Syntaxin8Modulates The Post-Synthetic Trafficking Of TrkA Receptor And The Inflammation-Related Pain Transmission

[Background and objective]Nerve growth factor (NGF), as the first identified neuotrophin, promotes the survival, maintenance and neurite outgrowth of sensory and sympathetic neurons. NGF signaling occurs through two entirely distinct receptor types, p75NTR and TrkA receptors. While p75NTR bind to all neurotrophins with similar affinity, TrkA receptors display selectivity toward NGF. NGF binds the extracellular domain of TrkA initiating receptors phosphorylation and activating downstream signal transduction cascades including Ras/Mitogen-Activated Protein Kinase (MAPK) and PI3-kinase (PI3K)/protein kinase B (Akt) pathways. Therefore NGF-mediated signal transduction and biological functions appear to be determined at least partially by the levels of its specific TrkA receptors located in the plasma membrane. The cell-surface levels of TrkA depend on two opposite processes, newly synthesized TrkA receptors insertion into the plasma membrane and the cell-surface localized TrkA endocytosis. So far the removal of TrkA receptors from the plasma membrane by endocytosis has been studied in details, for example GIPC (GAIP-interacting protein, C terminus) promotes TrkA internalization into early endosome via binding with the juxtamembrane domain of TrkA receptor; Rab7, Endophilin B1and Nedd4-2modulate TrkA degradative pathway. However, the mechanism underlying insertion of newly synthesized TrkA receptors into the plasma membrane is less well understood.To uncover novel TrkA-interacting proteins and learn more about the mechanisms that modulate the TrkA surface location, the yeast two-hybrid assay was performed to screen a rat DRG cDNA library using the intracellular domain of TrkA as the bait. Several candidate TrkA-associated proteins were identified, one of which was STX8. STX8is a family member of Q-SNARE which mediates membrane fusion through their SNARE homology coiled-coil domain interacting with the cognate R-SNARE partners to form organelle specific docking and fusion complexes in eukaryotic cells. Syntaxins as the Q-SNARE members have been reported to interact with a wide range of receptors and dynamically regulate their trafficking events. For example, syntaxin16mediates recycling of CFTR channels, a cyclic AMP-dependent chloride channel and regulates its surface levels in polarized epithelial cells; syntaxin6affects the post-Golgi transport and cell-surface levels of vascular endothelial growth factor receptor2(VEGFR2). Recent study suggests that STX8binds CFTR channels and regulates CFTR trafficking, as well as its chloride channel activity.In light of the interaction between STX8and TrkA and the property of STX8that modulate protein trafficking as a member of Q-SNARE, we questioned the possibility that STX8might regulate TrkA intracellular trafficking. Here our study show evidence that STX8could regulate TrkA cell-surface levels via promoting TrkA trafficking from Golgi to plasma membrane. In vitro and in vivo functional studies found that STX8could modulate NGF-induced DRG neurons survival and inflammation-related pain transmission. Our results suggest that STX8is a novel modulator of TrkA receptor trafficking.[Methods and results]1. STX8associates with TrkA but not TrkB receptorsA physical interaction between STX8and TrkA was assessed by co-immunoprecipitation (co-IP) assay with homogenates from co-transfected human embryonic kidney293(HEK293) cells and rat dorsal root ganglions (DRG). We found that not only exogenous but also endogeneous TrkA, but not TrkB receptors, could co-immunoprecipitate with STX8. Immunohistochemical (IHC) analysis indicated that TrkA and STX8co-expressed in DRG neurons. Also in cultured DRG neurons punctate staining of STX8was found to be colocalized with TrkA, Taken together, these data indicate that STX8could interact with TrkA receptors in DRG neurons and the interaction is specific to TrkA but not to its homolog TrkB receptors.2. STX8regulates the surface levels of TrkA but not TrkB receptorsThe gain-of-function and loss-of-function analyses were used to determine the effect of STX8on TrkA or TrkB surface levels by ratiometric fluorescence assay and the biotinylation assay in transfected PC12cells. We observed that STX8over-expression could significantly increase the cell surface levels of TrkA but not TrkB receptors. Conversely, introducing STX8ATM, which lacks STX8hydrophobic transmembrane anchor as a dominant negative form and siRNA knocking down STX8could significantly decrease the surface levels of TrkA, but not TrkB. And we confirmed the effect of STX8on TrkA surface levels in cultured DRG neurons.3. TrkA C-terminal is necessary and sufficient for TrkA/STX8interactionTo identify the domain of the TrkA receptor responsible for the STX8association, we generated a series of mutants in which various domains were deleted from full-length TrkA respectively and TrkB chimera mutants with relative domain replacing with that of TrkA. The co-IP assay in HEK293cells indicated that TrkAACT, which carried the CT deletion lost association with STX8and that TrkBAct in which CT domain replacing with that of TrkA obtained association with STX8. These observations suggested that the CT domain is necessary and sufficient for TrkA to interact with STX8. In addition, the ratiometric fluorescence assay indicated that STX8overexpression increased the surface levels of the TrkBAct mutants, which suggested that STX8regulates TrkA surface levels via its interaction with the TrkA CT domain.4. STX8facilitates TrkA surface targeting via the post-biosynthetic pathwayAs the surface levels of TrkA depend on insertion and endocytosis, opposite receptor trafficking processes, we next sought to determine which process STX8regulates. We first performed a ratiometric fluorescence assay measuring the internalization of TrkA receptor as described in "Experimental Procedures" to investigate the effect of STX8on TrkA internalization. We found that after15min of NGF stimulation, approximately50±2%of surface TrkA was internalized and the internalization of TrkA remained unaffected under STX8overexpression or knock-down condition (Fig.4A,B), suggesting that STX8is not involved in TrkA internalization. A cleavable surface biotinylation assay was performed in615cells to further confirm this result that STX8did not be involved in TrkA internalization. At last, we found that blocking internalization by DN-dynamin did not affect the STX8-mediated regulation of TrkA surface levels. Therefore, these results suggest that STX8modulates TrkA surface levels via the post-biosynthetic but not the endocytic pathway.5. STX8promotes TrkA export from the GolgiTo further investigate whether STX8regulates TrkA ER-to-Golgi trafficking or post-Golgi sorting, we first examined whether STX8overexpression or knockdown affected the TrkA localization in the ER or the Golgi by detecting the immunofluorescence colocalization between TrkA and the ER marker calnexin or the trans-Golgi network (TGN) marker TGN38. The colocalization CCs between TrkA and calnexin showed no significant difference between the three groups. However, the colocalization of TrkA and TGN38was poorer in the high STX8expression group, while it was better in the STX8-depleted group. These data suggested that STX8regulated TrkA location in Golgi apparatus. These results were further confirmed using a sucrose density gradient centrifugation assay.6. STX8modulates NGF-induced downstream signalling and DRG neuron survivalWe examined the effect of STX8on NGF-induced signal transduction by detecting NGF-mediated activation of TrkA, Erk1/2and Akt and found that STX8regulates NGF-induced downstream signalling by modulating the TrkA cell surface levels. DRG neurons depend on neurotrophin signalling for their survival. We first filtered NGF-dependent and BDNF-dependent DRG neurons and then detected apoptotic neurons by TUNEL staining and caspase3detection. The results indicated that STX8regulates the survival of NGF-dependent but not BDNF-dependent DRG neurons.7. STX8depletion in TrkA-positive DRG neurons relieves inflammatory pain in vivoThe NGF-TrkA pathway appears to play a pivotal role in the generation and maintenance of inflammatory pain. To examine whether STX8depletion in DRG neurons has analgesic potential in a rat inflammatory pain model, we knock down STX8in rat DRG by recombinant adeno-associated virus serotype6(rAAV6)-mediated RNA interference and performed the formalin test. We found that STX8knockdown reduced the rats paw licking behaviour recorded in phase Ⅱ. The results suggest that STX8depletion could induce analgesic effects in the formalin-induced inflammatory pain response.[Conclusion]1. STX8modulates cell surface levels of TrkA receptor by promoting TrkA trafficking from Golgi targeting plasma membrane.2. STX8modulates cell surface levels of TrkA by associating with the CT domain of TrkA receptor but not TrkB.3. STX8modulates NGF-mediated biological functions.