The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation. The transient receptor potential (TRP)1 superfamily of cation channels consists of a large number of recently identified molecules that share sequence homology with the Drosophila protein named after a phototransduction mutant called trp. According to sequence similarities, the TRP channels are further divided into subfamilies, such as TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) (see reviews in Refs. 1 and 2). These channels are involved in diverse cellular functions including receptor and store-operated Ca 2ϩ entry (3), Ca 2ϩ transport (4, 5), trace metal detection (6), and temperature (7-9) and osmolality (10, 11) sensations. The activation mechanisms for most of the TRP channels remain to be elucidated. Specific ligands have been found for TRPC3, TRPC6, TRPC7, TRPV1, TRPV4, TRPM2, TRPM4, TRPM5, TRPM7, and TRPM8. These include endogenous substances, such as lipids (diacylglycerol (12), anandamide (13, 14), and phosphatidylinositol 4,5-bisphosphate (15)), nucleotides (ADP-ribose (16) (23,24), 2APB was soon found to directly block native store-operated channels (25-27), sarco/ endoplasmic reticulum Ca 2ϩ -ATPase pumps (28), mitochondrial permeability transition pore (29), and a few other ion channels (30). The mechanism of action for 2APB is likely to be complex. In addition to inhibition, low concentrations of 2APB enhanced the activity of store-operated channels (26). At greater than 50 M, 2APB activated a Ca 2ϩ -permeable nonselective cation channel with a 50-picosiemens single channel conductance and very low open probability in rat basophilic leukemia cells (31).2APB has been perceived as a general inhibitor of TRP channels (1). However, except for TRPC3 (24,32), the effects of this drug...

Slit regulates migration of not only neurons, but also nonneuronal cells, such as leukocytes and cancer cells. Slit effect on cancer cell migration has not been well-characterized. In this study, we used several different assays to examine Slit effect on breast cancer cell migration in vitro. We show that ubiquitin-specific protease 33 (USP33)/VDU1, originally identified as a von Hippel-Lindau tumor suppressor (VHL) protein-interacting deubiquitinating enzyme, binds to the Robo1 receptor, and that USP33 is required for Slit responsiveness in breast cancer cells. Slit induces redistribution of Robo1 from intracellular compartments to the plasma membrane in a USP33-dependent manner. Slit impairs directional migration of breast cancer cells without affecting their migration speed. This inhibitory effect is Robo-mediated and USP33-dependent. These data uncover a previously unknown function of USP33 and reveal a new player in Slit-Robo signaling in cancer cell migration.cell migration and motility ͉ metastasis ͉ Slit-Robo signaling C ell migration is a fundamental process critical for not only embryonic development but also homeostasis in adult animals. A number of molecular cues guide axons and migrating neurons (1-4). Recent studies suggest that molecular mechanisms modulating migration of cells in different tissues/organs are conserved. For example, guidance cues, receptors, and the intracellular signaling pathways for neuronal migration are also used for cells outside of the nervous system, ranging from immune cells, myoblasts, and endothelial cells to tumor cells (4-7). The Slit gene was first identified in Drosophila, and subsequent studies indicated that secreted proteins of the Slit family and their receptors of the Roundabout (Robo) family play important roles in neuronal guidance (8-13). The Slit genes are frequently inactivated in cancer (14-19).Accumulating evidence supports that chemokines and their receptors play important roles in tumorigenesis and cancer metastasis, including chemotactic invasion and migration of cancer cells (20,21). In some cases, cancer cells show increased expression of chemokine receptors not expressed in normal nontumor cells, providing a plausible explanation for distant metastasis to organs that secrete corresponding chemokine ligands (22). Studies from our group and other groups have shown that Slit suppresses chemokine-directed chemotaxis of leukocytes and breast cancer cells (23-25) and inhibits medulloblastoma cell invasion (26). These observations suggest a potential therapeutic strategy in controlling aberrant cell migration during cancer metastasis. Cellular and molecular mechanisms underlying Slit signaling in cancer cells remain to be elucidated.To dissect the Slit-Robo signaling pathway, we carried out yeast two-hybrid screens by using the intracellular domain of Robo1 as bait (27). From such screening, we have identified ubiquitin-specific protease 33 (USP33)/von Hippel-Lindau tumor suppressor protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein ...

Commissural axons cross the ventral midline of the neural tube in a Slit-dependent manner. The underlying molecular mechanisms remain to be elucidated. Here we report that the deubiquitinating enzyme USP33 interacts with the Robo1 receptor. USP33 is essential for midline crossing by commissural axons and for their response to Slit. Our results reveal a previously unknown role of USP33 in vertebrate commissural axon guidance and in Slit signaling.

The transient receptor potential canonical type 5 (TRPC5) channel is a member of the channels that has been implicated in neurite extension and growth cone morphology of hippocampal neurons. Although homomeric TRPC5 channels are activated following stimulation of G(q/11)-coupled receptors, the exact mechanism for this activation remains unresolved. Using two-electrode voltage clamp recordings, we show that the activity of TRPC5 channels expressed in Xenopus oocytes is dependent on the presence of Ca2+ at the extracellular as well as the cytoplasmic side of the plasma membrane. TRPC5 was activated by the stimulation of coexpressed M5 muscarinic receptors or by ionomycin. The TRPC5 activity was detectable with the presence of submillimolar levels of extracellular Ca2+, but it was eliminated by the injection of 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid into the oocytes. Lanthanum could substitute for extracellular Ca2+ to support TRPC5 activity. Coexpression of Ca2+-binding protein 1 (CaBP1), but not calmodulin (CaM), inhibited the TRPC5 activity, without affecting the cell surface expression of TRPC5 proteins. Using in vitro binding assays, we demonstrated direction interactions between CaBP1 and TRPC5. The CaBP1-binding sites at the C terminus of TRPC5 are closely localized, but not identical, to CaM-binding sites. We conclude that TRPC5 is a Ca2+-regulated channel, and its activity is negatively controlled by CaBP1.

No abstract

Intracellular vesicular transport is important for photoreceptor function and maintenance. However, the mechanism underlying photoreceptor degeneration in response to vesicular transport defects is unknown. Here, we report that photoreceptors undergo apoptosis in a zebrafish β-soluble N-ethylmaleimide-sensitive factor attachment protein (β-SNAP) mutant. β-SNAP cooperates with N-ethylmaleimide-sensitive factor to recycle the SNAP receptor (SNARE), a key component of the membrane fusion machinery, by disassembling the cis-SNARE complex generated in the vesicular fusion process. We found that photoreceptor apoptosis in the β-SNAP mutant was dependent on the BH3-only protein BNip1. BNip1 functions as a component of the syntaxin-18 SNARE complex and regulates retrograde transport from the Golgi to the endoplasmic reticulum. Failure to disassemble the syntaxin-18 cis-SNARE complex caused BNip1-dependent apoptosis. These data suggest that the syntaxin-18 cis-SNARE complex functions as an alarm factor that monitors vesicular fusion competence and that BNip1 transforms vesicular fusion defects into photoreceptor apoptosis.

A switch in the response of commissural axons to the repellent Slit is crucial for ensuring that they cross the ventral midline only once. However, the underlying mechanisms remain to be elucidated. We have found that both endocytosis and recycling of Robo1 receptor are crucial for modulating Slit sensitivity in vertebrate commissural axons. Robo1 endocytosis and its recycling back to the cell surface maintained the stability of axonal Robo1 during Slit stimulation. We identified Arf6 guanosine triphosphatase and its activators, cytohesins, as previously unknown components in Slit-Robo1 signalling in vertebrate commissural neurons. Slit-Robo1 signalling activated Arf6. The Arf6-deficient mice exhibited marked defects in commissural axon midline crossing. Our data showed that a Robo1 endocytosis-triggered and Arf6-mediated positive-feedback strengthens the Slit response in commissural axons upon their midline crossing. Furthermore, the cytohesin-Arf6 pathways modulated this self-enhancement of the Slit response before and after midline crossing, resulting in a switch that reinforced robust regulation of axon midline crossing. Our study provides insights into endocytic trafficking-mediated mechanisms for spatiotemporally controlled axonal responses and uncovers new players in the midline switch in Slit responsiveness of commissural axons.

A common pathological hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis, is cytoplasmic mislocalization and aggregation of nuclear RNA-binding protein TDP-43. Perry disease, which displays inherited atypical parkinsonism, is a type of TDP-43 proteinopathy. The causative gene DCTN1 encodes the largest subunit of the dynactin complex. Dynactin associates with the microtubule-based motor cytoplasmic dynein and is required for dynein-mediated long-distance retrograde transport. Perry disease-linked missense mutations (e.g., p.G71A) reside within the CAP-Gly domain and impair the microtubule-binding abilities of DCTN1. However, molecular mechanisms by which such DCTN1 mutations cause TDP-43 proteinopathy remain unclear. We found that DCTN1 bound to TDP-43. Biochemical analysis using a panel of truncated mutants revealed that the DCTN1 CAP-Gly-basic supradomain, dynactin domain, and C-terminal region interacted with TDP-43, preferentially through its C-terminal region. Remarkably, the p.G71A mutation affected the TDP-43-interacting ability of DCTN1. Overexpression of DCTN1G71A, the dynactin-domain fragment, or C-terminal fragment, but not the CAP-Gly-basic fragment, induced cytoplasmic mislocalization and aggregation of TDP-43, suggesting functional modularity among TDP-43-interacting domains of DCTN1. We thus identified DCTN1 as a new player in TDP-43 cytoplasmic-nuclear transport, and showed that dysregulation of DCTN1-TDP-43 interactions triggers mislocalization and aggregation of TDP-43, thus providing insights into the pathological mechanisms of Perry disease and other TDP-43 proteinopathies.