Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

Abstract

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca 2؉ -permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca 2؉ -permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca 2؉ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca 2؉ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.E stablishment 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 targetderived extracellular cue necessary for outgrowth (1). Upon binding to its extracellular receptor, NGF activates the phosphoinositide 3-kinase (PI3K) signaling pathway, which is essential for the survival of developing neurons, and the mitogen-activated protein kinase (MAPK) pathway, which promotes differentiation and neurite outgrowth (2, 3). These signaling pathways have numerous downstream effectors in developing neurons, including several Ca 2ϩ -permeable transient receptor potential (TRP) channels (4-8).Thermosensitive TRP channels from the vanilloid subfamily (thermoTRPV channels) consist of four nonselective Ca 2ϩ -permeable cation channels, TRP vanilloid 1 (TRPV1) to TRPV4, originally described as pain and temperature sensors in adult sensory neurons (9-12). Recent evidence suggests, however, that only TRPV1 functions as a molecular sensor of heat and painful stimuli in vivo, while the function of TRPV2 to -4 remains unclear (13-16). TRPV2 and TRPV4 have also been detected in developing peripheral neurons, suggesting that they may play a role in growth programs during development (17, 18). Consistent with this notion, TRPV2 has been implicated in axon outgrowth (18), and critical mutations in TRPV4 result in peripheral axonal neuropathies (19,20). Despite these initial findings, the details by which thermoTRPV channels influence neuronal development remain unknown.Here we explore the molecular mechanisms by which thermoTRPV channels contribute to neurotrophin-mediated peripher...

TRP Channels in Skin Biology and Pathophysiology

“…Furthermore, heat sensitivity appears not to be conserved in human TRPV2 [83]. There is evidence that TRPV2 plays a role in neurite outgrowth, particularly in response to mechanical stretch of neurons [81] or nerve growth factor stimulation [84]. Whether this has implications for nerve regeneration in the skin has yet to be explored.…”

Structure of the Full-Length TRPV2 Channel by Cryo-EM

“…Ca 2 þ imaging experiments were performed with membrane-permeable Fluo4 (Fluo4-AM, Life Technologies) as described previously 14 . In short, F11 cells were co-transfected with the TRPV2 protein and red fluorescent protein (RFP), which identified transfected cells.…”

“…It has been suggested that post-translational modifications, lipid-protein interactions and protein-protein interactions may regulate the function of these channels 2 . Specifically, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) levels in the membrane may modulate TRPV2 activity 17 , and direct phosphorylation of TRPV2 by extracellular-signal-regulated kinases has been shown to regulate TRPV2 expression and activity 14 . A detailed structural analysis of TRPV2 may provide deeper insight into the cellular function of TRPV2, which has been elusive since its discovery 7,12,18 .…”

Transient receptor potential vanilloid 2 activation by focal mechanical stimulation requires interaction with the actin cytoskeleton and enhances growth cone motility

“…In addition, Ca 2+ influx via NMDA receptors regulates Rho‐GTPase activity through Lim1/ IQGAP1/Cdc42 complexes, which facilitate actin poly‐merization in cultured cerebellar granule cells (28–30). Moreover, TRPV2 phosphorylation in the early endosome is regulated by the nerve growth factor (NGF)/ tropomyosin receptor kinase A (TrkA) signaling path‐way, and its activation induces Ca 2+ release from endo‐somes to enhance axonal outgrowth (31). As described above, [Ca 2+ ] i transients induced by extracellular factors are important for growth cone motility, axonal out‐growth, and circuit formation in the spinal cord; however, it remains unknown whether Ca 2+ transients triggered by mechanically evoked TRPV2 activation contribute to growth cone motility or axonal outgrowth.…”