated

Ca2+ influx may contribute to TRPV2 translocation to the cell surface. Expression

of a dominant-negative Ca2+-impermeable TRPV2 mutant rescued muscular

dystrophy in rodent models, highlighting the importance of TRPV2-mediated Ca2+

signals in disease progression (52).

Additional serum factors and ligands that induce TRPV2 translocation to the plasma membrane have been identified, including neuropeptide head activator, fMetLeuPhe peptide involved in macrophage migration, RGA protein and lysophospholipids (53-56). These ligands and proteins are proposed to mediate TRPV2 trafficking via similar signaling pathways such as PI3K, CaM kinase and cyclic AMP. However, regulated trafficking of TRPV2 has remained controversial, as groups have been unable to replicate previous results. For example, Penna et al. found that while IGF-1 may affect TRPV2 activity, it has no

effect on plasma membrane levels of the channel (47). Additionally, IGF-1 had no

effect on the subcellular distribution of endogenous TRPV2 in DRG-derived F11

cells (27). Similarly, in macrophages TRPV2 was identified in the endoplasmic

reticulum (ER) under basal conditions, while upon stimulation, TRPV2 was

detected at the plasma membrane. It is presumed that the channel must traffic to

the plasma membrane via vesicles; however TRPV2 immunoreactivity is only

11 observed at the cell surface or in the ER (56). Electrophysiological studies have

identified a TRPV2-like current in endosomes from HEK293 cells and proteomics

experiments have detected endosomal TRPV2 by mass spectrometry (57, 58),

further suggesting that TRPV2 may predominantly localize to intracellular

membranes.

Discrepancies amongst different groups may have occurred for a number

of reasons. Groups have employed different expression systems, TRPV2

constructs and antibodies to detect plasma membrane localization of the

channel. Additionally, TRPV2 modulators are non-specific and can potentially

affect other proteins involved in cation influx. Regulated plasma membrane

expression and the subcellular distribution of TRPV2 remain unclear. A

comprehensive study in multiple cell types with validated tools is necessary to

fully understand how the subcellular localization of TRPV2 affects its function.

1.7 Involvement of TRPV2 in nervous system function and axon extension

While knockout of TRPV2 in mice did not result in changes in sensory transduction, an interesting phenotype was observed in which a large percentage of the TRPV2 knockout mice were subject to perinatal lethality (10). Embryos from TRPV2 knockout mice were smaller than their wild type counterparts (10), suggesting that perinatal lethality of the TRPV2 knockout mice may be due to a defect during embryonic development. Furthermore, in situ hybridization showed that TRPV2 message is expressed in motor and sensory neurons beginning at embryonic day 9.5 in mouse (33). Expression is broad in immature neurons;

TRPV2 expression becomes restricted to specific subsets of neurons as the

12 embryo ages and the mice progress into the post-natal stage (33). Importantly,

TRPV2 expression in immature neurons is associated with axon growth (33).

Expression of a dominant negative TRPV2 pore deletion mutant, as well as

TRPV2-specific shRNA, reduced axon length in cultured embryonic mouse DRG sensory neurons. Introduction of TRPV2-specific shRNA to chick embryos in vivo also inhibited axon extension (33). These studies indicate that TRPV2 is a vital protein during embryonic development and that its expression affects axon extension in developing peripheral neurons; however the molecular mechanisms by which TRPV2 is regulated to enhance neurite outgrowth during neuronal development remain unclear.

TRPV2 expression may also increase after injury of peripheral neurons.

Upregulation of TRPV2 immunoreactivity was observed in sympathetic neurons after sciatic nerve axotomy (24). It was proposed that the increase in TRPV2 contributes to pain sensation after nerve injury. However, in the context of the role of TRPV2 in axon extension, it is possible that TRPV2 functions in regeneration and overgrowth of sensory neurons. Further studies to delineate the role of TRPV2 in neurite extension and neuronal regeneration are needed to clarify its role in these processes.

1.8 Neurotrophins and their receptors

TRPV2 was recently identified as a molecular player in peripheral neuron development (33). Neurotrophins are essential extracellular cues that promote survival and differentiation of developing peripheral neurons (59). During nervous system development, developing neurons innervate their targets to make specific

13 connections, leading to the formation of functional circuits. The mechanisms by

which these connections are made were further clarified in the late 1940s and

early 1950s, when it was found that after implantation of a mouse tumor in a

developing chick embryo, sensory neurons from the chick embryo began to

innervate the tumor (60). This led to the hypothesis that the mouse tumor

secreted a diffusible factor that attracted the chick neurons to grow and

differentiate (61). This target-derived factor was later isolated from mouse

salivary glands (62) and identified as nerve growth factor (NGF). Subsequently,

the chemo-attractive hypothesis was formed, predicting that developing neurons

grow toward a target-derived signal; cells that receive the signaling molecule at

an optimal concentration survive and grow, while those that are exposed to lower

concentrations are subject to cell death (63). This would lead to the formation of

specific neural connections while preventing overgrowth of innervating neurons.

In the periphery, morphogens and neurotrophins such as NGF are typically

secreted by non-neuronal targets such as muscle fibers and skin tissue, while in

the central nervous system neurotrophins are typically secreted by target neurons (64). In vivo studies have shown that NGF is essential for survival of certain peripheral neurons during development. Application of NGF-specific antibodies that reduce the effective concentration of NGF, as well as genetic deletion of NGF in the mouse, leads to reduced survival of peripheral neurons during development (65-67). Subsequent to the discovery of NGF, two other major diffusible neurotrophic factors involved in neuronal cell growth and survival have been identified: brain derived neurotrophic factor (BDNF) and neurotrophin

14 3 (NT3) (64). NGF remains the prototypical and most extensively characterized

neurotrophin.

Neurotrophins bind to and signal through two receptor types expressed on

the plasma membranes of developing neurons: Tropomyosin Related Kinase

(Trk) receptors and p75 neurotrophin receptor (p75NTR) (68, 69).

Three Trk receptors have been identified: TrkA, TrkB and TrkC. Each Trk receptor has selective affinity for the three major neurotrophins: NGF binds to

TrkA; BDNF binds to TrkB; NT3 binds to TrkC (Figure 1.4) (59, 68). Trk receptors are receptor tyrosine kinases (RTKs). They contain a membrane- spanning region, an extracellular neurotropin binding domain and an intracellular kinase domain. Trk receptors can exist in the as monomers or dimers. Upon ligand binding, the receptors dimerize, activating the kinase domain and autophosphorylation of intracellular Tyr residues, which leads to the formation of adapter complexes on the intracellular portion of the receptor. This results in activation of three RTK-associated signaling pathways: extracellular signal-related kinase (ERK; MAPK); PI3K-Akt; phospholipase Cγ1-PKC (PLC)

(59). These signaling pathways function mainly to promote cell survival, differentiation and neurite outgrowth in developing neurons by altering the functionality of proteins in the acute setting and altering transcription of certain target to maintain long-term effects (59).

Signaling pathways in neurons are difficult to study in vivo; therefore,

PC12 cells have been used as a model system for studying the signaling pathways involved in neuronal cell differentiation. PC12 cells are derived from a

15 rat adrenal tumor (70). After exposure to NGF, PC12 cells halt proliferation and

differentiate into cells that resemble sympathetic neurons (70). Differentiation

includes expression of neuronal specific markers such as β-III tubulin,

of neurotransmitters, increased expression of voltage gated channels, leading to

increased excitability, and distinct morphological changes, including the

formation of long neurite extensions (Figure 1.5) (70). PC12 cells express TrkA

and p75-NTR, both receptors that have affinity for NGF (70). Upon binding to its

high affinity receptor TrkA, NGF initiates signaling pathways that promote

survival and differentiation in PC12 cells. NGF activates the prototypical growth

factor signaling pathways including PI3K/Akt signaling and MAPK signaling

(Figure 1.6). The PI3K/Akt pathway is essential for survival in developing

neurons. Inhibition of this pathway using pharmacological inhibitors or genetic

deletion of key PI3K/Akt signaling mediators leads to neuronal cell death (Figure

1.6) (71). MAPK is necessary for neuronal cell differentiation. Inhibition of MAPK

signaling prevents developing neurons from forming neurite extensions (Figure

1.6) (71). PI3K/Akt signaling also contributes to differentiation, but it is not essential for differentiation. For example, PI3K/Akt signaling affects neurite branching (72, 73).

Although NGF activates major growth factor signaling pathways, its phenotypic effects on PC12 cells appear to be unique. For example, treatment of

PC12 cells with epidermal growth factor (EGF), which also activates PI3K/Akt signaling and MAPK signaling, does not cause differentiation (74). This is due to differences in the duration of signals induced by EGF compared to NGF.

16 Activation of the MAPK signaling pathway with EGF peaks at 10 min and reverts

to baseline by 30 min. Activation of MAPK by NGF also peaks at 10 min but

remains sustained for hours to days (74). This is due to concurrent activation of the phospholipase C (PLC) pathway. PLC signaling attenuates an inhibitor of

MAPK signaling, allowing sustained MAPK activation and differentiation. Since

EGF does not activate PLC signaling, MAPK is inhibited after several minutes of

EGF exposure, even in the presence of growth factor (74).

p75NTR is a member of the tumor necrosis factor (TNF) family of receptors

(59, 68, 69). It has similar affinity for all three major neurotrophins, and its affinity for neurotrophins is low relative to the Trk receptors (Figure 1.4) (59). The receptor contains no known enzymatic activity and is thought to modulate adapter proteins and Trk receptors during neurotrophin signaling (68). The effects of p75NTR activation in developing neurons can be diverse depending on the Trk receptors expressed by the cell and the adapter proteins present and available. These effects include promotion of cell survival or cell death, in addition to modulation of proliferation and neurite outgrowth (68).

1.9 Retrograde transport of neurotrophin signals

A major question regarding neurotrophin signaling was how target-derived

NGF binding to its receptor at distal terminals of neurons could affect signaling in the nucleus, which could be several millimeters away in the soma. Neurotrophin signals are thought to be transported from the terminals to the soma via endosomal vesicles (75, 76). In general, newly internalized vesicles mature to early endosomes followed by late endosomes and eventually lysosomes, where

17 endosomal components are degraded. Rab GTPases are essential for

maturation of endocytic vesicles (77) (Figure 1.7). Key Rab proteins in endocytic processes of neurotrophins and their receptors include Rab5, which is involved in maturation of early endosomes, and Rab7, which is key for late endosome and lysosomal maturation (77, 78) (Figure 1.7).

In neurons, binding of NGF to TrkA causes receptor activation as well as

internalization of the NGF/TrkA complex into endosomal vesicles (Figure 1.8).

Within newly internalized endosomes derived from the distal axon of developing

neurons, NGF can remain bound to TrkA, and the complex can continue to

activate the MAPK signaling pathway along the axon (75, 76). Rather than

maturing and fusing with lysosomal membranes, these so-called signaling

endosomes are long-lived and act as platforms that allow for local signaling

needs to be met within the potentially long neuronal extensions (76) (Figure 1.8).

The cytoplasmic face of the endosomal membrane associates with MAPK

scaffolding proteins to concentrate MAPK signaling molecules and allow for

efficient transmission of the signal downstream of NGF (79). Signaling

endosomes are transported along microtubules within axonal projections by

microtubule associated motor proteins and eventually reach the nucleus in the

cell body where they can alter (76) (Figure 1.8).

The molecular identity of signaling endosomes remains controversial,

although a considerable amount of evidence indicates that the endosomal

population responsible for retrograde transport of NGF-TrkA signals is Rab7-

positive late endosomes (75, 76). Expression of dominant-negative (DN) Rab7 in

18 neurons halts retrograde transport of neurotrophin-containing endosomes (80).

Additionally, Rab7-positive endosomes are transported at a faster rate than

Rab5-positive endosomes, suggesting that late endosomes are more efficiently moved along the axon (81). A proteomic analysis of TrkA-containing endosomes identified Rab7 and Rab7 effector proteins in the same endosomal pool, indicating that TrkA-positive endosomes are closely associated with Rab7 (82).

Furthermore, pathogenic mutations to Rab7 result in dysregulated neurotrophin signaling and axonal neuropathies (83, 84).

In addition to Rab7-positive late endosomes, evidence also implicates

Rab5-positive early endosomes in retrograde transport of neurotrophin signals.

Treatment of PC12 cells with NGF leads to colocalization of TrkA with early endosomal markers Rab5 and EEA1 (85). Additionally, NGF immunoreactivity has been identified in Rab5-positive endosomes in primary DRG neurons (86).

Taken together, the conflicting results regarding the molecular nature of signaling endosomes in developing neurons signifies that neurotrophin signals may be transported in both early and late endosomes. Different time points and experimental conditions might dictate in which compartment neurotrophins are detected. NGF/TrkA may progress through different endosomal populations as they mature, leading to detection of the complex in multiple compartments. It is also possible that different neuronal cell types employ different mechanisms for processing neurotrophins. For example, sympathetic neurons may possess different trafficking mechanisms for NGF/TrkA compared to sensory neurons, or central neurons may process neurotrophins and their receptors differently than

19 peripheral neurons. Nonetheless, a great deal of evidence indicates that proper

function of signaling endosomes is necessary for differentiation and survival of

developing neurons (75).

1.10 Ca2+ as a second messenger in neurotrophin signaling

A major second messenger that contributes to neurotrophin signaling is intracellular Ca2+ (87). NGF signaling influences expression, localization and expression of Ca2+ handling proteins, including Ca2+-permeable ion channels, to promote neuronal survival and differentiation during development (88-93). Ca2+

transients have been implicated in various stages and processes governing

neuronal development (87, 94). Ca2+ signals are especially important for the

different processes of differentiation, including neuronal subtype specification,

neurotransmitter specification and neurite growth and branching (95). As a

second messenger, Ca2+ is particularly suited to regulate several different processes due to its versatility. Ca2+ possesses high temporal and spatial specificity, due to the expression of numerous Ca2+ handling proteins and their different biophysical characteristics and subcelluar distributions (87).

Intracellular Ca2+ signals can mediate effects in numerous different ways.

Ca2+ signals can have acute effects in regulating the cytoskeleton within growing

axons and dendrites, as well as long-term effects, for example by altering gene

transcription (95). Ca2+ is particularly important for the formation, growth and

turning of neuronal extensions. Ca2+ can mediate growth or retraction of neurites,

depending on the location of the signal, the nature of the signal and the

developmental stage of the neuron (87). For example, in the growth cone, Ca2+

20 entry through mechanosensitive ion channels in the plasma membrane slows

axon growth, while Ca2+ release from intracellular stores within the growth cone enhances growth (96).

Overall, numerous Ca2+ channels are involved in neurotrophin signaling and neurite outgrowth during neuronal cell development. Silencing or deletion of one single Ca2+ channel reduces but does not fully attenuate NGF-induced neurite extension, suggesting that these multiple Ca2+channels act in concert to modulate different local processes in a specific manner to fine-tune Ca2+ signals,

leading to neurite extension and formation of specific neural connections.

1.11 TRP channels in neurite outgrowth and neurotrophin signaling

Amongst the Ca2+ channels involved in neurotrophin signaling, members

of the TRP family of Ca2+-permeable ion channels have emerged as important players in neurotrophin-mediated neurite outgrowth. TRPC channels were first

identified as molecular players in neurite outgrowth. It was found that BDNF

activated TRPC3 currents in hippocampal neurons (97). Activation occurred

through the PLC signaling pathway, and affected growth cone motility. It was

proposed that TRPC3 activation lead to local depolarizations in the growth cone,

activating voltage-gated Ca2+ channels and causing a Ca2+ increase in the

growth cone (97). Later, a BDNF-modulated TRPC-like current was detected in

the growth cone of xenopus neurons (92). This current was reduced by

knockdown of xenopus TRPC1 (xTRPC1). Inhibition of xTRPC1 inhibited growth

cone turning but did not affect BDNF-induced neurite outgrowth (92). These

21 studies suggested that TRPC channels serve as rapid effectors of neurotrophin

signaling to guide growth cones during neurite outgrowth.

TRPC channels have also been implicated in neurotrophin signaling in

PC12 cells, where they influence neurite outgrowth. Treatment of PC12 cells with

NGF increases the expression of TRPC1, TRPC3 and TRPC6 (90, 91, 93, 98).

Increased expression and activity of these channels is associated with increased

neurite extension in PC12 cells. TRPC5 is thought to have an opposite effect on

neurite growth. NGF treatment decreases TRPC5 expression. Expression of a

dominant negative TRPC5 mutant increases NGF-induced neurite extension,

while overexpression of function TRPC5 decreases neurite outgrowth (93, 99).

TRPC5 is also expressed in the growth cone of hippocampal neurons, where it

acts as a negative regulator of neurite extension (100). Overall, it is thought that

the expression profile and subcellular localization of particular TRPC channels

are important for their effects in neurite formation and extension. Local TRPC-

mediated Ca2+ changes can have opposite effects, depending on where they occur in the cell.

TRPV channels are also involved in neurotrophin signaling. Although not abundantly present in developing neurons, TRPV1 is highly expressed in TrkA- positive adult sensory neurons (101). Activation of TrkA by NGF in the adult is

associated with inflammatory signaling and increased pain sensation (102). This

occurs through sensitization of TRPV1. Exposure of adult sensory neurons to

NGF increases TRPV1 expression (103), increases TRPV1 plasma membrane

levels (104, 105) and sensitizes TRPV1 to its numerous activators, including

22 temperature (103). Increased expression and sensitivity of TRPV1 is consistent

with the finding that NGF exposure leads to hyperalgesia (102).

TRPV4 is present in both developing and adult sensory neurons (88, 106).

In the adult, TRPV4 has been reported to act as a temperature and mechanical

sensor (107), while its role in development is less clear. NGF regulates TRPV4

expression in developing sensory neurons and PC12 cells (88). Increased

TRPV4 expression is associated with enhanced neurite outgrowth (88). It was proposed that TRPV4 activity alters the actin cytoskeleton, leading to neurite outgrowth (88). Finally TRPV2 has also been detected in both developing and adult sensory neurons. During development, TRPV2 was shown to enhance axon outgrowth (33), although the molecular mechanisms by which it does so remain unclear. Overall, while TRPV channels, specifically TRPV2 and TRPV4, have been implicated in neurotrophin-mediated neurite outgrowth, the mechanistic details by which they contribute are unknown.

1.12 Purpose of this study

Upon its discovery in 1999, TRPV2 was proposed to have two separate physiological functions: 1) Noxious heat sensitivity, with a temperature threshold of 52°C (4); 2) Growth factor sensitivity (50). TRPV2 was proposed to act as the molecular source of a growth factor-sensitive Ca2+ current in non-excitable cells.

Currently TRPV2 is not thought to function as a heat sensor in vivo, as TRPV2-

null mice have normal responses to temperature compared to WT controls (10).

However TRPV2 has been proposed to play a role in numerous growth factor

regulated processes, such as insulin release, myocyte growth and degeneration,

23 cell migration and macrophage phagocytosis (46, 51, 54, 56, 108). These

studies have not been without controversy, as results exploring the regulation of

TRPV2 by growth factors have not been reproducible (22, 28, 47). Much of the controversy stems from the lack of specific tools to modulate or detect endogenous TRPV2 protein (22).

In this dissertation, we employ newly developed tools to characterize regulation of endogenous TRPV2 protein by growth factors. In Chapter 2, we generate and validate monoclonal antibodies suitable for detection of endogenous TRPV2. Using these newly generated tools, we tested the controversial hypothesis that growth factor stimulation leads to increased TRPV2 levels at the plasma membrane and found that TRPV2 localizes to intracellular membranes in both the absence and presence of growth factors. In Chapter 3, we determine how TRPV2 is regulated in developing peripheral neurons. Our results show that TRPV2 is regulated by MAPK signaling downstream of NGF to promote neurite outgrowth.

24 1.13 Figures

Figure 1.1. The mammalian Transient Receptor Potential (TRP) family. The mammalian TRP superfamily is categorized into 6 subfamilies based on sequence homlogy: TRPM, TRPP, TRPML, TRPA, TRPC and TRPV. TRP channels are expressed in a wide range of excitable and non- excitable cell types and are implicated in numerous different physiological and pathological processes. The TRPV subfamily has been the most extensively studied due to its putative role in temperature and pain sensation.

25

Figure 1.2. Function of TRPV subfamily members. TRPV1-4 are known as thermoTRPs and are activated at the indicated temperature thresholds in heterologous expression systems. TRPV5 and TRPV6 are highly Ca2+ selective and function in epithelial Ca2+ handling. TRPV1 functions as a temperature sensor in vivo while the role of TRPV2-4 in sensory transduction remains unclear.

26

Figure 1.3. Domain structure of thermoTRPV channels. Representation of a thermoTRPV monomer. The transmembrane domain contains 6 membrane-spanning alpha helices. TM5, TM6 and the TM5-6 linker form the pore of the channel. The soluble N-terminus is larger than the C- terminus and contains a structurally conserved ankyrin repeat domain (ARD) with 6 ankyrin repeats. The smaller C-terminus possesses a conserved TRP box domain.

27

Figure 1.4. Neurotrophins and their receptors. There are three major neurotrophins in the mammalian nervous system: NGF, BDNF and NT3. Each binds with high affinity to a specific receptor tyrosine kinase (from the tropomyosin related kinase family; Trk) at the surface of developing neurons: NGF binds to TrkA; BDNF to TrkB and NT3 to TrkC. Binding of neurotrophin to its respective Trk receptor activates canonical growth factor signaling cascades including PI3K and MAPK/ERK. Additionally, each neurotrophin binds with relatively low affinity to the p75 neurotrophin receptor (p75 NTR). p75 NTR is from the TNF-α family of receptors and complements Trk receptor signaling.

28

Figure 1.5. Effect of NGF on PC12 cell morphology. Phase contrast micrographs of PC12 cells cultured in the absence (left) and presence (right) of NGF (100 ng/mL; 72 hours). Cells treated with NGF display distinct morphological changes, including the formation of neurite extensions.

29

Figure 1.6. Schematic representing the major signaling pathways activated by NGF. Binding of NGF to its extracellular receptor TrkA initiates receptor dimerization, autophosphorylation and activation of PI3K and MAPK signaling pathways. PI3K signaling is necessary for survival of developing neurons, while MAPK signaling is required for neurite outgrowth.

30

Figure 1.7. Cartoon depicting receptor mediated endocytosis. When receptor binds to ligand, the receptor/ligand complex is internalized via clathrin-dependent mechanisms. The clathrin-coated vesicle sheds its coat and can mature to an early endosome. This process is dependent on Rab5 activity. The early endosome then matures to a late endosome, involving loss of Rab5 and gain of Rab7, which fuses with lysosomes and leads to cargo and receptor degradation. Additionally, the receptor can be recycled back to the plasma membrane via recycling endosomes. CV, clathrin-coated vesicle; EE, early endosome; LE, late endosome; RE, recycling endosome; Lys, lysosome; Endo/lys, endolysosome.

31

Figure 1.8. Cartoon depicting long-distance retrograde transport of

NGF/TrkA containing signaling endosomes. When NGF binds to its extracellular receptor at the terminals of developing neurons, the receptor-ligand complex is internalized into an endosomal compartment. These endosomes do not acidify and NGF can remain bound to TrkA in the endosomal lumen. Additionally, MAPK scaffolding proteins associate with the endosomal membrane to concentrate MAPK signaling components near the activated NGF/TrkA complex. The endosomes are transported in the retrograde direction via microtubule- associated motor proteins. Eventually, the endosomes reach the soma, where the active MAPK signaling pathway can alter gene transcription in the nucleus.

32 1.14 Tables

Table 1.1. Modulators of TRPV2 activity.

33

Chapter 2

Effect of growth factors, including IGF-1, on

translocation of TRPV2 to the plasma membrane

Portions of this chapter were published in:

Cohen MR, Huynh KW, Cawley D, Moiseenkova-Bell VY. Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies. PLoS One. 2013;8(12):e85392.

34 2.1 Introduction

The transient receptor potential (TRP) family of nonselective cation channels contains 28 recently identified mammalian homologs grouped into six subfamilies based on sequence homology: vanilloid (TRPV), canonical (TRPC), melastatin

(TRPM), ankyrin (TRPA), mucolipin (TRPML), and polycystin (TRPP) (109). TRP channels are proposed to function in a broad range of processes, although the exact cellular function of several TRP channels remains elusive. Considerable challenges in elucidating the function of TRP channels include the absence of the specific activators, inhibitors and antibodies for each individual family member

(110). The controversial function of TRPV subfamily members provides a good example of this current problem in TRP field.

The TRPV subfamily consists of six members (TRPV1-6) (109). TRPV1 has been the most comprehensively studied TRP channel due to its role in noxious pain sensation (111). Capsaicin, the active ingredient in chili peppers, is a specific activator of TRPV1 and was used for identification and characterization of the channel properties (7). Specific activators and inhibitors, in addition to

TRPV1 knockout mice, have consistently indicated that TRPV1 acts as a heat and pain sensor in vivo (8). TRPV2 shares nearly 50% sequence identity with

TRPV1 and was cloned concurrently by two laboratories (4, 50). One group identified TRPV2 as an insulin-like growth factor-1 (IGF-1) sensitive Ca2+ channel. Upon exposure to IGF-1, heterologously expressed TRPV2 was shown to move from intracellular membranes to the cell surface, where it mediated Ca2+ influx (50). However, later studies indicated that, while IGF-1 signaling may affect

35 TRPV2 activity, it does not affect surface expression of the channel (47, 112).

TRPV2 was also originally shown to function as a noxious heat sensor in a heterologous expression system (4). Later, TRPV2 was proposed to function in osmo- and as well (29). However, recently generated TRPV2 knockout mice display normal sensory transduction, suggesting that TRPV2 does not function as a noxious heat and mechanical sensor in vivo (10). Additionally these mice were subject to perinatal lethality, indicating that TRPV2 has another, as yet unknown function (10).

The physiological function of endogenous TRPV2 has remained controversial due to the lack of pharmacological and biochemical tools to study this channel

(113). Unlike TRPV1, TRPV2 is not modulated by vanilloids, such as capsaicin

(4). Putative activators and inhibitors of TRPV2 such as 2-aminoethoxydiphenyl borate (2-APB) and SFK96365 affect other TRP channel family members and non-selective cation permeation pathways (39). The only other tools for exploring the endogenous function of the channel have been commercially available polyclonal antibodies generated against small linear peptides derived from

TRPV2. Based on these available tools, TRPV2 has been proposed to play a major functional role in diseases such as muscular dystrophy, cardiomyopathy, prostate cancer, bladder cancer, glioblastoma development and diabetes (46, 51,

54, 114, 115). Recently, TRPV2 has also been proposed to be involved in immune response mechanisms, neuronal development and insulin secretion (33,

46, 108). However, these results have not been without controversy (113).

Differences in commercially available polyclonal antibodies utilized in many of

36 these studies may be especially problematic for studying endogenously expressed TRPV2.

Since pharmacological effectors of TRPV2 are non-specific and endogenous

TRPV2 ligands are unknown, efforts to understand the proposed endogenous function of TRPV2 have been hindered. Validation of currently available antibodies and generation of antibodies suitable for detection of endogenously expressed TRPV2 would provide the best path towards accelerating understanding the cellular function of this . Here we generated for the first time TRPV2 monoclonal antibodies raised against full-length, tetrameric

TRPV2. These antibodies, together with commercially available TRPV2 polyclonal antibodies, were characterized for detection of recombinant and endogenously expressed TRPV2. We found that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenously expressed TRPV2 by western blot and immunocytochemistry, commercially available antibodies raised against synthetic and recombinant linear peptides derived from TRPV2 failed to detect endogenously expressed TRPV2 in cell lines or rodent tissues. Importantly, we tested the effects of IGF-1 on endogenous

TRPV2 translocation to the plasma membrane using our newly generated and validated monoclonal antibodies. We found that IGF-1 had little to no effect on surface expression of TRPV2, suggesting that TRPV2 predominantly localizes to intracellular membranes. We expect that these results will allow for further investigation of the role endogenously expressed TRPV2 plays in cell function and disease.

37 2.2 Materials and Methods

2.2.1. Ethics Statement - All animal studies were approved by the Case

Western Reserve University Institutional Animal Care and Use Committee.

2.2.2 Plasmids - The following cDNAs were kindly provided by the indicated

investigators: rat TRPV2 ankyrin repeat domain, Rachelle Gaudet (Harvard

Medical School); rat TRPV2 C-terminus with MBP-tag, Sharona Gordon

(University of Washington); rat TRPV1 and TRPV2, David Julius (University of

California San Francisco).

2.2.3. Protein expression and purification - Full-length 1D4-tagged rat

TRPV2 was cloned into a YepM plasmid and overexpressed in BJ5457 S.

cerevisiae (ATCC). TRPV2 membranes were prepared and solubilized in 0.087%

wt/v lauryl maltose-neopentyl glycol (MNG) (Anatrace), 20 mM HEPES (pH 8.0),

150 mM NaCl, 5% glycerol, 1.0mM DTT and 1 mM PMSF for one hour at 4°C.

The insoluble fractions were pelleted by ultracentrifugation at 100,000g, 4°C for

45 minutes. The soluble fraction containing TRPV2 protein was incubated

overnight at 4°C with CNBr-activated Sepharose 4B coupled with 1D4 antibody.

The column was packed and washed with washing buffer consisting of 0.006%

decyl maltose-neopentyl glycol (Anatrace), 20 mM HEPES (pH 8.0), 150 mM

NaCl, and 1 mM DTT. The TRPV2 protein was eluted with 3 mg/ml 1D4 peptide

(Genescript USA) in washing buffer, concentrated and subjected to size-

exclusion chromatography (SEC). Rat TRPV2 ankyrin repeat domain (ARD) and

MBP-tagged rat TRPV2 C-terminus were expressed in BL21 (DE3) cells and

purified as previously reported (48, 116).

38 2.2.4. TRPV2 antibody generation - Mouse monoclonal antibodies against

full-length rat TRPV2 protein were obtained using standard methods (117).

TRPV2 antibodies were purified from hybridoma supernatants by protein G affinity chromatography.

2.2.5. Commercially available antibodies - The following commercially available antibodies were used: α-VRL-1 SC-22520 and α-phospho-Akt (Santa

Cruz), α-VRL-1 PC421 (Calbiochem), α-TRPV2 ACC-032 (Alomone Labs), α-

Na,K-ATPase α1, α-pan-Akt and α-β-Actin ().

2.2.6. Cell culture and transfection - The following cell lines were kind gifts from the indicated investigators: HeLa cells, Marvin Nieman (Case Western

Reserve University); F11 cells, Sharona Gordon (University of Washington) (48).

CHO-K1 cells were obtained from ATCC. Cells were maintained in a humidified atmosphere at 37°C and 5% CO2. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high (Invitrogen) supplemented with 10% FBS (Cellgro), 100 unit/mL penicillin and 100 µg/mL streptomycin

(Invitrogen). F11 cells were cultured in F12 medium (Invitrogen) supplemented with 10% FBS, HAT supplement (Invitrogen), 100 unit/mL penicillin and 100

µg/mL streptomycin. CHO-K1 cells were cultured in F12 medium supplemented with 10% FBS, 100 unit/mL penicillin and 100 µg/mL streptomycin.

Transfection of plasmid was performed using polyethyleneimine (PEI;

Polysciences) as a carrier. A solution containing serum-free DMEM, 20 mM

HEPES pH 7.5, and 3 µg PEI per 1 µg plasmid was incubated for 10 min at room temperature to allow DNA:PEI complexes to form. The mixture was then added

39 directly to growth medium overlaying the cells. After a 4-hour incubation period at

37°C, fresh growth medium was added to cells. Cells were harvested or fixed 16-

24 hours after transfection.

TRPV2 siRNA (rat VRL-1 SMARTpool) and non-targeting siRNA were

obtained from Dharmacon. Transfection of siRNA was performed using

Lipofectamine 2000 (Invitrogen) as a carrier following the manufacturer’s

protocol.

For protein extraction, cells were incubated in lysis buffer (150 mM NaCl, 2

mM EDTA, 50 mM Tris-HCl (pH 7.5), 1% Triton-X100 and a protease inhibitor

cocktail) for 30 min on ice. Lysates were cleared at 20,000 x g for 20 min. Protein

concentrations were determined by BCA assay.

2.2.7. Mouse tissue lysate generation and immunoprecipitation - Male mice (P24) were deeply anesthetized with isofluorane and decapitated. Brains and hearts were removed and homogenized by 10 strokes with a dounce homogenizer in lysis buffer. Homogenates were incubated on ice for 30 minutes and cleared by centrifugation at 20,000 x g for 20 min and 100,000 x g for 30 min to remove unbroken cells and insoluble membrane fragments. The supernatant was pre-cleared with 50 µl protein A/G agarose beads. 2.5 mg of pre-cleared lysate was incubated with 10 µg anti-TRPV2 antibody for 2 hours at 4°C. Next,

50 µl protein A/G agarose was added for 2 hours at 4°C to capture TRPV2 antibody complexes. Protein A/G agarose beads were washed 3 times in lysis buffer and proteins were eluted with Laemmli sample buffer. Samples were

40 boiled at 95°C for 5 min. Immunoprecipitation of TRPV2 was analyzed by

western blot with IR-dye-labeled TRPV2 2D6 antibody.

2.2.8. Western blot analysis - Lysates and immunoprecipitates were loaded

onto 10% Tris-Glycine gels (Invitrogen) and run at 135 V for 90 min. Proteins

were transferred to nitrocellulose at 40 V for 2.5 hours at 4°C. Protein transfer

and equal loading was confirmed by Ponceau S staining. Membranes were

blocked in TBS-T with 10% (w/v) non-fat dry milk powder and incubated with

primary antibody for 1 hour at room temperature. Primary antibodies were

detected with IR-dye-labeled secondary antibodies (LiCor; 0.1 µg/ml) using the

Odyssey imaging system (LiCor). 2D6 and 17A11 were used at 1 µg/ml, TRPV2

ACC-032 (Alomone) at 2 µg/ml, VRL-1 PC421 (Calbiochem) at 5 µg/ml and VRL-

1 SC-22520 (Santa Cruz) at 10 µg/ml.

2.2.9. Immunocytochemistry - HeLa cells and CHO cells were seeded on

glass coverslips in 24 well plates at a density of 1×104 cells per well. The following day, cells were transfected with plasmid for 24 hours. Cells were washed in PBS, fixed in 4% paraformaldehyde and blocked and permeabilized in

PBS containing 0.3% Triton-X100 (PBS-T) and 1.5% normal goat serum. For analysis of TRPV2 antibody recognition of TRPV2-1D4, TRPV2 monoclonal antibodies, cells were incubated in PBS-T containing TRPV2 antibody for 1 hour followed by Alexa 488-labeled goat anti-mouse for 1 hour. After 5 washes in

PBS, cells were incubated with Alexa 568-labeled 1D4 antibody for 1 hour. For polyclonal TRPV2 antibodies, cells were incubated with TRPV2 antibody and

1D4 antibody for 1 hour followed by Alexa 488-labeled donkey anti-mouse and

41 Alexa 594-labeled donkey anti-rabbit or donkey anti-goat. Coverslips were mounted onto glass slides using Vectashield mounting medium.

F11 cells were seeded in 35 mm iBidi dishes at a density of 3×104 cells per dish. The following day, cells were washed in PBS containing 100 µM CaCl2, fixed in 4% paraformaldehyde and blocked and permeabilized in PBS-T containing 100 µM CaCl2 and 1.5% normal goat serum. After blocking and permeabilization, cells were incubated in primary antibody for 1 hour at room temperature. After 3 washes in PBS, cells were incubated in secondary antibody for 1 hour.

For IGF-1 experiments, F11 cells, HeLa cells and CHO-K1 cells transiently expressing TRPV2, or F11 cells, which endogenously express TRPV2, were treated with serum-free medium for 6 hours. Vehicle or IGF-1 (20 ng/ml) was added to the cells at 37°C. Cells were then fixed and immunostained for TRPV2 with 17A11 antibody.

Images were obtained using a Leica TCS SP2 confocal microscope.

Brightness and contrast of images were adjusted using Photoshop (Adobe). Only linear changes were applied.

2D6 and 17A11 were used at 2 µg/ml, TRPV2 ACC-032 (Alomone) at 4

µg/ml, VRL-1 PC421 (Calbiochem) at 10 µg/ml and VRL-1 SC-22520 (Santa

Cruz) at 20 µg/ml.

2.2.10. Biotinylation of cell surface proteins - CHO-K1 cells transiently expressing TRPV2 and F11 cells were cultured to approximately 75% confluence in 10 cm dishes and serum-deprived for 6 hours. After the indicated treatments,

42 cells were washed 3 times in PBS and biotinylation reagent (Sulfo-Link NHS-LC-

biotin, Pierce, 0.5 mg/ml in PBS) was added at 37°C. Following a 30 min

incubation period, the biotin reagent was removed and cells were washed twice

in PBS containing 100 mM glycine and twice in PBS alone. Next, lysates were

prepared as described previously and incubated with 50 µl of streptavidin-

agarose beads (Invitrogen) for 1 hour at 4°C to capture biotinylated proteins. The

beads were pelleted and washed 3 times in lysis buffer. Captured proteins were

eluted with Laemmli sample buffer, boiled at 95°C for 5 min, and analyzed by

western blot with indicated antibodies.

2.3 Results

2.3.1. Translocation of overexpressed TRPV2 in response to growth

factors - We initially sought to characterize the mechanisms by which TRPV2

translocates from intracellular membranes to the plasma membrane in response

to growth factor stimulation. In order to do so, we employed a TRPV2 construct

with a C-terminal 1D4 epitope tag. The 1D4 epitope consists of the last nine C-

terminal residues of rhodopsin and binds specifically to the 1D4 monoclonal

antibody. By using this construct, we were confident that 1D4 immunoreactivity

corresponded with TRPV2 protein (118). Additionally, we employed F11 cells as a model cell line for studying TRPV2 trafficking. F11 cells are derived from DRG neurons and are thought to act as a native-like expression system for TRPV channels (48). F11 cells also endogenously express TRPV2 (119). TRPV1-1D4

or TRPV2-1D4 were transfected in F11 cells under normal growth conditions and

immunostained for 1D4. As expected, TRPV1 displayed a distribution consistent

43 with localization to the plasma membrane (Figure 2.1a). TRPV2

immunoreactivity in contrast appeared to localize predominantly within

intracellular membranes (Figure 2.1a). This was surprising, since the F11 cells

were cultured in the presence of serum, which is rich with growth factors.

TRPV1-1D4 was expressed at the plasma membrane, indicating the 1D4 epitope

tag did not prevent translocation of TRPV channels to the cell surface.

Although regulated plasma membrane translocation of TRPV2 was

characterized upon discovery of the TRPV2 gene (50), this hypothesis has

remained controversial, as certain groups have failed to observe growth factor

regulated changes in its subcellular distribution (27, 47). As such, we attempted

to further characterize the effects of growth factors on translocation of TRPV2 to

the cell surface. IGF-1 treatment failed to induce translocation of TRPV2-1D4 to

the plasma membrane in F11 cells (Figure 2.1b). Additionally, TRPV2-1D4

remained in intracellular membranes after IGF-1 treatment of HeLa cells (Figure

2.2). We found that TRPV2-1D4 colocalized with endoplasmic reticulum (ER)

marker calreticulin in both the absence and presence of IGF-1 in HeLa cells

(Figure 2.2), indicating that overexpressed TRPV2 localizes predominantly to the

ER.

Overexpression of TRP channels can lead to accumulation of the channel

protein in multiple organelles. For example, overexpressed TRPP2 accumulates predominantly in the ER as well as the plasma membrane in some cases (120,

121). Therefore, it remains important to assess the effects of growth factors on trafficking of endogenously expressed TRPV2. Much of the work to determine the

44 function of TRPV2 depends on commercially available polyclonal antibodies.

Many of these antibodies are poorly validated and are of poor quality and have led to controversial and erroneous results (110). We therefore characterized and validated the most commonly used commercially available antibodies against

TRPV2. Additionally, we generated monoclonal antibodies using full-length, tetrameric TRPV2 as an antigen.

2.3.2. Detection of recombinant TRPV2 and determination of TRPV2 binding region - TRPV2 is an integral membrane protein that functions as a homotetramer (113). Each monomer has an approximate molecular weight of 86 kDa and consists of 6 transmembrane spanning helices, 6 N-terminal ankyrin repeats and a short C-terminus (Figure 2.3a) (113). Divergence in the sequence between TRPV2 and other TRPV subfamily members resides in the distal C- terminus; therefore most available polyclonal TRPV2 antibodies were generated against synthetic or recombinant peptides derived from the C-terminus. The commercially available antibodies against C-terminal peptides from mouse and rat TRPV2 used in this study include PC421 (Calbiochem) and ACC-032

(Alomone). Additionally, a polyclonal antibody against an N-terminal peptide from human TRPV2 was tested (SC-22520; Santa Cruz) (Figure 2.3a).

To determine the specificity of these antibodies against TRPV2, western blots were performed against full-length recombinant rat TRPV2 as well as the soluble N-terminal ankyrin repeat domain and C-terminus fused to maltose binding protein. Full-length recombinant TRPV2 was recognized by four of the five antibodies tested (Figure 2.3b). SC-22520 did not recognize full-length rat

45 TRPV2. SC-22520 was raised against a peptide derived from human TRPV2 and is expected to have lower reactivity with rat TRPV2; it recognized the N-terminal

ARD but not full-length TRPV2 (Figure 2.3b). As expected, PC421 and ACC-

032, which were generated against a peptide from the distal C-terminus, recognized both recombinant full-length TRPV2 and the C-terminus (Figure

2.3b). Mouse monoclonal antibodies 2D6 and 17A11 generated in our laboratory also recognized full-length recombinant TRPV2 and the C-terminus (Figure

2.3b). The expected binding site of each antibody tested is mapped in the schematic in Figure 2.3a.

Since TRPV1 shares nearly 50% sequence identity with TRPV2, we tested our TRPV2 monoclonal antibodies for cross-reactivity with TRPV1 by heterologously overexpressing 1D4-tagged TRPV1 and TRPV2 in HeLa cells.

2D6 recognized TRPV2 but not TRPV1 in HeLa cells (Figure 2.3c). 17A11 showed very little immunoreactivity with TRPV2 expressed in HeLa cells and non-specific reactivity with a protein of approximately 120 kDa (Figure 2.3c).

These results show that, of our monoclonal antibodies, 2D6 was the most suitable candidate for detection of endogenous TRPV2 by western blot.

2.3.3. Recognition of endogenously expressed TRPV2 - An important test to validate antibody recognition of endogenously expressed protein is to determine specificity against tissues or cells in which the target protein has been reduced or deleted. F11 cells are derived from dorsal root ganglion (DRG) sensory neurons and provide a native-like environment for the study of thermoTRPV channels (48). RT-PCR analysis demonstrated that both rat and

46 mouse TRPV2 mRNA are present in F11 cells (119). To determine recognition of

endogenous TRPV2, F11 cells were transfected with siRNA to silence TRPV2

expression. Using 2D6, we observed a nearly 3 fold reduction in band intensity at

the molecular weight corresponding to TRPV2 in cells treated with TRPV2 siRNA

compared non-targeting siRNA (Figure 2.4a). ACC-032 recognized numerous bands that were not sensitive to TRPV2 siRNA, while SC-22520, PC421 and

17A11 showed no immunoreactivity with F11 cell extracts (Figure 2.4b). Out of the antibodies we tested, 2D6 is the best suited for detection of endogenous

TRPV2 by western blot.

2.3.4. Immunoprecipitation of TRPV2 from mouse brain and heart -

Determination of endogenous TRPV2 binding partners will aid in understanding the cellular function of the channel. An essential tool for identifying TRPV2 binding partners is an antibody suitable for immunoprecipitation of endogenous

TRPV2. Based on quantitative studies of TRP channel mRNA levels in mouse organs, TRPV2 transcript was determined to be present in the highest amounts in the DRG, brain, and heart (122). Therefore, we tested the efficiency of our monoclonal antibodies and the commercially available polyclonal TRPV2 antibodies for immunoprecipitation of endogenous TRPV2 from mouse brain and heart lysates. We found very little TRPV2 immunoreactivity in whole tissue extracts, suggesting that endogenously expressed TRPV2 protein is present in these tissues at very low levels (Figure 2.5). The commercially available polyclonal antibodies, in addition to 17A11, failed to precipitate TRPV2 from mouse brain and heart (Figure 2.5). 2D6 was able to precipitate TRPV2 from

47 mouse brain and heart (Figure 2.5). 2D6 antibody detected a higher molecular weight band that may correspond to TRPV2 dimer. These results indicate that

2D6 antibody is best suited for immunoprecipitation of TRPV2 and will likely provide a useful tool for determining physiological TRPV2 binding partners.

Additionally, co-immunoprecipitation results obtained for endogenously expressed TRPV2 with the polyclonal antibodies we tested should be interpreted with caution.

2.3.5. Detection of TRPV2 by immunocytochemistry - Polyclonal antibodies have been utilized to determine the factors that regulate trafficking of heterologously and endogenously expressed TRPV2 (50). Therefore, employing properly validated antibodies to study the subcellular distribution of TRPV2 will be extremely important for understanding TRPV2 function in the cell. We tested the monoclonal antibodies generated in our laboratory as well as the commercially available polyclonal antibodies for recognition of TRPV2 by immunocytochemistry using 1D4-tagged TRPV2 expressed in HeLa cells. We found that 17A11 reacted specifically with 1D4 positive cells and co-localized with 1D4 at the subcellular level (Figure 2.6a). Additionally, 17A11 showed no cross-reactivity with 1D4-tagged TRPV1 (Figure 2.6b). 2D6 showed little immunofluorescence (data not shown). The commercially available polyclonal antibodies displayed immunoreactivity with both 1D4 positive and negative cells, and only partially co-localized with 1D4 (Figure 2.6a). PC421 showed limited immunofluorescence and did not co-localize with 1D4 at the subcellular level.

Fluorescence corresponding to SC-22520 partially co-localized with 1D4 at the

48 cell interior but also appears at the plasma membrane, whereas 1D4

fluorescence is only present in intracellular membranes (Figure 2.6a). ACC-032

clearly did not co-localize with 1D4 fluorescence at the subcellular level (Figure

2.6a). Our results indicate that 17A11 is the best candidate for specific

recognition of endogenously expressed TRPV2.

2.3.6. Effect of IGF-1 on cell surface expression of TRPV2 - Under basal

conditions, TRPV2 localizes to the endoplasmic reticulum (ER); upon exposure

to growth factors and mechanical stimulation, it is thought that TRPV2

translocates to the plasma membrane (50, 51). In addition, increased surface expression of TRPV2 has been observed and implicated in several diseases, including muscular dystrophy, cardiomyopathy, and cancer (51, 54). Since

TRPV2 shows activity in the absence of ligand, the subcellular localization of the channel is likely to be important for its function (47).

We employed our newly developed and characterized TRPV2 monoclonal antibodies to test the effects of IGF-1 on the subcellular distribution of TRPV2 transiently expressed in CHO cells as in the original studies of TRPV2 translocation (50). Treatment of CHO cells with IGF-1 increased phospho-Akt levels indicating activation of the IGF-1 signaling pathway (Figure 2.7a). Cell surface biotinylation assays revealed a limited amount of heterologously expressed TRPV2 resides on the plasma membrane of CHO cells; however no significant changes in plasma membrane levels of TRPV2 were detected after exposure to IGF-1 for up to 30 minutes (Figure 2.7b, c). Two bands were detected with 2D6 antibody in the biotinylated fraction (Figure 2.7b). The higher

49 molecular weight band likely corresponds to a TRPV2 glycosylation product.

Additionally, immunocytochemistry revealed that most TRPV2 resides in an

intracellular compartment, likely the ER, in both the absence and presence of

IGF-1 (Figure 2.7d). We also tested the effects of IGF-1 on endogenous TRPV2

localization in F11 cells. IGF-1 treatment increased phospho-Akt levels in F11

cells (Figure 2.8a). Cell surface biotinylation and immunocytochemistry revealed

that little to no TRPV2 is present at the cell surface in the absence and presence

of IGF-1 (Figure 2.8b, c). Overall, these results are consistent with previous reports suggesting that while growth factor signaling may affect TRPV2 activity, it does not affect its plasma membrane expression (27, 47, 112).

2.4. Discussion

Progress in understanding the cellular function of TRPV2 has been slowed by the lack of tools available to study the channel (113). Studies of TRPV2 knockout mice have revealed that while TRPV2 may not have a major role in sensory transduction, it may perform some other important function (10). Surface expression of TRPV2 was originally shown to be modulated by growth factors

(50). Additionally, increased plasma membrane expression of the channel has been implicated in the pathophysiology of a number of diseases (51, 54).

Regulated trafficking of TRPV2 to the cell surface remains controversial, as groups have employed different tools for studying channel translocation (113).

Our initial analysis revealed that overexpressed TRPV2 localized to intracellular membranes in multiple cell types in both the absence and presence of growth factors.

50 Conflicts in understanding TRPV2 trafficking may be based in the fact that

TRPV2 antibodies raised against linear peptides were not suitable for immuno-

detection of TRPV2. In order to study trafficking mechanisms of endogenously

expressed TRPV2, we extensively characterized the commonly used

commercially available TRPV2 polyclonal antibodies to determine if they were

suitable for this goal. The antibodies we screened were PC421 (Calbiochem),

SC-22520 (Santa Cruz) and ACC-032 (Alomone). PC421 is a commonly used

antibody in TRPV2 studies (119, 123). It was generated against peptide derived

from the distal C-terminus of mouse and rat TRPV2

(KNSASEEDHLPLQVLQSP). This was the same epitope that was employed in

the studies of TRPV2 identification (4). We found that PC421 recognizes recombinant TRPV2 by western blot but it was not able to detect endogenous

TRPV2 from F11 cells. Additionally, it failed to immunoprecipitate TRPV2 from mouse brain and heart lysates. Importantly, PC421 showed limited reactivity with

TRPV2 by immunocytochemistry and possible cross-reactivity with another

protein in the cell.

SC-22520 and other TRPV2 antibodies from Santa Cruz are also

frequently utilized and were generated against an N-terminal peptide from human

TRPV2 (115, 124-126). SC-22520 did not detect full-length recombinant or

endogenous TRPV2 by western blot and failed to immunoprecipitate TRPV2 from

mouse tissues. Like PC421, SC-22520 also showed limited detection of TRPV2

by immunocytochemistry and appeared to cross-react with an unknown plasma

membrane protein.

51 ACC-032, like PC421, was generated against a linear epitope from the rodent TRPV2 distal C-terminus (KKNPTSKPGKNSASEE). This was the same epitope used to generate antibodies that were employed to identify TRPV2 as a growth factor sensitive channel (50). ACC-032 was able to detect full-length recombinant TRPV2 by western blot. However it detected multiple bands from

F11 cell lysates that were not sensitive to TRPV2 siRNA. Additionally, it was not able to immunoprecipitate TRPV2 from mouse tissues and did not co-localize with TRPV2 at the subcellular level by immunofluorescence.

To overcome the difficulties and controversies in studying TRPV2 regulation, we generated monoclonal antibodies against full-length tetrameric

TRPV2. After extensive screening, we found two monoclonal antibodies fit for detection of endogenously expressed TRPV2 (2D6 and 17A11). Both 2D6 and

17A11 were able to recognize recombinant full-length TRPV2 by western blot.

2D6 was the best candidate for western blot detection as it showed robust detection of heterologously expressed TRPV2 from HeLa cells and no cross- reactivity with the highly homologous TRPV1. Importantly, when endogenous

TRPV2 expression was reduced in a neuronal cell line with siRNA, detection of

TRPV2 with 2D6 was also diminished. Additionally, 2D6 was the only antibody we tested able to immunoprecipitate TRPV2 from mouse tissues. 17A11 was best suited for detection of TRPV2 by immunocytochemistry. 17A11 reacted specifically with cells expressing 1D4-tagged TRPV2 and co-localized with 1D4 immunofluorescence at the subcellular level. Moreover, this antibody, for the first time, was able to reveal the endogenous localization of TRPV2 in a neuronal cell

52 line. Our analyses indicated that 2D6 is the best available antibody for detection of TRPV2 by western blot and immunoprecipitation while 17A11 is most suited for immunocytochemistry.

Much of the work to delineate the function of TRP channels relies on commercially available antibodies. Many of these antibodies are of poor quality and therefore have led to controversial and erroneous results (110). An extensive characterization of these antibodies is needed in order to determine their efficacy in detection of specific TRP channels. Additionally, our approach using tetrameric

TRPV2 as antigen may be applicable for generating antibodies against other

TRP channels of unclear function.

It has been shown previously that IGF-1 regulates heterologously overexpressed and endogenous TRPV2 trafficking to the plasma membrane

(50). Although there is agreement that PI3K signaling modulates TRPV2 activity, its effects on regulated insertion of TRPV2 into the plasma membrane remains controversial (27, 47, 112). Since trafficking of TRPV2 appears to be important for its function, we used our newly validated TRPV2 monoclonal antibodies to test the hypothesis that growth factors, specifically IGF-1, increase surface expression of the channel.

Both cell surface biotinylation assays and immunocytochemistry indicated that IGF-1 had no effect on TRPV2 surface expression when transiently expressed in CHO-K1 cells. This is in direct contradiction with the original report that IGF-1 increases TRPV2 surface expression but in agreement with later studies showing that growth factor signaling only affects channel activity and not

53 its plasma membrane levels (47, 50, 112). Furthermore, we tested the effect of

IGF-1 on plasma membrane expression of endogenous TRPV2 in DRG-derived

F11 cells. Likewise, we found that little to no TRPV2 was present on the cell surface in the absence or presence of IGF-1.

Overall, our results indicate that TRPV2 primarily resides in intracellular membranes and its subcellular distribution is not sensitive to IGF-1 treatment. It will be important to use these newly characterized antibodies to determine if the expression and distribution of TRPV2 changes in disease states such as muscular dystrophy, cardiomyopathy and cancer.

54 2.5. Figures

Figure 2.1. Treatment of F11 cells with IGF-1 does not induce translocation of overexpressed TRPV2 to the plasma membrane. (A) F11 cells cultured under normal growth conditions were transfected with 1D4-tagged TRPV1 or TRPV2, then fixed and stained with 1D4 antibody. (B) F11 cells were transiently transfected with TRPV2-1D4. Cells were then cultured in serum-free medium for 12 hours followed by a 30 min treatment with IGF-1 (25 ng/ml). Cells were then fixed and stained with 1D4 antibody. Three representative images are shown for cells cultured in the absence and presence of IGF-1. Scale bar represents 10 µm.

55

Figure 2.2 IGF-1 treatment does not induce translocation of overexpressed TRPV2 to the plasma membrane in HeLa cells. HeLa cells were transfected with TRPV2-1D4, then cultured in serum-free medium for 12 hours followed by treatment with IGF-1 (25 ng/ml) for 30 min. Cells were then fixed and stained for TRPV2-1D4 (green) and calreticulin (red), an ER marker. Scale bar represents 10 µm.

56

Figure 2.3. Immuno-detection of recombinant TRPV2 and mapping of the TRPV2 binding region. (A) Schematic of the domain arrangement for a TRPV2 monomer with the approximate epitope sites for indicated TRPV2 antibodies. ARD, ankyrin repeat domain; TM, transmembrane domain. (B) Western blots with indicated TRPV2 antibodies against purified full-length rat TRPV2, purified rat TRPV2 ankyrin repeat domain and purified rat TRPV2 C-terminus. (C) Western blots with indicated TRPV2 antibodies against extracts from HeLa cells transiently transfected with empty vector, TRPV1-1D4 and TRPV2-1D4.

57

Figure 2.4. TRPV2 monoclonal antibody recognizes endogenous

TRPV2 by western blot. F11 cells were transfected with control or TRPV2 siRNA (200 nM, 48 hr) followed by western blot analysis with (A) anti-

TRPV2 2D6 or (B) indicated polyclonal antibodies. Quantification of the band corresponding to the molecular weight of TRPV2 in (A) was performed using LiCor Odyssey software. TRPV2 band intensity was normalized to actin. Error bars represent s.e.m. for 3 separate experiments.

58

Figure 2.5. Immunoprecipitation of endogenous TRPV2. Immunoprecipitation of TRPV2 with 10 µg of indicated antibodies from 2.5 mg mouse brain lysate and 2.5 mg mouse heart lysate. TRPV2 was detected by western blot with IR dye-labeled 2D6 antibody. Input represents 100 µg of total protein. Membranes from overexpressing recombinant rat TRPV2 were loaded as a control.

59

Figure 2.6. Immunostaining with TRPV2 antibodies. (A) HeLa cells transiently expressing TRPV2-1D4 immunolabeled with indicated TRPV2 antibodies (green) and 1D4 antibody (red). Scale bar represents 10 µm. (B) HeLa cells transiently expressing TRPV1-1D4 immunolabeled with TRPV2 17A11 (green) and 1D4 antibody (red). Scale bar represents 10 µm.

60

61

Figure 2.7. Regulation of TRPV2 trafficking by insulin-like growth factor-1 in CHO-K1 cells. (A) CHO-K1 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. (B) CHO- K1 cells transiently expressing TRPV2 were treated with IGF-1 (20 ng/ml) for indicated times. Surface proteins were biotinylated in intact cells at 37°C. Cells were then lysed and biotinylated proteins were captured with streptavidin agarose. Captured proteins were resolved by SDS-PAGE and detected by western blot with the indicated antibodies. Surface proteins represent the biotinylated fraction and the total lysate represents 5% of total protein. (C) TRPV2 band intensity of the biotinylated fraction was measured using LiCor Odyssey software. Intensities were normalized to biotinylated Na/K ATPase band intensities. Error bars represent S.E.M. from 3 separate experiments. Differences are not statistically significant. (D) CHO-K1 cells transiently expressing TRPV2 treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

62

63

Figure 2.8. Effect of IGF-1 on endogenous TRPV2 trafficking in F11 cells. (A) F11 cells were treated with IGF-1 (20 ng/ml) for the indicated times and immunoblotted with a phospho-Akt specific antibody. Membranes were then stripped and re-probed with a pan-Akt antibody. (B) Biotinylation of surface proteins from F11 cells was performed following the procedure from Figure 2.9b. (C) F11 cells treated with vehicle (PBS) or IGF-1 (20 ng/ml) for 20 min were fixed and immunolabeled for TRPV2 (17A11 antibody). Images are representative of 3 separate experiments. Scale bar represents 10 µm.

64

Chapter 3

MAPK/ERK regulates TRPV2 downstream of NGF

to enhance neurite outgrowth

Portions of this chapter were published in:

Cohen MR, Johnson WM, Pilat JM, Kiselar J, DeFrancesco-Lisowitz A, Zigmond RE, Moiseenkova-Bell VY. 2015. NGF regulates TRPV2 via ERK signaling to enhance neurite outgrowth in developing neurons. Mol Cell Biol, In Press.

65

3.1 Introduction

Establishment of precise neural connections during nervous system

development is essential in forming functional circuits. Neurite outgrowth allows

for connection and communication between developing neurons and their

targets. In the developing peripheral nervous system, nerve growth factor (NGF) is a target-derived extracellular cue necessary for outgrowth (59). Upon binding to its extracellular receptor, NGF activates the phosphoinositide 3 kinase (PI3K) signaling pathway, which is essential for survival of developing neurons, and the mitogen activated protein kinase (MAPK) pathway, which promotes differentiation and neurite outgrowth (70, 71). These signaling pathways have numerous downstream effectors in developing neurons, including several Ca2+-permeable

TRP channels (89-93).

Thermosensitive TRP channels from the vanilloid subfamily (thermoTRPV

channels) consist of four non-selective Ca2+-permeable cation channels (TRPV1-

4) originally described as pain and temperature sensors in adult sensory neurons

(4-7). Recent evidence suggests however that only TRPV1 functions as a

molecular sensor of heat and painful stimuli in vivo, while the function of TRPV2-

4 remains unclear (8, 10, 11, 127). TRPV2 and TRPV4 have also been detected

in developing peripheral neurons, suggesting that they may play a role in growth

programs during development (33, 88). Consistent with this notion, TRPV2 has

been implicated in axon outgrowth (33), and critical mutations in TRPV4 result in

peripheral axonal neuropathies (128, 129). Despite these initial findings, the

66 details by which thermoTRPV channels influence neuronal development remain

unknown.

Here we explore the molecular mechanisms by which thermoTRPV channels contribute to neurotrophin-mediated peripheral neuron development.

We found that amongst the thermoTRPV proteins, TRPV2 and TRPV4 are abundantly present in embryonic sensory neurons, and application of NGF specifically increased TRPV2 protein levels. TRPV2 upregulation occurred through the MAPK signaling pathway, which is essential for neuronal differentiation (70, 71), to enhance neurite outgrowth in a Ca2+-dependent manner. Generation of TRPV2 monoclonal antibodies (130) allowed us to determine that endogenous TRPV2 co-localized with Rab7, a late endosomal marker associated with retrograde trafficking, in embryonic DRG neurons.

Exposure to NGF leads to the formation of signaling endosomes containing TrkA and active MAPK components (80, 86). Consistently, we found co-localization of

TRPV2 with both TrkA and phosphorylated Erk1/2, suggesting that TRPV2 populates signaling endosomes and is directly modulated by ERK. In line with this hypothesis, we found that Erk2 phosphorylates TRPV2 in vitro and identified sites on the soluble N- and C-terminus of TRPV2 critical for phosphorylation by

Erk2. Mutation of these sites reduced NGF-induced neurite growth and altered

TRPV2 protein expression and Ca2+ signals, indicating that phosphorylation of

TRPV2 by ERK is essential for enhancement of Ca2+ signaling and neurite outgrowth. Thus, we propose a mechanism by which ERK regulates Ca2+

signaling via TRPV2 to augment neurite outgrowth in developing neurons.

67 3.2 Materials and Methods

3.2.1. Chemicals and antibodies - The following antibodies were used:

anti-1D4 (118) (1 µg/ml for western blot and immunocytochemistry), anti-TRPV2

2D6 (2 µg/ml for western blot) and anti-TRPV2 17A11 (10 µg/ml for immunocytochemistry) mouse mAbs were generated in our laboratory (130); anti-

β-Actin mouse mAb (Cell Signaling Technology, Danvers, MA, #3700, western blot for 1:1000), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) XP Rabbit mAb (Cell Signaling Technology, #4370, 1:1000 for western blot, 1:200 for immunocytochemistry), anti-p44/42 MAPK (Erk1/2) mouse mAb (Cell Signaling

Technology #9107, 1:1000 for western blot), anti-Akt pan mouse mAb (Cell

Signaling Technology, #2920, 1:1000 for western blot); anti-phospho-Akt (Santa

Cruz Biotechnology, Dallas, TX), anti-Na,K-ATPase (Cell Signaling Technology

#3010, 1:100 for western blot); anti-TRPV1 (NeuromAb clone N221/17, 1:500 for western blot, 1:100 for immunocytochemistry) and anti-TRPV3 (NeuromAb clone

N15/4, 1:1000 for western blot, 1:100 for immunocytochemistry); anti-TRPV4

(AbCam Inc., Cambridge, MA, #39260, 1:200 for western blot and immunocytochemistry); anti-TrkA (Millipore #AB9354, Billerica, MA, 1:100 for immunocytochemistry); anti-Rab7 (Santa Cruz Biotechnology, SC-6563, 1:100 for immunocytochemistry).

NGF-7s, Wortmannin and LY294002 were purchased from Sigma (St.

Louis, MO). U0126 was obtained from Cell Signaling Technology. PhosStop phosphatase inhibitor and EDTA-free complete protease inhibitor were

68 purchased from Roche (Indianapolis, IN). Peptide-N-glycosidase F (PNGaseF) is

from New England Biolabs (Ipswich, MA).

3.2.2. Cell culture and transfection - All cells were cultured in 95% O2,

5% CO2 at 37°C in a humidified atmosphere. Pheochromocytoma 12 (PC12) cells were a kind gift of Dr. John Mieyal (Case Western Reserve University).

PC12 cells were maintained in Ham’s F12 Kaughn nutrient mix (F12K; Life

Technologies, Carlsbad, CA) with 15% horse serum (Life Technologies), 5% fetal bovine serum (FBS; GE Healthcare, Piscataway, NJ), 100 U/ml penicillin and 100

µg/ml streptomycin (Life Technologies). For NGF treatments, cells were changed from normal growth medium to differentiation medium, which consisted of F12K,

1% horse serum, 0.5% FBS and 100 ng/ml NGF. HEK293T, HeLa cells and F11 cells were cultured and transfected as described previously (130).

Transfections of PC12 cells with plasmid DNA were performed using

Lipofectamine LTX reagent (Life Technologies) according to the manufacturer protocol. Transfection of siRNA was performed using RNAiMax Lipofectamine

(Life Technologies) according to the manufacturer protocol. Control siRNA was obtained from Santa Cruz (SC-44235). siRNA targeting TRPV2, Erk1 and Erk2 were obtained from Dharmacon (TRPV2, L-091197-02; Erk1, L-100592-02; Erk2,

L-096054-02). In case of experiments employing PI3K or MEK inhibitors, cells were pre-treated with vehicle (DMSO) or inhibitors for 30 min prior to NGF treatment.

3.2.3. Dissociation and culture of primary E18 DRG neurons - E18

DRGs from Sprague-Dawley rats were obtained from Brain Bits (Springfield, IL).

69 Cells were dissociated by treatment with 0.1 U collagenase and 0.8 U dispase

(Roche) in Hibernate A medium without Ca2+ or Mg2+ (Brain Bits) for 1 hr at 37°C.

Next, the tissue was resuspended in Hibernate AB medium (Brain Bits) and triturated with a p200 pipette. The cells were centrifuged at 200 × g and resuspended in Nb4Activ (Life Technologies) containing NGF (25 ng/ml). Cells were then seeded onto poly-D- (Sigma) coated glass coverslips in 6 well plates. Cells were cultured for 5 days prior to fixation for immunofluorescence.

Half of the medium was changed on day 2. In case of experiments employing

PI3K or MEK inhibitors, cells were treated with vehicle (DMSO) or inhibitors at the time of plating.

3.2.4. Plasmids - Rat TRPV2 in pcDNA3.1 was obtained from Dr. David

Julius (University of California – San Francisco). TRPV2 was engineered with a

C-terminal 1D4 epitope (TETSQVAPA) as described previously (130). Control and TRPV2 shRNA were expressed in the pGFP-C-shLenti (Origene, Rockville,

MD). In addition to expressing shRNA under the U6 promoter, this plasmid also allowed for expression of GFP under the CMV promoter.

3.2.5. Site-directed mutagenesis - To generate TRPV2 mutants, changes were introduced using mutated oligonucleotides for E/K (E599K

Forward 5’-CTGGATGCCTCCCTAAAGCTGTTCAAGTTCACC-3’; Reverse 5’-

GGTGAACTTGAACAGCTTTAGGGAGGCATCCAG-3’. E609K Forward 5’-

ACCATTGGTATGGGGAAGCTGGCTTTCCAG-3’; Reverse 5’-

CTGGAAAGCCAGCTTCCCCATACCAATGGT-3’); or S/A (S6A Forward 5’-

ATGACTTCAGCCTCCGCCCCCCCAGCTTTCAGGCTGGAG-3’; Reverse 5’-

70 CTCCAGCCTGAAAGCTGGGGGGGCGGAGGCTGAAGTCAT-3’; S37A

Forward 5’- CAGGAACCGCCCCCCATGGAGGCACCATTCCAGAGGGAGGAC-

3’; Reverse 5’-

GTCCTCCCTCTGGAATGGTGCCTCCATGGGGGGCGGTTCCTG-3’; S47A

Forward 5’-CAGAGGGAGGACCGGAATTCCGCCCCTCAGATCAAAGTGAAC-

3’; Reverse 5’-

GTTCACTTTGATCTGAGGGGCGGAATTCCGGTCCTCCCTCTG-3’; S760A

Forward 5’-CAGGTCCTCCAGGCCCCCACAGAGACC-3’; Reverse 5’-

GGTCTCTGTGGGGGCCTGGCGGACCTG-3’) and WT TRPV2-1D4 as a

template. The TRPV2 mutant constructs were obtained by PCR using the

Accumprime polymerase kit from Life Technologies. The mutants were confirmed

by DNA sequencing.

3.2.6. Cytosolic Ca2+ measurements - Intracellular Ca2+ measurements were obtained using the fluorescent Ca2+ indicator Fluo4-AM (Life Technologies).

Cells were co-transfected with RFP to identify transfected cells. Experiments

were performed in extacellular solution (ECS) containing (in mM): 120 NaCl, 3

KCl, 2 CaCl2, 2 MgCl2, 30 glucose, 11 sorbitol, and 20 HEPES (pH 7.3). Cells were loaded with 5 µM Fluo4-AM in ECS at room temperature for 30 min,

followed by two 5 min washes in ECS. All measurements were obtained at room

temperature. Emitted fluorescence was measured from single cells using a C1

Plus confocal system on a Nikon Eclipse Ti-E microscope (Nikon Instruments

Inc., Melville, NY) at wavelengths of 495 nm (excitation) and 520 nm (emission)

71 at a frequency of 1 image every 3 sec. Fluorescence intensity was analyzed

using Image J software.

3.2.7. Western blot analysis - Protein extracts were prepared in lysis

buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EGTA, 1% Triton X-100, and

protease and phosphatase inhibitors) as described previously (130). 50 µg of

protein extract was separated by SDS-PAGE using 4-20% Tris Glycine gels (Life

Technologies) and transferred to nitrocellulose membranes. Following block with

10% non-fat dry milk in TBST, membranes were probed with primary antibody

followed by IR-dye conjugated secondary antibodies (LiCor, Lincoln, NE).

Immunoreactivity was detected using the Odyssey Infrared Imaging System

(LiCor). Quantification of band intensities was performed using the LiCor

Odyssey software. For quantification of TRPV2 band intensity, both the upper

and lower bands were included.

3.2.8. Removal of N-linked glycans - To remove N-linked glycans, PC12 cell protein extracts were treated with PNGaseF in G7 reaction buffer and 1%

NP-40 (New England Biolabs) for 1hrat 37°C.

3.2.9. RNA extraction and cDNA synthesis - RNA from cultured F11 and PC12 cells was extracted using Trizol (Life Technologies) following the manufacturer’s instructions. Briefly, 1 ml of Trizol was added to each well of a 6 well dish and incubated at room temperature for 5 min. 200 µl of chloroform

(Fisher Scientific) was added to the mixture, vigorously shaken for 15 sec, and incubated at room temperature for 5 min. Samples were centrifuged at 12,000 × g for 15 min at 4 °C. The upper aqueous phase was transferred to a tube

72 containing 0.5 ml isopropanol, shaken by hand for 30 s, and incubated at room

temperature for 10 min. Following centrifugation at 12,000 × g for 15 min at 4°C,

the supernatant was removed. The pellet was washed in 75% RNase-free ethanol (Fisher Scientific, Waltham, MA) and centrifuged at 7,500 × g for 5 min at

4°C. Ethanol was removed from the tube and the pellet was air dried for 10 min on ice. The pellet containing extracted RNA was then resuspended in 100 µl

DEPC-treated water. cDNA synthesis was completed using SuperScript II

reverse transcriptase (Life Technologies) with 1 µg of RNA as template following

manufacturer’s instructions.

3.1.10. Semiquantitative RT-PCR of TRPV2 and GAPDH - PCR amplification reaction was completed in 50 µl final volume using 5 µl of cDNA from synthesis described above, 10 µl 5× goTaq Buffer, 1 µl (1 µM final

concentration) primers (GAPDH Foward 5’-ATGGTGAAGGTCGGTGTG-3’;

GAPDH Reverse 5’-GCCTCTCTCTTGCTCTCAGT-3’ TRPV2 forward 5’-

ATGACTTCAGCCTCCAGCC-3’ TRPV2 reverse 5’-

TCCAGCGCAGGTATTCTAGC-3’), 1 µl dTNP mix (200 µM final concentration),

0.25 µl GoTaq2 (Promega, Madison, WI), and 29.25 µl H2O.

The PCR protocol consisted of denaturation at 94°C for 5 min and 34 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s and extension at 68°C for 1.5 min. 20 µl of the PCR reaction was analyzed on 1.2% agarose gels and visualized with ethidium bromide staining. Quantification of band intensity was completed using ImageJ.

73 3.2.11. Immunofluorescence - Cells were fixed and stained as described previously (130). Confocal images were obtained using a Leica TCS SP2 laser scanning confocal microscope with a HCX PL APO CS 40.0 × 1.25 oil UV objective. Argon (488 nm) and helium/neon (594 nm) lasers were used for excitation and images were obtained using Leica Confocal Software version

2.61. Brightness and contrast were adjusted using Adobe Photoshop. Only linear changes were made. Any enhancements were applied across images in an identical manner.

3.2.12. Cell surface biotinylation - Cell surface biotinylation experiments were performed as described previously (130). Briefly, PC12 cells were treated with NGF for 24 hr. Cells were washed in PBS and incubated with EZ-Link Sulfo-

NHS biotin (Thermo Scientific, Waltham, MA) for 30 min at 37°C. The reaction was quenched with 100 mM glycine in PBS followed by wash in glycine-free

PBS. Cells were then lysed and incubated with streptavidin-agarose (Life

Technologies) to capture biotinylated proteins. The biotinylated fraction was analyzed by western blot.

3.2.13. Morphology analysis of PC12 cells - PC12 cells were plated on

6 well plates coated with 50 µg/ml rat tail collagen type I (Life Technologies). 24 hr later, cells were transfected with either GFP, WT TRPV2 or DN TRPV2 as described above. 24 hr post-transfection, growth medium was changed to differentiation medium, which consisted of F12K, 1% horse serum, 0.5% FBS and 100 ng/ml NGF for 72 hr. To identify cells overexpressing TRPV2, cells were fixed and immunostained with 1D4 antibody. Cells expressing GFP were used as

74 vector-transfected controls. For knockdown experiments, cells were transiently

transfected with control or TRPV2 shRNA (Origene). shRNA constructs also

expressed GFP, which allowed for detection of transfected cells. Each condition

was imaged in duplicate using a Leica DMI 6000 B fluorescence microscope for

three independent experiments. Fluorescence images from 25 random fields

were collected. The number of cells bearing a neurite was determined by visual

examination of the field and counting the total number of 1D4- or GFP-positive

cells and 1D4- or GFP-positive cells that possessed a neurite at least twice the

length of the cell body diameter. Neurite length was manually determined using

ImageJ software. Neurite length was measured for all cells in the visual field for which the entire neurite was visible and the extension did not contact another cell. Cells from aggregates were disregarded.

3.2.14. In vitro kinase assay - Recombinant TRPV2 was expressed and purified as described previously (49). 500 ng of MBP (Sigma) or recombinant

TRPV2 were incubated with or without 100 ng of recombinant Erk2 (Sigma) in 1x

Erk2 kinase buffer (in mM: 25 MOPS pH 7.2, 12.5 glycerol-2-phosphate, 25

MgCl2, 5 EGTA, 2 EDTA, 0.25 DTT). The kinase reaction was initiated by addition of 25 µM cold ATP (Promega) and 10µCi γ-32P (Perkin-Elmer, Waltham,

MA). The reaction was terminated 45 min after initiation by addition of SDS-

PAGE gel loading buffer (Bio-Rad, Hercules, CA) containing 50 mM β- mercaptoethanol. Samples were run on a 4-20% Tris Glycine gel (Invitrogen), stained using GelCode Blue stain (Thermo Scientific), dried over night, and exposed to autoradiography film (Denville Scientific, South Plainfield, NJ).

75 3.2.15 - Isolation of TRPV2 from HEK293T cells and mass spectrometry analysis. HEK293T cells were transfected with 1D4-tagged

TRPV2 for 48 hr, followed by cell lysis and immunoprecipitation with sepharose beads conjugated to 1D4 antibody. Captured proteins were treated with

PNGaseF, eluted with 1D4 peptide (10 mg/ml; Genscript) and resolved using

SDS-PAGE. Presence of TRPV2 protein was confirmed by western blot. Protein was digested in-gel using the Asp-N (Promega, Madison, WI) (100 ng, 4h at

37oC) and modified trypsin (Promega, Madison, WI) (100 ng, overnight at 37oC) dual enzyme approach. For phosphorylation analysis, the proteolytic peptide mixture was further enriched for phosphopeptides using TiO2 column (Pierce,

Thermo Scientific).

LC-MS experiments were carried out on an Orbitrap Elite mass

spectrometer (Thermo Electron, San Jose, CA) interfaced with a Waters

nanoAcquity UPLC system (Waters, Taunton, MA). To analyze possible

posttranslational modifications (PTM) of TRPV2 protein approximately 300 ng of

proteolytic peptide mixture of TPRV2 was loaded onto a trap column (180 µm ×

20 mm packed with C18 Symmetry, 5 µm, 100 Å (Waters, Taunton, MA)) to pre-

concentrate the sample and wash away excess salts. The reverse phase

separation was performed on a reversed phase column (75 µm x 250 mm nano

column, packed with C18 BEH130, 1.7 µm, 130 Å (Waters, Taunton, MA)) using

a gradient of 2 to 45% mobile phase B (0.1% formic acid and acetonitrile (ACN))

and mobile phase A (100% water/0.1 % formic acid) over a period of 60 min at

37 °C and a flow rate of 300 nl/min. Peptides eluting from the column were

76 directed to a nano-electrospray source with the capillary voltage of 2.5 kV. All mass spectra were obtained from data-dependent experiments. MS and MS/MS spectra were acquired in the positive ion mode with the full scan MS recorded for eluted peptides (m/z range of 350–1800) in the FT mass analyzer at a resolution

R of 120,000 followed by MS/MS of the 20 most intense peptide ions scans in the ion trap (IT) mass analyzer.

The resulting MS2 data were initially searched against TRPV2 protein database using Mass Matrix software to identify all specific sites of modification

(131). In particular, MS2 spectra were searched for nonspecific peptides of

TRPV2 using mass accuracy values of 8 ppm and 0.7 Daltons for MS1 and MS2 scans respectively, with the allowed variable modifications including carbamidomethylation for cysteines, oxidative modifications for methionines amino acid, phosphorylation for serine, threonine and tyrosine, and asparagine deamidation. All detected MS2 spectra for each site of phosphorylation were manually verified.

3.2.16. Statistical analyses - All results are presented as mean ± s.e.m.

Significance of differences was determined by ANOVA followed by Tukey’s post- hoc test for multiple comparisons unless otherwise indicated. Statistical significance was accepted at p values: * p<0.05; ** p<0.01; *** p<0.001.

3.3 Results

3.3.1. TRPV2 is expressed in developing neurons and regulated by

NGF - To assess the expression of thermoTRPV channels in development of peripheral neurons, embryonic day 18 (E18) rat DRG neurons were

77 immunostained with antibodies against TRPV1-4. TRPV1 and TRPV3

immunoreactivites were sparse in βIII-tubulin positive neurons, while TRPV2 and

TRPV4 immunoreactivites were more abundant (Figure 3.1a). TRPV4 appeared

almost exclusively in the cell bodies of neurons, while TRPV2 was present in

both cell bodies and neuronal extensions (Figure 3.1a). We also tested for

expression of TRPV1-4 in NGF-sensitive PC12 cells. The neuroendocrine PC12

cell line is derived from a rat adrenal chromaffin tumor and is commonly used as

a model for studying NGF signaling and neuronal cell differentiation (70, 132).

We failed to detect TRPV1 protein by western blot analysis in PC12 cell lysates

in both the absence and presence of NGF. Specificity of the TRPV1 antibody

was tested by using mouse kidney extracts, which produced a band at

approximately 100 kDa, corresponding to the predicted molecular weight of

monomeric TRPV1 protein (Figure 3.1b). TRPV3 and TRPV4 proteins were

detected in PC12 cells but their levels were unaffected by NGF treatment (Figure

3.1b). In contrast, TRPV2 protein content increased substantially after NGF treatment (Figure 3.1b). An increase in phosphorylated Erk1/2 (pErk1/2) levels confirmed activation of NGF signaling (Figure 3.1b).

TRPV2 has previously been implicated in axon outgrowth during peripheral neuron development (33); however the mechanisms by which TRPV2 is regulated during neuronal development remain unknown. We therefore further explored how NGF regulates TRPV2. RT-PCR confirmed the presence of TRPV2 mRNA in PC12 cells (Figure 3.2a). F11 cells, which are derived from embryonic

DRG neurons and endogenously express TRPV2 (119), were used as a positive

78 control (Figure 3.2a). Our TRPV2 monoclonal antibody (130) displayed

immunoreactivity with two bands in PC12 cell extracts. One band corresponded

to the predicted molecular weight of rat TRPV2 (~86 kDa) while the other ran at a

higher molecular weight (~100 kDa) (Figure 3.2b). The specificity of the TRPV2 antibody was confirmed using siRNA directed against TRPV2. Cells transfected with TRPV2 siRNA displayed decreased band intensity for both the 86 kDa and

100 kDa bands compared to control cells (Figure 3.2b). We had previously observed two bands in cells heterologously expressing TRPV2 and predicted that the upper band may represent glycosylated TRPV2 (130). Treatment of PC12 cell protein extracts with PNGaseF, which cleaves N-linked glycans, eliminated the higher molecular weight band, indicating that this band represents N- glycosylated TRPV2 (Figure 3.2c). These results confirm that both bands recognized by the TRPV2 antibody correspond to TRPV2 protein.

A time course analysis revealed that treatment of PC12 cells with NGF led to an increase in TRPV2 protein content beginning at 6 hr and peaking between

24 and 48 hr (Figure 3.2d). TRPV2 protein content remained elevated for at least 72 hr of NGF treatment (Figure 3.2d). Semiquantitative RT-PCR experiments showed that TRPV2 mRNA content also increased at the 6 and 12 hr time points, after which mRNA levels reverted back to baseline (Figure 3.2e).

Immunofluorescence revealed a comparable time course for upregulation of

TRPV2 protein (Figure 3.2f). TRPV2 localized to developing neurites after NGF treatment, similar to its distribution in embryonic DRG neurons (Figure 3.2f).

Overall, these results confirm the presence of TRPV2 protein in NGF-sensitive

79 developing sensory neurons and show that treatment of PC12 cells with NGF

results in a sustained increase in TRPV2 protein.

3.3.2. TRPV2 enhances NGF-induced neurite outgrowth - Intracellular

Ca2+ signals are involved in morphological changes mediated by NGF (95). Since

NGF signaling is tightly linked to neurite outgrowth in PC12 cells, we next

explored the contribution of TRPV2 activity to NGF-induced neurite extension.

Activation of TRPV2 by membrane stretch was previously shown to increase

intracellular Ca2+ levels and axon outgrowth in developing neurons (33). Studying

endogenous TRPV2 activity and TRPV2-mediated Ca2+ changes has remained difficult due to limited specific pharmacological tools available to activate or inhibit the channel (22, 133). TRPV2 may also display channel activity in the absence of any specific agonist or activator (33, 47). Recently probenecid was described as a specific TRPV2 activator (43). TRPV2-null neurons however display indistinguishable Ca2+ responses to probenecid compared to wild type

(WT) neurons (10). We also did not observe probenecid-activated changes in

cytosolic Ca2+ in cells expressing TRPV2 (data not shown). We therefore chose

to employ a genetic approach to test the effects of TRPV2 activity on NGF-

induced neurite outgrowth (133). A dominant negative TRPV2 construct (DN

TRPV2), where two key Glu residues in the pore region of the channel were mutated to Lys (E599K; E609K), was used in this study (Figure 3.3a). These Glu residues are conserved in rat and mouse TRPV2, and mutation of these residues in mouse TRPV2 to Lys has been shown to decrease Ca2+ permeation through

the pore of the channel (47, 52).

80 After mutation of the pore Glu residues, an HA tag was engineered onto

the protein to allow for specific immunodetection of DN TRPV2. To determine if

DN TRPV2 co-assembles with WT TRPV2, a co-immunoprecipitation assay was

performed. HEK293T cells were transfected with DN TRPV2-HA, WT TRPV2-

1D4 or DN TRPV2 plus WT TRPV2 and subjected to immunoprecipitation

(Figure 3.3b). We found that DN TRPV2 reciprocally co-immunoprecipitated with

WT TRPV2 (Figure 3.3b). Additionally, singly transfected lysates were mixed ex vivo to confirm that the interaction represented co-assembly of heteromeric tetramers rather than an interaction between homomeric tetramers. WT TRPV2 and DN TRPV2 failed to co-IP after mixing lysates, further suggesting that the mutant channel co-assembles with the WT TRPV2 protein (Figure 3.3b). DN

TRPV2-HA also colocalized with WT TRPV2-1D4 in HeLa cells, further indicating

DN TRPV2 can form a complex with WT TRPV2 in the cell (Figure 3.3c).

In order to test if DN TRPV2 inhibited the activity of endogenously

expressed TRPV2, F11 cells were employed, since F11 cells endogenously

express TRPV2 (119) and allow for efficient transfection. DN TRPV2 expressed

at similar levels compared to WT TRPV2 in F11 cells (Figure 3.4a). F11 cells

transfected with DN TRPV2 displayed reduced cytosolic Ca2+ responses after treatment with 2-APB, a TRPV2 activator (35, 37), compared to cells expressing vector alone (Figure 3.4b). Overexpression of WT TRPV2 in F11 cells augmented the Ca2+ increases in response to 2-APB (Figure 3.4b).

Furthermore, F11 cells overexpressing WT TRPV2 displayed distinct

morphological changes, including the formation of neurites, compared to cells

81 expressing empty vector or DN TRPV2 (Figure 3.5a). Approximately 25% of F11 cells bear a neurite under normal culture conditions; overexpression of wild type

(WT) TRPV2 increased the percentage of neurite-bearing F11 cells (Figure 3.5b) and the length of neurites (Figure 3.5c) in F11 cells compared to vector controls in the absence of applied neurotrophin. In contrast, expression of DN TRPV2 in

F11 cells failed to enhance the percentage of cells with neurites (Figure 3.5b) or increase neurite length (Figure 3.5c) compared to vector controls.

We next tested the effects of WT TRPV2 and DN TRPV2 overexpression on NGF-mediated neurite outgrowth in PC12 cells. Overexpression of WT

TRPV2 and DN TRPV2 in PC12 cells had no effect on the percentage of neurite- bearing cells in the absence of applied NGF (Figure 3.6a, b). However, WT

TRPV2 overexpression increased the percentage of cells possessing a neurite

(Figure 3.6b) and the length of neurites (Figure 3.6a, c) compared to GFP-

transfected controls after 3 days in culture in the presence of NGF. Expression of

DN TRPV2 failed to increase the percentage of cells with neurites (Figure 3.6b).

Importantly, neurite length in the presence of NGF was significantly decreased

for cells expressing DN TRPV2 compared to GFP controls (Figure 3.6a, c).

We also tested the effect of silencing TRPV2 expression on NGF-induced

neurite outgrowth using shRNA. The shRNA constructs also expressed GFP,

which indicated transfected cells. Transfection of TRPV2 shRNA efficiently

knocked down TRPV2 overexpressed in HEK293T cells (Figure 3.7a). Since a

smaller percentage of PC12 were transfected, immunofluorescence was

employed to assess knockdown of endogenous TRPV2 protein. Transfection of

82 TRPV2-specific shRNA reduced endogenous TRPV2 immunofluorescence in

PC12 cells compared to cells transfected with control shRNA (Figure 3.7b, c).

Expression of TRPV2 shRNA had no effect on the percentage of neurite bearing

PC12 cells after NGF treatment (Figure 3.7d); however, cells expressing TRPV2

shRNA showed a significant decrease in neurite length compared to cells

expressing control shRNA (Figure 3.7e). Overall, this data indicates that

increased expression of functional TRPV2 significantly contributes to

enhancement of neurite length in response to NGF signaling.

3.3.3. NGF-induced increase in TRPV2 protein is mediated by MAPK

signaling - Binding of NGF to its extracellular receptor TrkA initiates intracellular signaling cascades that promote cell survival and differentiation. The PI3K signaling pathway is necessary for NGF-induced survival of PC12 cells while the

MAPK signaling pathway is essential for differentiation and neurite outgrowth in

PC12 cells (70, 71). Previous studies have suggested that the function of TRPV2

is regulated by growth factors via the PI3K signaling pathway (47, 50, 108). We

therefore tested the effects of PI3K inhibitors LY294002 and wortmannin on

NGF-regulated TRPV2 expression. After 24 hr treatment of PC12 cells with NGF,

an increase in TRPV2 protein was observed; PI3K inhibitors did not have a

significant effect on NGF-induced upregulation of TRPV2 protein levels (Figure

3.8a). Both LY294002 and wortmannin reduced phosphorylation of Akt (pAkt) in

both the absence and presence of NGF, indicating suppression of PI3K activity

(Figure 3.8a). Inhibition of MAPK signaling with the MEK1/2 inhibitor U0126

significantly reduced the NGF-induced upregulation of TRPV2 protein in PC12

83 cells (Figure 3.8b). U0126 decreased pErk1/2 levels, confirming inhibition of the

MAPK pathway (Figure 3.8b). Treatment with U0126 also decreased TRPV2 mRNA levels in PC12 cells after 6 hr treatment with NGF compared to vehicle controls (Figure 3.8c). Consistent with previous reports (134, 135), U0126 decreased the percentage of neurite-bearing cells and neurite length after NGF treatment compared to vehicle control and LY294002 (Figure 3.8d).

The major effector of neurite outgrowth mediated by MAPK downstream of

MEK1/2 is Erk1/2. To determine if Erk1/2 specifically regulates upregulation of

TRPV2, we employed siRNA to reduce Erk1/2 expression. Transfection of PC12 cells with Erk1/2 siRNA reduced Erk1/2 protein levels approximately 40-50% compared to control siRNA (Figure 3.8e). Similar to the MEK1/2 inhibitor, transfection of Erk1/2 siRNA attenuated NGF-induced upregulation of both

TRPV2 protein (Figure 3.8e) and mRNA (Figure 3.8f) in PC12 cells.

Additionally, Erk1/2 siRNA reduced NGF-induced neurite outgrowth compared to control siRNA (Figure 3.8g). Activation of MAPK/ERK signaling by NGF in PC12 cells peaks after minutes of exposure (74). We observed increases in pErk1/2 levels after 10 min treatment with NGF, whereas TRPV2 protein levels did not significantly increase until 6 hr exposure to NGF (Figure 3.8h). This shows that activation of MAPK/ERK signaling precedes upregulation of TRPV2 expression, and TRPV2 is regulated downstream of Erk1/2.

Importantly, we also observed a decrease in TRPV2 immunofluorescence in primary embryonic DRG neurons cultured in the presence of U1026 for 5 days compared to cells cultured with DMSO or LY294002 (Figure 3.8i). These results

84 indicate that the MAPK signaling pathway, which promotes neurite outgrowth,

contributes to NGF-induced upregulation of TRPV2 expression in both PC12

cells and primary developing sensory neurons.

3.3.4. NGF does not induce TRPV2 translocation to the plasma

membrane in PC12 cells - Growth factors such as insulin-like growth factor-1

(IGF-1) were initially thought to induce translocation of TRPV2 to the plasma

membrane through the PI3K signaling pathway, where it mediates a Ca2+ current that contributes to growth factor signaling (50). Using our monoclonal TRPV2 antibody however we previously found that IGF-1 had no effect on plasma membrane translocation of overexpressed and endogenous TRPV2 (130). Since we observed that PI3K signaling had no effect on upregulation of TRPV2, we next tested if NGF treatment increased plasma membrane levels of TRPV2 in

PC12 cells. Cell surface biotinylation experiments failed to detect TRPV2 at the surface of PC12 cells in both the absence and presence of NGF (Figure 3.9a). In contrast, TRPV3 was present in the biotinylated fraction in both the absence and presence of NGF, and surface levels of TRPV3 did not change in response to

NGF treatment (Figure 3.9b). Additionally, we found that TRPV2 colocalized with

Rab7, a late endosomal marker, after NGF treatment (Figure 3.9c), further indicating that TRPV2 does not translocate to the cell surface after NGF stimulation. Consistent with results from our laboratory and others showing that

IGF-1 and other growth factors have no effect on the surface levels of TRPV2

(47, 112, 130), these data show that while NGF treatment increases TRPV2 content, it does not induce translocation of the channel to the cell surface. The

85 absence of TRPV2 in the biotinylated fraction indicates that TRPV2

predominantly localizes to intracellular membranes in PC12 cells.

3.3.5. ERK phosphorylates TRPV2 to enhance neurite outgrowth -

When NGF binds and activates TrkA at the terminals of developing neurites, the

NGF-TrkA complex is internalized into endosomes and transported along

microtubules toward the cell body in a retrograde manner, where it continues to

signal through PI3K and MAPK (75). Activated Erk1/2 is closely associated with these TrkA-containing endosomes, where it can facilitate local signaling throughout the developing neuronal processes (86). Since we observed that

TRPV2 colocalized with an endosomal marker in PC12 cells, and TRPV2 immunoreactivity is present in both the cell body and β-III tubulin containing neurites of embryonic DRG neurons (see Figure 3.1a), we next tested if TRPV2 is associated with these NGF signaling endosomes in embryonic DRG neurons.

TRPV2 immunoreactivity showed substantial colocalization with Rab7 (Figure

3.10a), which is essential for retrograde transport of Trk-containing signaling endosomes in developing neurons (80). Partial colocalization was also observed between TRPV2 and both TrkA (Figure 3.10b) and pErk1/2 (Figure 3.10c) in embryonic DRG neurons. These results suggested that ERK might directly modulate TRPV2 protein within the neurites of developing neurons.

To test if ERK phosphorylates TRPV2, recombinant activated Erk2 was incubated with γ32P-ATP and either myelin basic protein (MBP), a known ERK

substrate, or pure tetrameric TRPV2 (49). TRPV2 and MBP were both

phosphorylated in the presence, but not absence, of activated Erk2 (Figure

86 3.10d), suggesting that TRPV2 is a viable ERK substrate and ERK may directly

phosphorylate TRPV2 through NGF signaling.

Post-translational modifications of TRPV2 have not been extensively

characterized. To directly test if phosphorylation of TRPV2 by ERK affects NGF-

induced neurite outgrowth, we first needed to determine the possible TRPV2

residues that ERK phosphorylates. A mass spectrometry analysis of TRPV2

expressed in HEK293T cells revealed several phosphorylation sites. Four

identified Ser residues were within proline-directed motifs (Ser/Thr-Pro) and

predicted to act as ERK substrates (Ser6, Ser37, Ser47, Ser760; Figure 3.11a-

d). All four sites are within flexible regions of the distal N- and C-termini of the

protein (Figure 3.11d). Mutation of all four sites to Ala (S6A, S37A, S47A,

S760A; TRPV2∆4S) substantially reduced phosphorylation of TRPV2 by Erk2 in vitro (Figure 3.12a).

We next tested if phosphorylation of TRPV2 by ERK affected NGF- induced neurite outgrowth in PC12 cells. We employed a protocol in which cells were transfected with vector control, WT TRPV2 or TRPV2∆4S followed by a 30 min pretreatment with either vehicle or U0126 and 72 hr treatment with NGF.

U0126 significantly reduced the percentage of cells possessing a neurite for all

transfection conditions (Figure 3.12b, c). Overexpression of TRPV2∆4S did not significantly affect the percentage of cells possessing a neurite compared to WT

TRPV2; however neurite length for cells transfected with TRPV2∆4S was

significantly reduced compared to WT TRPV2 in the absence of U0126 (Figure

3.12b, d). Interestingly, U0126 reduced neurite length for vector control and WT

87 TRPV2 transfected cells but not cells expressing TRPV2∆4S (Figure 3.12d).

Pretreatment with U0126 reduced expression of exogenously expressed WT

TPRV2 in PC12 cells after NGF exposure (Figure 3.12e). Additionally,

expression of TRPV2∆4S was significantly lower than WT TRPV2 in the absence

of inhibitor, and levels of TRPV2∆4S were unaffected by U0126 (Figure 3.12e).

Silencing Erk1/2 had similar effects on neurite outgrowth mediated by WT

TRPV2 and TRPV2∆4S, namely Erk1/2 siRNA reduced protein levels (Figure

3.12f) and NGF-induced neurite outgrowth for exogenously expressed WT

TRPV2 but not TRPV2∆4S (Figure 3.12g, h). Erk1/2 siRNA efficiently reduced

Erk1/2 protein levels under these conditions (vector, 68.2 ± 0.66 % of control siRNA; WT TRPV2, 69.6 ± 0.61 % of control siRNA; TRPV2∆4S, 66.1 ± 0.44% of control siRNA). These results indicate that while inhibition of ERK signaling decreases expression of WT TRPV2 and reduces neurite outgrowth mediated by

WT TRPV2, it has little to no effect on TRPV2∆4S. This is consistent with the

hypothesis that ERK phosphorylates TRPV2 at these N- and C-terminal Ser

residues downstream of NGF to enhance neurite outgrowth.

Since we previously associated TRPV2-mediated Ca2+ changes with

neurite outgrowth, we predicted that expression of TRPV2∆4S might decrease

TRPV2-mediated Ca2+ transients. Ca2+ imaging analysis showed that the response of TRPV2∆4S to 2-APB was significantly reduced compared to WT

TRPV2 in F11 cells (Figure 3.12i). Additionally, U0126 reduced the response of

WT TRPV2 expressing cells to 2-APB, but not cells expressing TRPV2∆4S

(Figure 3.12i). Overall, these results indicate that phosphorylation of TRPV2 by

88 ERK enhances TRPV2-mediated expression and Ca2+ signals to increase neurite

outgrowth downstream of NGF.

3.4 Discussion

TRPV2 was discovered as a growth factor regulated Ca2+-permeable ion channel in mouse fibroblasts (50). The channel was also originally proposed to act as a noxious temperature detector in sensory neurons (4). TRPV2 knockout mice however display normal sensory transduction, including temperature sensation, suggesting that TRPV2 does not function as a noxious heat sensor in vivo (10, 127). Additionally, the mechanisms by which growth factors regulate endogenous TRPV2 function remain unclear (22, 112).

TRPV2 is ubiquitously expressed, in contrast to the other thermoTRPV proteins (136), further signifying that it plays some other important physiological role. Endogenously expressed TRPV2 has primarily been detected in intracellular membranes, as opposed to the cell surface, including endosomes (57, 58, 137).

It has been proposed that TRPV2 displays constitutive basal activity and to date there are no confirmed endogenous channel modulators, while exogenous

TRPV2 activators and inhibitors are promiscuous Ca2+ channel modulators (22).

The channel has been implicated in numerous different physiological and pathological processes, including growth factor signaling, macrophage migration, cancer cell metastasis, heart disease and neuronal cell development (22, 33, 50-

52, 108, 136). Nonetheless, the function of endogenous TRPV2 remains controversial due to the lack of reliable tools available to specifically modulate and detect the channel protein (22).

89 Generation of monoclonal TRPV2 antibodies has allowed us to begin

exploring the endogenous function of TRPV2 (130). Strong evidence points

towards a role for TRPV2 in development of peripheral neurons (10, 33). Global

knockout of TRPV2 in mice resulted in perinatal lethality (10). Furthermore,

TRPV2 mRNA was detected in the mouse spinal cord at E9.5, before the

expression of other thermoTRP channels (33). Using our newly generated antibodies, we observed TRPV2 protein in embryonic DRG neurons and PC12 cells, both well-established models for studying peripheral neuron development and neurotrophin signaling (138). Treatment of PC12 cells with NGF, which is essential for survival and differentiation of developing peripheral neurons, led to a

sustained increase in TRPV2 protein content without affecting plasma membrane

levels of TRPV2. Since evidence indicates that TRPV2 displays constitutive

basal activity (33, 47), upregulation of TRPV2 protein content is likely a major

mechanism of functional regulation of the channel by NGF.

Ca2+ is an important second messenger in NGF-mediated neuronal cell differentiation (95). Increases in cytoplasmic Ca2+ levels are associated with cytoskeletal rearrangements leading to morphological changes, alterations in gene transcription and changes in cell excitability (95). Here, we observed that overexpression of Ca2+-impermeable TRPV2 mutant in PC12 cells reduced NGF-

induced neurite outgrowth. Silencing TRPV2 expression also decreased neurite

length in PC12 cells after NGF treatment, further indicating that NGF-induced

upregulation of functional TRPV2 likely alters intracellular Ca2+ levels in developing neurons, leading to increases in neurite extension. Inhibiting or

90 silencing TRPV2 did not completely impair neurite outgrowth in PC12 cells, consistent with evidence indicating that multiple Ca2+-permeable channels are involved in neurite outgrowth (96). This data is in line with a previous report showing that silencing or inhibiting TRPV2 reduces axon growth in primary DRG neurons (33). Abnormal peripheral neuron development may account for the embryonic abnormalities and eventual perinatal lethality observed in TRPV2-null mice (10).

We further explored the molecular details by which TRPV2 is regulated by

NGF. NGF signaling begins at the axon terminal of developing neurons. Binding of NGF to its receptor TrkA at the cell surface causes receptor dimerization, autophosphorylation and activation of signaling pathways including PI3K and

MAPK (75). After NGF binding, the NGF-TrkA complex is internalized into endosomes, where TrkA can continue to signal as it is transported along microtubules toward the neuronal cell body (75, 86). Although PI3K signaling has previously been linked to TRPV2 trafficking and activity (47, 50), we found no connection between PI3K activity and NGF-induced upregulation of TRPV2 protein levels. In contrast, pharmacological inhibition of MAPK signaling significantly reduced TRPV2 protein levels in both primary developing neurons and PC12 cells. Additionally, siRNA-mediated inhibition of MAPK signaling reduced TRPV2 expression in PC12 cells.

TrkA-containing endosomes are thought to be long-lived and enriched in

NGF signaling components such as MAPKs (75, 86). These endosomal platforms allow for NGF-TrkA to meet local signaling needs within neuronal

91 extensions and affect gene transcription once the endosomes reach the nucleus

in the cell body (76). We found that the Ca2+-permeable TRPV2 displays a

punctate staining pattern in the extensions of embryonic DRG neurons. TRPV2

colocalized with Rab7, a marker for late endosomes, in addition to both TrkA and

pErk1/2 in neurite extensions, suggesting that these important neurotrophin

signaling components populate similar endosomal pools as TRPV2. Additionally,

we observed that Erk2 phosphorylates TRPV2 in vitro, suggesting that ERK may

directly modulate TRPV2, and thus intracellular Ca2+ signaling within developing

neurites.

Post-translational modifications of TRPV2 are not well characterized,

although we identified four Ser residues on its distal N- and C-terminus of the

channel predicted to be ERK substrates (139). Two of these predicted residues

(Ser 6 and Ser 760) were identified as phosphorylation sites for TRPV2 from nervous system tissues in a phosphoproteomic study (140), while two other sites

(Ser 37 and Ser 47) were identified in macrophages (141). Mutation of these predicted ERK sites reduced phosphorylation of TRPV2 by Erk2 in vitro, as well as NGF-mediated neurite outgrowth, TRPV2 protein expression and 2-APB induced Ca2+ transients. Overall, this data is consistent with a mechanism by

which ERK phosphorylates TRPV2, increasing channel expression and Ca2+

signals to enhance neurite outgrowth in developing neurons.

Based on our studies, we propose that MAPK signaling regulates TRPV2

downstream of NGF by altering expression of TRPV2 and directly through

phosphorylation of the channel by ERK. Our data indicates that TRPV2 localizes

92 to Rab7-positive late endosomes, which are involved in retrograde trafficking on

NGF and TrkA (80). TRPV2 activity within endosomes would mediate a Ca2+ flux

from the endosomal lumen to the of the neurite. The close association

between TRPV2 and NGF-TrkA within signaling endosomes allows for direct

modulation of TRPV2 protein by MAPK signaling components within developing

neuronal processes (86). ERK directly phosphorylates TRPV2 on the cytoplasmic

N- and C-termini of the channel, leading to increased TRPV2 protein content,

alterations in local Ca2+ signaling within neurites and augmented neurite

outgrowth (Figure 3.13). Localization of TRPV2 to signaling endosomes provides

an efficient and versatile mechanism for regulating Ca2+ signaling within long neurites of developing neurons without directly changing neuronal excitability

(142). Downstream effects of TRPV2 activity remain to be explored, although one possibility is that TRPV2-mediated Ca2+ signals derived from endosomes might mediate endosomal maturation and fusion (143).

In addition, TRPV2 expression has been shown to be upregulated in DRG neurons after peripheral nerve injury (24, 144). Injury is thought to activate

intrinsic neuronal growth programs, including neurotrophin signaling, to promote

axon regrowth (145). Neurotrophin release in response to injury also leads to

inflammatory signaling, hyperalgesia and aberrant sprouting in peripheral

sensory neurons, resulting in potentially debilitating neuropathic pain (103, 146-

152). Increased expression of TRPV2 was thought to contribute directly to pain sensation after nerve injury (24). Nevertheless, based on recent TRPV2 genetic deletion studies (10) coupled with our current findings, it is possible that TRPV2

93 functions directly in regeneration of sensory neurons after injury. Further studies to delineate the role of TRPV2 in neuronal sprouting and growth are needed to determine if TRPV2 represents a molecular player and potential therapeutic

target in neurite outgrowth related to injury-induced neuropathic pain.

94 3.5. Figures

Figure 3.1. NGF upregulates TRPV2 in models of developing neurons. (A) E18 DRG neurons immunostained with antibodies against the thermoTRPV channels (TRPV1, TRPV2, TRPV3 and TRPV4; green) and β-III tubulin (red). Scale bar represents 100 µm. (B) Western blot analysis from 50 µg of PC12 cell lysates using the indicated thermoTRPV antibodies plus phosphorylated Erk1/2 (pErk1/2), total Erk1/2 and β-Actin. Cells were cultured in differentiation medium in the absence or presence of NGF (100 ng/ml) for 12 hr. Since PC12 cells lacked TRPV1 immunoreactivity, mouse kidney lysate was loaded as a positive control.

95

96 Figure 3.2. NGF treatment results in sustained upregulation of TRPV2 expression in PC12 cells. (A) mRNA expression of TRPV2 in F11 cells and PC12 cells was examined by RT-PCR using TRPV2 specific PCR primers. (B) Western blot analysis using TRPV2 monoclonal antibody of PC12 cells transfected with 200 nM control siRNA or TRPV2 siRNA for 48 hr. Bar graph represents relative TRPV2 band intensities. TRPV2 band intensity for control siRNA was defined as 1. Data represent mean ± s.e.m. p<0.05 by unpaired t- test. (C) PC12 cell protein extracts were treated with and without PNGaseF and immunoblotted with the TRPV2 and β -Actin antibodies. (D) PC12 cells were treated with NGF (100 ng/ml) for indicated times. 50 µg of whole cell lysates were then analyzed by western blot with TRPV2 and β -Actin antibodies. Line graph represents relative TRPV2 protein for each time point. The β -Actin-normalized TRPV2 band intensity at the 0 hr time point was defined as 1. *p<0.05 relative to the 0 hr NGF time point by one-way ANOVA followed by Dunnet post hoc test. Data represent mean ± s.e.m for 3 independent experiments. (E) RT-PCR analysis from PC12 cells treated with NGF (100 ng/ml) for indicated times. RNA was extracted and reverse transcribed to cDNA, followed by PCR amplification using TRPV2 and GAPDH specific primers. Line graph represents relative TRPV2 mRNA for each time point. The GAPDH-normalized TRPV2 band intensity at the 0 hr time point was defined as 1. Data represent mean ± s.e.m. of 3 independent experiments. *p<0.05 relative to the 0 hr NGF time point by one- way ANOVA followed by Dunnet post hoc test. (F) PC12 cells were treated for indicated times with NGF (100 ng/ml) then fixed and immunostained with anti- TRPV2 antibody. Scale bar represents 25 µm.

97

Figure 3.3. Dominant negative TRPV2 co-assembles with WT TRPV2. (A) Cartoon representation of a TRPV2 dimer. Arrows indicate the location of the conserved Glu resides that were mutated to Lys to generate DN TRPV2. (B) HEK293T cells were transfected with DN TRPV2-HA alone, WT TRPV2-1D4 alone or DN TRPV2-HA plus WT TRPV2-1D4 and subjected to immunoprecipitation with anti-1D4 or anti-HA antibodies. To determine specificity of tetrameric complexes, singly transfected DN TRPV2-HA and WT TRPV2-1D4 lysates were mixed ex vivo and subjected to immunoprecipitation (mix). Left panel represents schematic of experimental design; right panel shows representative western blots following IP with 1D4 and HA antibodies. (C) HeLa cells transfected with both DN TRPV2-HA and WT TRPV2-1D4 were fixed and stained with HA and 1D4 antibodies. Scale bar represents 10 µm.

98

Figure 3.4. Expression of DN TRPV2 reduces the Ca2+ response of F11 cells to 2-APB. (A) F11 cells were transiently transfected with either empty vector, WT TRPV2 or DN TRPV2 then subjected to western blot analysis with indicated antibodies. (B) Intracellular Ca2+ levels were measured by Fluo4 fluorescence using time-lapse microscopy. F11 cells expressing empty vector (n=80), WT TRPV2 (n=72) or DN TRPV2 (n=61) were treated with 2-APB (100 µM) at the 45 s time point (indicated by arrow). Data represent the mean normalized Fluo4 intensity ± s.e.m.

99

Figure 3.5. TRPV2 overexpression enhances neurite outgrowth in F11 cells. (A) Representative micrographs of F11 cells co-transfected with GFP and either empty vector, WT TRPV2 or DN TRPV2 for 24 hours. Cells were imaged for GFP fluorescence from 25 random fields per experiment. Scale bar represents 100 µm. (B) Percentage of cells with neurites was determined from images in (A). A neurite was defined as an extension at least the length of the cell body diameter. Data represent mean ± s.e.m from three independent experiments. (C) Neurite length was determined from images in (A) for cells expressing vector (N=188), WT TRPV2 (N=271) and DN TRPV2 (N=177). Extensions from aggregated cells and extensions that contacted another cell were disregarded. Data represent mean ± s.e.m.

100

Figure 3.6. TRPV2 activity enhances NGF-induced neurite outgrowth in PC12 cells. (A) Representative micrographs of PC12 cells treated with NGF (100 ng/ml; 72 hr) after transfection with GFP

(control) or 1D4-tagged WT TRPV2 or DN TRPV2. After NGF treatment, cells were fixed and stained with 1D4 antibody followed by Alexa488-labeled secondary antibody. Scale bar represents 100

µm. (B) PC12 cells were transfected with GFP, WT TRPV2 or DN

TRPV2 and grown in the absence or presence of NGF (100 ng/ml;

72 hr) then imaged for fluorescence as in (A). Data represents mean

± s.e.m. from 3 independent experiments. (C) Neurite length (µm) for

PC12 cells transfected with GFP (n=141), WT TRPV2 (n=122) or DN

TRPV2 (n=67) and treated with NGF (100 ng/ml; 72 h).. Data represents mean ± s.e.m. from 3 independent experiments.

101

Figure 3.7. Silencing TRPV2 expression impairs NGF-induced neurite outgrowth. (A) Western blot analysis with indicated antibodies of HEK293T cells transiently transfected with rat TRPV2 and either control or TRPV2 shRNA. (B) PC12 cells were transfected with control shRNA or TRPV2 shRNA. 24 hr later, cells were treated with NGF (100 ng/ml) for 72 hr. Cells were then fixed and immunostained for TRPV2 (red). GFP indicates a transfected cell (green) and nuclei were stained with Hoescht (blue). (C) Mean TRPV2 fluorescence intensity ± s.e.m. for GFP positive cells (control shRNA n=30; TRPV2 shRNA n=19). p<0.001 by unpaired t test. (D) PC12 cells were transfected with control or TRPV2 shRNA followed by 72 hr treatment with NGF (100 ng/ml). 50 random images were obtained for GFP fluorescence and analyzed for percentage of cells with neurites. Data represents the mean ± s.e.m. percentage of neurite-bearing, GFP-positive cells from 3 independent experiments. n.s. not significant (p=0.94) by unpaired t test. (E) Mean ± s.e.m. neurite length of PC12 cells transfected with control shRNA (n=362) or TRPV2 shRNA (n=454) and treated with NGF for 72 h. p<0.001 by unpaired t test.

102

103 Figure 3.8. MAPK signaling mediates upregulation of TRPV2. (A) PC12 cells were pretreated with DMSO, LY294002 (5 µM) or wortmannin (20 nM) for 30 min, followed by treatment with NGF (100 ng/ml; 24 hr) as indicated. TRPV2, pAkt, total Akt and β -Actin protein levels were detected by western blot. The β -Actin normalized TRPV2 band intensity for DMSO treated cells in the absence of NGF was defined as 1. Bar graph represents mean ± s.e.m. for 3 independent experiments. (B) PC12 cells were pretreated with DMSO or U0126 (10 µM) for 30 min, followed by treatment with NGF (100 ng/ml; 24 hr) as indicated. TRPV2, pErk1/2, total Erk1/2 and β -Actin protein levels were detected by western blot. The β -Actin normalized TRPV2 band intensity for DMSO treated cells in the absence of NGF was defined as 1. Bar graph represents mean ± s.e.m. for 3 independent experiments. (C) PC12 cells were pre-treated with DMSO or U0126 (10 µM) for 30 min, followed by treatment with NGF (100 ng/ml; 6 hr) as indicated. TRPV2 and GAPDH mRNA levels were determined by RT-PCR. The GAPDH normalized TRPV2 band intensity for DMSO-treated cells in the absence of NGF was defined as 1. Bar graph represents mean ± s.e.m. for 3 independent experiments. (D) Bar graphs representing percentage of cells with neurites and neurite length (µm) for PC12 cells cultured in the presence of NGF and either vehicle (DMSO), LY294002 (5 µM) or U0126 (10 µM). Data represent mean ± s.e.m. for 3 independent experiments. (E). PC12 cells were transfected with control or Erk1/2 siRNA for 48 hr followed by 12 hr treatment with NGF as indicated. TRPV2, pErk1/2, total Erk1/2 and β-Actin protein levels were detected by western blot. The β-Actin normalized TRPV2 or total Erk1/2 band intensities for control siRNA transfected cells in the absence of NGF were defined as 1. Bar graphs represents mean ± s.e.m. for 3 independent experiments. (F) PC12 cells were transfected and treated as in (E) and TRPV2 and GAPDH mRNA levels were determined by RT-PCR. The GAPDH normalized TRPV2 band intensity for control siRNA transfected cells in the absence of NGF was defined as 1. Bar graph represents mean ± s.e.m. for 4 independent experiments. (G) Bar graphs representing percentage of cells with neurites and neurite length (µm) for PC12 cells transfected with control or Erk1/2 siRNA and cultured in the presence of NGF for 72 hr. Data represent mean ± s.e.m. for 5 independent experiments. (H) PC12 cells were treated with NGF (100 ng/ml) for indicated times followed by western blot analysis with TRPV2, pErk1/2, total Erk1/2 and β-Actin antibodies. Line graphs represent relative β -Actin normalized TRPV2 and total Erk1/2 normalized pErk1/2 levels. Band intensities at the 0 NGF time point were defined as 1. Data represent mean ± s.e.m. for 3 independent experiments. *p<0.05 relative to the 0 NGF time point by one-way ANOVA followed by Dunnet post hoc test. (I) E18 DRG neurons were cultured in the presence of DMSO, LY294002 (25 µM) or U1026 (10 µM) for 5 days, then immunostained for TRPV2 (green) and β -III tubulin (red). All images were obtained with identical microscope settings. Scale bar represents 100 µm.

104

Figure 3.9. NGF does not induce translocation of TRPV2 to the plasma membrane in PC12 cells. (A) PC12 cells were cultured in the absence or presence of NGF (100 ng/ml) for 24 hr. Surface proteins were biotinylated in intact cells at 37°C. Cells were then lysed and biotinylated proteins were captured with streptavidin agarose. Captured proteins were resolved by SDS-PAGE and detected by western blot with TRPV2 and Na/K ATPase antibodies. Na/K ATPase was used as a positive control. Surface proteins represent the biotinylated fraction and input represents 5% of total extracted protein. (B) Cell surface biotinylation experiment performed as in (A). TRPV3 and Na/K ATPase antibodies were used for western blot analysis. (C) PC12 cells were cultured in the absence or presence of NGF (100 ng/ml) for 24 hr, followed by fixation and staining for TRPV2 (green) and Rab7 (red). Scale bar represents 10 µm.

105

Figure 3.10. TRPV2 is phosphorylated by ERK. E18 DRG neurons were cultured for 5 days in the presence of NGF then fixed and immunostained for TRPV2 (green) and (A) Rab7 (red), (B) TrkA (red) or (C) pErk1/2 (red). Arrow points to region of zoomed images represented in bottom panels. Scale bar represents 5 µm. (D) Recombinant MBP or TRPV2 (500 ng) were incubated in the absence or presence of recombinant active Erk2 (100 ng) and g32P-ATP as indicated. Phosphorylation of MBP or TRPV2 was detected by autoradiography. The presence of recombinant proteins was confirmed by coomassie stain.

106

107

Figure 3.11. Mass spectrometry analysis of heterologously expressed TRPV2 to determine phosphorylation sites. (A) MS/MS spectra of the phosphorylated form of TSASSPPAFR peptide (2-11) observed as a doubly protonated ion at the m/z of 550.74. The presence of y6-y8, and b5-98 and b9-98 fragment ions with 79.97 Da mass shift shows that modification of this peptide occurred at S6. Asterisk indicates the fragment ions that were modified by 79.97 Da. (B) MS/MS spectra of the modified form of QEPPPMESPFQRE peptide (30- 42) observed as a doubly protonated ion at the m/z of 834.34. The presence of y6-y7 fragment ions with 79.97 Da mass shift, and y8-y11 ions with the mass shift of 79.97 Da and 16 Da shows that phosphorylaton and oxidation of this peptide occurred at position S37 and M35, respectively. Asterisk indicates the fragment ions that were modified by 79.97 Da. (C) MS/MS spectra of the phosphorylated form of DRNSSPQIKVNL peptide (43-54) observed as a doubly protonated ion at the m/z of 725.85. The presence of y8-y10, and b5-b10 fragment ions with 79.97 Da mass shift shows that modification of this peptide occurred at S47. Asterisk indicates the fragment ions that were modified by 79.97 Da. (D) MS/MS spectra of the phosphorylated form of DHLPLQVLQS peptide (751-760) observed as a doubly protonated ion at the m/z of 615.30. The presence of y1-y7 fragment ions with 79.97 Da mass shift and unmodified b5-b9 fragment ions shows that modification of this peptide occurred at S760. Asterisk indicates the fragment ions that were modified by 79.97 Da. (E) Domain structure of a TRPV2 monomer. TRPV2 consists of large soluble N- and C-termini. The N- terminus contains six ankyrin repeats that form the ankyrin repeat domain. The four predicted ERK phosphorylation sites (indicated by arrows) reside on the distal N- and C-terminus.

108 109

Figure 3.12. Phosphorylation of TRPV2 by ERK enhances NGF-induced neurite outgrowth in PC12 cells. (A) 1 µg of recombinant TRPV2 or WT TRPV2 and TRPV2∆4S isolated from HEK293T cells were incubated with activated Erk2 and γ32P-ATP. Phosphorylation of the TRPV2 proteins was determined by autoradiography and the presence of protein was confirmed by coomassie stain. (B) Representative images of PC12 cells transfected with GFP plus either empty vector, or 1D4-tagged WT TRPV2 or TRPV2∆4S, and treated with NGF (100 ng/ml) in either the absence or presence of U0126 (10 µM) for 72 hr. (C) Percentage of cells with neurites for PC12 cells imaged in (B). Data represent mean ± s.e.m. for 3 independent experiments. (D) Mean neurite length ± s.e.m. for cells as imaged in (B). Vector control + DMSO n= 265; vector control + U0126 n= 126 ; WT TRPV2 + DMSO n= 181 ; WT TRPV2 + U0126 n= 93; TRPV2∆4S + DMSO n= 195 ; TRPV2∆4S + U0126 n= 119. (E) Western blot analysis of PC12 cells transfected and treated as in (B-D) with indicated antibodies. Bar graph represents mean band intensities ± s.e.m. for 3 independent experiments. The β-Actin normalized TRPV2-1D4 band intensity for WT-TRPV2-expressing, DMSO-treated cells was defined as 1. (F) Western blot analysis with indicated antibodies for PC12 cells were transfected with control or Erk1/2 siRNA for 24 hr, followed by 24 hr transfection with GFP plus vector, or 1D4-tagged WT TRPV2 or TRPV2∆4S and 72 hr treatment with NGF. Bar graph represents mean band intensities ± s.e.m. for 3 independent experiments. The β- Actin normalized TRPV2-1D4 band intensity for cells transfected with control siRNA and WT TRPV2 was defined as 1. (G) Bar graph representing the percentage of cells with neurite for PC12 cells as transfected and treated in (F). Data represent mean ± s.e.m. for 3 independent experiments. (H) Mean neurite length ± s.e.m. for cells as transfected and treated in (F). Vector, control siRNA n=145; Vector, Erk1/2 siRNA n=151; WT TRPV2, control siRNA n=162; WT TRPV2, Erk1/2 siRNA n=123; TRPV2∆4S, control siRNA n=138; TRPV2∆4S, Erk1/2 siRNA n=103. (I) Intracellular Ca2+ transients measured by Fluo4 fluorescence using time-lapse microscopy. F11 cells expressing WT TRPV2 or TRPV2∆4S were treated with 2-APB (100 µM) at the 45 s time point (indicated by arrow). Data represent the mean normalized Fluo4 intensity. Bar graph represents peak in normalized Fluo4 fluorescence intensity determined for each condition. WT TRPV2 + DMSO n=177; WT TRPV2 + U0126 n=129; TRPV2∆4S + DMSO n=77; TRPV2∆4S + U0126 n=125. Data represent the mean ± s.e.m.

110

Figure 3.13. Model depicting the proposed mechanism by which NGF-activated MAPK signaling affects TRPV2 to enhance neurite outgrowth. Target-derived NGF binds to TrkA at the end of neurites. The complex is internalized into signaling endosomes and transported toward the nucleus in the cell body. At the nucleus, ERK signaling can increase TRPV2 expression. Within endosomes in the neurite shaft, TrkA activates ERK, which can phosphorylate TRPV2. Changes in Ca2+ due to increased TRPV2 protein expression and activity then enhance neurite outgrowth.

111

Chapter 4

Discussion and Future Directions

112 4.1 Summary

Two major findings resulted from this dissertation, both of which required the generation of new tools to study endogenously expressed TRPV2 protein: 1)

Contrary to previously published results (50, 51, 55, 56, 153, 154), TRPV2 translocation to the plasma membrane is not regulated by growth factor stimulation. This applied to both heterologously and endogenously expressed

TRPV2 in multiple expression systems and for multiple growth factors, including

IGF-1, NGF and serum. Future studies will explore the role of TRPV2 in regulating intracellular Ca2+ stores in different excitable and non-excitable cell types; 2) TRPV2 is regulated by NGF signaling downstream of MAPK/ERK to enhance neurite outgrowth during neuronal cell development. TRPV2 protein was detected in PC12 cells and embryonic DRG neurons, both of which are commonly used models for studying NGF signaling. NGF treatment increased

TRPV2 protein content in PC12 cells via the MAPK signaling pathway, which is essential for NGF-induced neurite outgrowth. Consistent with this finding, upregulation of TRPV2 promotes neurite outgrowth in developing neurons.

Lastly, ERK, the major effector of the MAPK pathway, phosphorylates TRPV2 to increase channel expression, TRPV2-mediated Ca2+ signals and neurite outgrowth downstream of NGF. Future studies aim to determine the mechanisms by which TRPV2-mediated Ca2+ signals serve to enhance neurite outgrowth in developing neurons.

4.2 Generation of monoclonal antibodies suitable for detection of endogenously expressed TRPV2

113 Despite that it shares nearly 50% sequence homology with other

thermoTRPV channels, TRPV2 is known as an orphan TRP since its cellular

function has remained unclear (22). A major barrier to studying TRPV2 function is a lack of specific tools to modulate TRPV2 activity or to detect TRPV2 in the cell. There are currently no known endogenous TRPV2 activators or inhibitors, and exogenous channel modulators are promiscuous, in that they can affect the activity of other Ca2+-permeable channels (22). Additionally, commonly used commercially available antibodies for TRPV2 are not suitable for immunodetection of endogenously expressed TRPV2 protein. These antibodies non-specifically cross-react with other proteins, causing the findings obtained with these antibodies to remain questionable.

Commercially available antibodies were generated against small linear peptides derived from the TRPV2 sequence. We initially used a similar approach, using the full soluble C-terminus of TRPV2 as an antigen to produce monoclonal

TRPV2 antibodies. This strategy ultimately failed, as the antibodies produced recognized the soluble C-terminus of TRPV2 but not the full-length protein.

Therefore, an alternative approach was employed, using full-length, tetrameric

TRPV2 as an antigen. With this strategy, several clones suitable for detection of endogenous TRPV2 by western blot, immunofluorescence and immunoprecipitation were generated. Importantly, these antibodies did not cross- react with the highly homologous TRPV1 protein, and did not appear to recognize any other protein in a non-specific manner.

114 These newly generated monoclonal antibodies were used to test the controversial hypothesis that growth factor stimulation causes translocation of

TRPV2 from an intracellular compartment to the plasma membrane via PI3K signaling (47, 50). Results indicated that TRPV2 is located in an intracellular compartment under basal conditions; IGF-1 treatment had no effect on the plasma membrane levels of heterologously expressed TRPV2 in multiple cell types, including CHO-K1 cells and HeLa cells despite activation of PI3K. TRPV2 immunoreactivity showed a reticuluar distribution and co-localized with an ER marker in HeLa cells. Importantly, IGF-1 also failed to change the subcellular distribution of endogenously expressed TRPV2, which also localized to an intracellular compartment, in the sensory neuron-derived F11 cell line.

Overall, using our newly generated monoclonal antibodies, we found that

TRPV2 primarily resides in intracellular compartments. Furthermore, growth factor stimulation did not change the subcellular distribution of the channel.

Regulated plasma membrane expression of TRPV2 has been shown to be important for the function of numerous cell types, including macrophages and cardiomyocytes (51, 108); however these studies were performed using antibodies derived from small linear peptides. Therefore, it will be important to use our new monoclonal antibodies to retest the hypothesis that TRPV2 translocates to the plasma membrane of macrophages and cardiomyocytes under stimulated or pathological conditions respectively.

In addition to TRPV2, other TRP channels still have unknown or unclear function. A similar strategy could be employed, using full-length tetrameric

115 channels as an antigen for the generation of antibodies for these channels. The

major limitation in this approach is that purification of these large tetrameric channel complexes requires precise biochemical conditions that would need to be optimized for each individual protein. Nonetheless purification of other TRP channels has been accomplished (155-157).

A current issue hindering biophysical studies of TRPV2 channel activity in a cellular system is the lack of a specific channel blocker or inhibitor. TRPV2 is inhibited by ruthenium red, a pan TRP channel blocker, as well as tranilast, an

anti-allergy drug (22, 158). However, these blockers also have off-target effects

that obstruct a clear interpretation of results. Since these new monoclonal

antibodies bind tightly and specifically to native tetrameric TRPV2, it is possible

that they could block or inhibit channel function. Future tests will be aimed at

determining the effects of antibodies 17A11 and 17H1 on the single channel

properties of purified TRPV2 reconstituted in liposomes (49), as well as

heterologously and endogenously expressed TRPV2 in cellular expression

systems.

Overall, using full-length tetrameric TRPV2 as an antigen allowed us to

produce monoclonal antibodies suitable for detection of TRPV2 by western blot,

immunofluorescence and immunoprecipitation. Using our newly generated antibodies, we were able to show TRPV2 primarily resides on intracellular membranes, and that IGF-1 signaling had no effect on regulated plasma membrane expression of TRPV2. These antibodies have allowed us to further

116 explore how TRPV2 is regulated during, and contributes to different cellular processes, including its role in neuronal cell development.

4.3 Regulation of TRPV2 by NGF signaling in developing neurons

ThermoTRPV channels were originally identified in adult sensory neurons

(4, 6, 7), although later they were also detected in embryonic peripheral neurons

(33, 88). TRPV2 mRNA was detected in developing mice at embryonic day 9.5, prior to expression of other thermoTRP channels (33). Silencing TRPV2 expression reduced axon length in developing sensory and motor neurons in culture and in vivo (33). TRPV4 has also been identified in developing peripheral neurons (5), and key mutations in TRPV4 can result in peripheral axonal neuropathies (129). To further explore the role of thermoTRPV channels in development of peripheral neurons, we tested for thermoTRPV protein expression in embryonic DRG neurons and PC12 cells. Our data shows that only

TRPV2 and TRPV4 are abundantly present in these cell types, and that amongst the thermoTRPVs, only TRPV2 expression is substantially upregulated by NGF exposure.

Treatment of PC12 cells with NGF led to a significant increase in TRPV2 protein, beginning at 6 hours at peaking between 24 and 48 hours. MAPK signaling, but not PI3K/Akt, contributed to NGF-induced upregulation of TRPV2.

MAPK signaling is essential for differentiation and neurite outgrowth in developing neuronal cells, which suggested that upregulation of TRPV2 protein may promote neurite outgrowth.

117 Consistent with this hypothesis, overexpression of WT TRPV2 in F11 cells increased the percentage of neurite bearing cells and the length of neurites, while overexpression of a dominant negative TRPV2 mutant failed to enhance neurite outgrowth. Interestingly, enhancement of neurite outgrowth in F11 cells by WT TRPV2 overexpression occurred in the absence of applied neurotrophin.

In PC12 cells, overexpression of WT TRPV2 also increased neurite outgrowth, but only after NGF was exogenously applied. It has been reported that F11 cells endogenously express and secrete neurotrophins such as NGF (159). That

TRPV2 increases neurite outgrowth only after application of NGF for PC12 cells would suggest that endogenously secreted neurotrophins may be acting in an autocrine or paracrine manner in F11 cells to facilitate the increased neurite outgrowth resulting from TRPV2 overexpression. Furthermore, dominant negative inhibition, as well as silencing TRPV2 expression, decreased neurite length in PC12 cells after NGF treatment. Inhibition of TRPV2 function or expression did not however affect the percentage of neurite-bearing cells, suggesting that while TRPV2 enhances neurite extension, it does not play a role in initiation of neurite formation. Taken together, this data shows that upregulation of functional TRPV2 channel by NGF serves to enhance neurite extension.

Previous studies have indicated that translocation of TRPV2 to the plasma membrane is mediated my PI3K signaling (50, 51, 55, 56, 153, 154). Once at the cell surface, PI3K signaling has also been shown to affect TRPV2 current (47).

Results presented here indicate that growth factors, including IGF-1 and NGF,

118 which both activate PI3K, had no effect on TRPV2 translocation to the plasma

membrane. Additionally, while NGF treatment leads to upregulation of TRPV2

expression, this does not occur through the PI3K pathway but rather through

MAPK signaling.

Although these results suggest that TRPV2 is not functionally regulated by

PI3K signaling, an effect of PI3K on TRPV2 channel activity and gating cannot be

ruled out. Current mediated by heterologously expressed TRPV2 in CHO cells was reduced by PI3K inhibitors (47) and recruitment of TRPV2 to phagosomes in activated macrophages was shown to be sensitive to LY294002 in addition to inhibitors of other pathways (108). In addition, phosphoinositides may mediate

Ca2+-dependent desensitization of TRPV2, which would link channel activity to

PI3K function (48).

That TRPV2 primarily localizes to intracellular membranes in neuronal cell

lines, coupled with the lack of specific TRPV2 modulators, makes testing the

effects of the PI3K pathway and phosphoinositides on channel gating technically

challenging. Patch clamp experiments of intracellular membranes, such as early

endosomes, have been performed to study intracellular TRP channels including

TRPV2 (57, 160). In order to test if PI3K signaling affects TRPV2 activity, these

intracellular patch clamp techniques could be employed testing the effects of

NGF and PI3K inhibitors on the single channel properties of TRPV2.

Interestingly, we found that while TRPV2 does not translocate to the

plasma membrane in the presence of NGF, it colocalizes with Rab7, a marker of

late endosomes, in PC12 cells and E18 DRG neurons. This is significant, since

119 Rab7 modulates NGF/TrkA signaling within endosomes of developing neurons

(83). When NGF binds to its receptor TrkA at the distal termini of developing

neurites, the complex is internalized into early and then late endosomes (75).

These endosomes are long-lived and do not to acidify, allowing for NGF to remain bound to TrkA and continue signaling (75, 76). Signaling endosomes are transported in a retrograde manner, where they can affect local signaling within the developing extension and eventually reach the soma, where the signaling components can regulate gene expression (75, 76, 86). Importantly, MAPK scaffolding proteins populate the cytoplasmic face of these endosomes, allowing for efficient local signaling through the MAPK pathway (79). Our observation that

TRPV2 colocalizes with Rab7 within neurites suggested a close association between the channel and other major NGF signaling components. Indeed we

also observed colocalization of TRPV2 with TrkA and activated Erk1/2 within the

neurites of E18 DRG neurons. This led us to hypothesize that TRPV2 might be

directly modulated by the MAPK/ERK pathway.

We were able to show that Erk2 directly phosphorylates TRPV2 in vitro,

and identified the residues within the N- and C-terminus of TRPV2 that are

phosphorylated by Erk2. Mutation of these residues to Ala reduced TRPV2

protein expression, TRPV2-mediated Ca2+ signals and NGF-induced neurite outgrowth in PC12 cells. These results are summarized in Figure 4.1.

Details by which phosphorylation by ERK affect TRPV2 function remain to be explored. We initially observed a transient increase in TRPV2 mRNA after

NGF treatment in PC12 cells, lasting only approximately 24 hours, while the

120 increase in TRPV2 protein is sustained for at least 72 hours. This suggests that

some other mechanism in addition to transcriptional upregulation of TRPV2

contributes to increased TRPV2 expression in response to NGF. Additionally,

inhibition of MAPK/ERK signaling and mutation of Ser within TRPV2

phosphorylated by ERK reduced protein levels of TRPV2 overexpressed in PC12

cells after NGF treatment. These results suggest that NGF signaling might

enhance TRPV2 protein stability via phosphorylation of TRPV2 by ERK.

In addition to promoting TRPV2 protein stability, phosphorylation of

TRPV2 by ERK might also alter channel activity. Phosphorylation is an important

regulation mechanism for other thermoTRPV channels, including TRPV1, where

phosphorylation at specific Tyr residues after NGF exposure sensitizes the channel in adult sensory neurons (104, 161, 162) and TRPV4, where phosphorylation affects channel activity and translocation to the plasma membrane (161, 162). Cytosolic Ca2+ imaging analysis showed that responses of

WT TRPV2 and TRPV2∆4S to activator 2-APB correlate with TRPV2 protein content. This would indicate that phosphorylation of TRPV2 by ERK has little effect on channel activity per se; however, little to no work has been performed to characterize the biophysical properties of TRPV2 especially in its native endosomal environment. Additionally, evidence suggests that TRPV2 shows constitutive activity in the absence of chemical activators. Therefore, it is possible that phosphorylation of TRPV2 by ERK affects basal activity of TRPV2, but not 2-

APB stimulated activity. This could be tested using specialized techniques for

121 patch clamping endosomal membranes that have been successfully employed

for studying other endolysosomal TRP channels (57, 160).

4.4 Remaining questions

4.4.1. Is TRPV2 regulated downstream of NGF/MAPK in vivo? Most of

the studies performed in this dissertation detailing the regulation mechanism of

TRPV2 by NGF were performed using PC12 cells. Additionally, we were able to

confirm key aspects of TRPV2 regulation in primary embryonic DRG neurons.

However it remains important to explore if TRPV2 is regulated by NGF/MAPK in

developing neurons in cultured primary neurons and in vivo. Studying the role of

NGF in differentiation of peripheral neurons has proven difficult since NGF

signaling is tightly linked to cell survival (71, 163). Inhibiting NGF signaling or deletion of key neurotrophin genes leads to neuronal death (163), preventing study of molecules involved directly in differentiation. As a result, we were unable to directly show that NGF exposure increases TRPV2 in primary DRG neurons in culture or in vivo. This can be overcome by deletion of BAX, a key gene in the apoptoic mechanism in the absence of NGF/TrkA (164). Knockout of BAX in mice prevents apoptosis upon removal or inhibition of NGF signaling, allowing for direct study of differentiation mechanisms (164). BAX knockout mice could be employed to determine if treatment of embryonic DRG neurons increases TRPV2 expression to enhance neurite outgrowth, similar to the results obtained using

PC12 cells.

4.4.2. Does TRPV2 activity directly affect endosomal Ca2+ levels? At

present, evidence linking TRPV2 activity to endosomal Ca2+ signals is correlative.

122 The link is based on the observation that overexpression of Ca2+-impermeable

DN TRPV2 hinders NGF-induced neurite outgrowth in PC12 cells. However we have yet to directly observe TRPV2-mediated Ca2+ signals derived from endosomal vesicles. We did not determine the source of Ca2+ signals in

response to 2-APB in this study, which remains an open question. Our model

assumes that endosomal [Ca2+] is higher than cytosolic [Ca2+], and so activation

of TRPV2 would lead to Ca2+ efflux from the endosomal lumen to the cytoplasm

(see Figure 3.13). Endosomal Ca2+ levels have remained difficult to measure

since most Ca2+ sensitive dyes are pH sensitive. Therefore measurements are

altered due to the acidic nature of endosomes.

Based on recent studies using a newly generated endosome-targeted pH-

insensitive Ca2+ biosensor, endosomes in non-excitable cells display

heterogeneity in their Ca2+ levels within single cells and across cells (165). The

[Ca2+] within endosomes of pancreatic β cells was estimated to range from

hundreds of nanomolar to 5 µM with higher Ca2+ levels in Rab7 positive late

endosomes compared to Rab5 positive early endosomes (165). This range of

[Ca2+] within the lumen of endosomes leaves open the possibility that Ca2+ could

also move from the cytoplasm to the endosomal lumen. If this is the case, then

Ca2+ permeation through TRPV2 may allow signaling endosomes to act as Ca2+

buffers to alter the amplitude as well as the temporal and spatial dynamics of

Ca2+ signals mediated by NGF. These same pH-insensitive Ca2+ biosensors could be employed to determine the effects of TRPV2 expression on Ca2+

123 dynamics within endosomal populations of PC12 cells and primary embryonic

DRG neurons.

4.4.3 How is TRPV2 targeted to endosomes in developing neurons?

TRPV2 lacks any sequence to target to specific subcellular compartments in its

primary structure. However targeting to certain compartments may require a

certain tertiary or quaternary fold that would be difficult to determine by sequence analysis alone. TRPV2 colocalized with the late endosomal marker Rab7 in both

PC12 cells and primary embryonic DRG neurons, suggesting that it populates the late endosomal compartment. How TRPV2 arrives at late endosomes remains an open question. The most direct route to late endosomes would involve endocytosis of the channel protein from the plasma membrane (see

Figure 1.7). However we failed detect endogenous TRPV2 at the plasma membrane in models of developing neurons using multiple detection techniques.

This would suggest that the cell employs an alternative pathway for directing

TRPV2 to late endosomes. It is possible that TRPV2 is targeted to endosomes from the ER/golgi. Vesicles that bud from the golgi can fuse with endosomal vesicles (166). In addition, ER membranes have been shown to form contacts with endosomal vesicles in developing neurons (167). This would allow for the possibility of exchange between the membrane components. A more details analysis of TRPV2 processing is necessary to determine exactly how the channel targets to endosomal membranes. A real-time analysis of GFP-labeled TRPV2 trafficking would shed light on this mechanism.

4.5 Potential mechanisms by which TRPV2 promotes neurite extension

124 Our data s that TRPV2 is involved in NGF signaling and that TRPV2

activity enhances neurite outgrowth. TRPV2 is a Ca2+-permeable channel, and therefore increased TRPV2 expression likely leads to changes in intracellular

Ca2+ signaling. Ca2+ is a versatile second messenger that can alter the structure and function of developing neurons. It has temporal and spatial specificity, allowing it to mediate acute effects on cell signaling at very specific subcellular locations (e.g. cytoskeletal rearrangements) as well as long-term effects, including changes in gene transcription (94). Multiple Ca2+ channels are involved in NGF signaling (87), as the NGF response requires integration of multiple local

Ca2+ signals to generate the proper response for neuron generation. In addition,

global Ca2+ levels can also influence overall effects in neuronal development

(94). TRPV2 has the potential to affect both local Ca2+ signals and global Ca2+

changes, and therefore may impact the downstream processes that Ca2+

governs. TRPV2 likely plays a part in the total of Ca2+ signaling, contributing to

the integration of external and internal signals during neuronal generation. How

TRPV2-mediated Ca2+ changes affect neuronal generation remains to be explored.

4.5.1 TRPV2-mediated Ca2+ signals might affect cytoskeletal structure in growing neurites - A distinct possibility is that TRPV2 activity plays a role in cytoskeletal rearrangements. TRPV channels have been shown to directly interact with cytoskeletal components including actin and tubulin (30-32).

Evidence indicates that TRPV channels can interact with tubulin dimers, as well as polymerized microtubules (31, 168). TRPV channels contain a cluster of basic

125 residues located on an on its C-terminus that interacts with acidic residues on tubulin dimers (30). This cluster of positively charged residues has

been shown to bind tubulin experimentally for TRP channels involved in neurite

extension and retraction, including TRPV1, TRPV4, TRPC1, TRPC5 and TRPC6

and is conserved in TRPV2 (30). Additionally, TRPV1 contains an N-terminal

tubulin binding site (169). Interaction of TRPV1 and tubulin may be dynamic,

depending on the conformation of the channel (30).

Post-translational modifications can alter the interaction between TRPV

channels and the cytoskeleton. For example, phosphorylation of TRPV1 by PKC

at Ser800 attenuates the interaction of TRPV1 with tubulin (170). Since

phosphorylation at this site sensitizes the channel, this links channel activity to

cytoskeletal interactions. Phosphorylation of TRPV4 also modulates its

interaction with the cytoskeleton. Both actin and tubulin interact with TRPV4 at

the same C-terminal site, which contains a predicted SGK1 phosphorylation site.

Phosphorylation of this site enhances actin binding and reduces tubulin binding.

Altering these interactions can lead to cell surface expansion and changes in cell

migration and motility (171). This has the potential to be important for the role of

TRPV channels in neurite outgrowth. Similar to TRPV2, TRPV4 expression also

may be upregulated in PC12 cells after NGF treatment, where it serves to

enhance neurite outgrowth (88). It is important to note that we failed to observe a

change in TRPV4 expression after NGF treatment in PC12 cells (see Figure 3.1).

Nonetheless, activation of TRPV4 in PC12 cells with 4αPDD causes

rearrangement of the actin cytoskeleton (88). Therefore, upregulation of TRPV4

126 activity may enhance neurite outgrowth by altering the cytoskeleton in the

developing neurite. Since Ca2+ and TRPV channel activity can modify the cytoskeleton, and TRPV2 localizes to developing neurites, TRPV2 activity may also lead to cytoskeletal rearrangements that promote neurite outgrowth. Further biochemical characterization of the interaction between actin, tubulin and TRPV2 is needed to determine if TRPV2 plays a similar role in cytoskeletal rearrangements. Additionally, the effects of TRPV2 activity can be test in situ by silencing TRPV2 expression and examining the effects on the actin and microtubule cytoskeleton by immunofluorescence.

4.5.2 Potential influence of TRPV2-mediated Ca2+ signals on Rab7

activity and signaling endosome function - After binding to NGF, the

NGF/TrkA complex is internalized as an active complex into the endocytic

pathway. Rab proteins are a family of membrane-associated Ras-like GTPases

that play a major role in endocytic transport and trafficking (77). Generally Rab

activity is associated with control of sorting, transport and maturation of endocytic

vesicles (77). The family members important for neurotrophin receptor trafficking

include Rab5, which regulates early endosome trafficking, Rab11, which

promotes receptor recycling, and Rab7, which is generally associated with late

endosome and lysosomal progression (86, 172, 173). In particular, Rab7-positive

late endosomes are associated with retrograde transport of NGF/TrkA in

developing peripheral neurons (75). When bound to GTP, Rab proteins are in the

active form while when bound to GDP, they are considered inactive. Although

Rabs have intrinsic GTPase activity, hydrolysis of GTP, and thus inactivation of

127 Rabs, is facilitated by Rab effector proteins called GTPase activating proteins

(GAPs) (77). GDP is then released and exchanged for GTP. This exchange is

promoted by guanine nucleotide exchange factors (GEFs) (77) (Figure 4.2).

Rab7 activity has previously been associated with NGF/TrkA signaling and

neurite outgrowth (83, 172). Dominant negative inhibition of Rab7 (constitutive

Rab7-GFP) is associated with enlargement of endosomal compartments, TrkA

accumulation in endosomes, enhanced TrkA and MAPK/ERK signaling and

increased neurite outgrowth (172). Rab7 activity promotes progression of late

endosomes to lysosomes, thus leading to receptor degradation. A reduction in

Rab7 activity would allow for longer-lived TrkA and thus enhanced neurotrophin

signaling. Indeed, pathogenic mutations that increase Rab7 lead to early

maturation of lysosomes and axonal neuropathies (83). This suggests that Rab7

controls the longevity of signaling endosomes and thus the duration of

neurotrophin signals. A longer-lived signal would allow for ERK signaling within

the neurites and soma of developing neurons, promoting neurite outgrowth (see

Figure 4.3).

How Rab7 activity is regulated is still unknown. Rab effector proteins are

subject to regulation by different second messengers, including intracellular Ca2+.

For example, Rab11 activity was reduced by local Ca2+ signals in Dictyostelium discoideum (174). This Ca2+ derived from Rab11-positive vacuoles bound to and increased activity of an EF-hand containing Rab-GAP that physically associated with and reduced activity of Rab11 (174). Similarly, an EF-hand-containing Rab-

128 GAP called TBC1D9 was identified as a Rab7-effector (175). Therefore, Rab7

activity may also be decreased by local Ca2+ signals that activate a Rab7-GAP.

We found that silencing TRPV2 expression, as well as dominant negative

inhibition of TRPV2-mediated Ca2+ signals, decreased NGF-mediated neurite

outgrowth. These effects are consistent with those observed in PC12 cells

overexpressing DN Rab7 (172), suggesting that TRPV2-mediated Ca2+ signals might lead to reduction in Rab7 activity and thus enhanced NGF/TrkA signaling

(83, 172) (Figure 4.3, 4). Preliminary data indicates that overexpression of WT

TRPV2, but not DN TRPV2, causes late endosomal enlargement. Additionally, silencing TRPV2 expression as well as overexpression of DN TRPV2 enhances

Rab7 activity. This would represent a positive feedback mechanism, whereby

NGF/TrkA signaling increases TRPV2 expression and signaling, and TRPV2 can in turn increase the effects of NGF signaling.

In addition to Rab7, TRPV2 shows a close association with Erk1/2 within endosomes. We showed that ERK could phosphorylate TRPV2, which contributes to enhanced TRPV2-mediated neurite outgrowth in PC12 cells. In addition to MAPK regulation of TRPV2, it is possible that TRPV2 activity in turn regulates MAPK function. Changes in cytoplasmic Ca2+ levels can increase the activity of the ERK cascade by multiple mechanisms (176). Ca2+ affects the

binding of ERKs to different cytoplasmic partners and also translocation of ERK

to the nucleus. Increased Ca2+ retains ERK in the cytoplasm, where it

preferentially phosphorylates cytoplasmic targets rather than nuclear targets

(177). This implies that TRPV2-mediated Ca2+ changes have the potential to

129 modulate ERK proteins, which may in turn alter TRPV2 expression and activity.

Feedback signaling between MAPK and TRPV2 remains to be explored.

4.5.3 Potential role of TRPV2 in neuronal regeneration - Peripheral nerve injury is thought to promote intrinsic neuronal growth programs, including neurotrophin signaling, associated with axon formation and extension during development (145). Neurotrophin release in response to injury also leads to inflammatory signaling, hyperalgesia and aberrant sprouting in peripheral sensory neurons, resulting in potentially debilitating neuropathic pain (145). A previous report showed that TRPV2 expression is upregulated after sympathetic nerve axotomy (24). It was interpreted that upregulation of TRPV2 contributes to hyperalgesia after nerve injury (24). However, it is now thought that TRPV2 does not play a major role in pain sensation (10). In the context of our results described in Chapter 3, it is possible that upregulation of TRPV2 in response to peripheral nerve injury may result in aberrant sprouting of sensory neurons in response to the newly upregulated neurotrophin signaling.

Aberrant structural plasticity of sensory neurons also occurs after injury to the spinal cord in addition to hyperexcitability and spontaneous activity (152, 178-

183). The primary deficit for patients that suffer from spinal cord injury (SCI) is locomotor impairment; however these patients also suffer from debilitating neuropathic pain (184). Abnormal structural plasticity after SCI has been well documented through observation that unmyelinated C and lightly myelinated Aδ primary afferent fibers sprout into the dorsal horn in rodent models and in after SCI (182, 185-191). These sprouting neurons express TrkA (192).

130 Evidence suggests that increased secretion of neurotrophins such as NGF after

SCI leads to pain development (186, 193). NGF is thought to act as an inflammatory mediator and a cue for the formation of new nociceptive inputs to the dorsal horn, which leads to pain signal amplification after injury (186, 193).

While potential molecular players that contribute to spontaneous activity and persistent hyperexcitability of sensory neurons after SCI have been identified

(152, 181-183), the processes governing aberrant structural plasticity in response to SCI remain poorly studied (178-180). Application of anti-NGF antibodies blocked sprouting of primary nociceptive neurons and reduced pain sensation after SCI (188). TRPV2 may represent a novel molecular player that promotes aberrant structural plasticity in primary sensory neurons. Understanding its role in signaling following injury to peripheral or central neurons may lead to discovery of new targets for post-injury pain management.

Understanding the role of TRPV2 in regeneration of injured neurons as well as aberrant sprouting of sensory neurons following injury would require the use of an in vivo mammalian model. Global TRPV2 knockout mice would not provide an ideal model, since these mice appear to display deficits in development (10). Therefore, a different approach would be necessary in order to manipulate TRPV2 expression in vivo. One viable alternative would be to employ antisense oligodeoxynucleotides (ODNs), which have been employed successfully to transiently inhibit expression of various proteins, including TRPV1 and TRPV4 channels, in DRG neurons (183, 194). Additionally, conditional

TRPV2 knockout mice have been generated to study the role of TRPV2 in

131 cardiomyocytes (195). This strategy could also be employed to specifically knock

down TRPV2 expression in different neuronal populations to determine the effect

on the channel on outgrowth following injury.

4.5 Concluding remarks

Although TRPV2 has remained an orphan-TRP, its role in developmental

physiology is important, as deletion of the TRPV2 gene leads to embryonic

abnormalities and perinatal death. While TRPV2 likely does not function as a

heat sensor per se, it plays a pivotal role in formational of sensory circuits.

Generation of new tools to study the endogenous function of TRPV2 accelerated

our understanding of the role of TRPV2 in growth factor signaling and neuronal

cell development. Further studies are needed to determine if TRPV2 represents a molecular target for regeneration of injured neurons. Additionally, further work is necessary to delineate the role of TRPV2 in the physiology of other cell types,

including cardiomyocytes, and macrophages.

132 4.6 Figures

Figure 4.1. Summary of findings. TRPV2 is upregulated downstream of NGF and MAPK to enhance neurite outgrowth.

133

Figure 4.2. Rab GTPase cycle. Rab GTPases switch between GTP- and GDP-bound forms. Conversion from GDP to GTP is caused by nucleotide exchange, which is catalyzed by a guanine nucleotide exchange factor (GEF). Conversion from GTP to GDP in contrast is mediated by GTP hydrolysis, which is facilitated by a GTPase activating protein (GAP). The GTP-bound form interacts with Rab effector molecules, while the GDP-bound form is considered inactive.

134

Figure 4.3. Possible mechanism proposing how increased TRPV2 expression might affect longevity of NGF signals. Increased TRPV2 expression has the potential to reduce Rab7 activity, leading to longer-lived NGF/TrkA containing endosomes, Erk1/2 signaling in the soma and enhanced neurite outgrowth. 2+ When TRPV2 expression or Ca permeability is disrupted, this relieves the inhibition on Rab7, leading to premature degradation of NGF/TrkA, reduced Erk1/2 signaling and reduced neurite outgrowth.

135

Figure 4.4. Mechanism predicting how TRPV2 activity might increase neurite outgrowth. Ca2+ permeates through the pore of TRPV2 from the endosomal lumen to the cytoplasm. The extra- endosomal Ca2+ binds to and activates an EF hand-containing Rab7 GAP, leading to Rab7 inhibition. The decrease in Rab7 activity leads to accumulation of TrkA in endosomes, enhanced TrkA and ERK signaling and increased neurite outgrowth.

136

Appendix

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reuse content provided the content was properly cited.

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repurpose PLoS content provided the original article is cited. In most cases, appropriate attribution can be provided by simply citing the original article.” http://journals.plos.org/plosone/s/content-license

2. Portions of Cohen et. al., Mol Cell Biol. 2015 Sep 28. pii: MCB.00549-15, were reproduced in Chapter 3. The American Society for Microbiology (ASM) has posted online that authors in ASM journal retain the right to reuse content in other publications provided the content was properly cited.

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