Latrotoxin-induced exocytosis in mast cells transfected with latrophilin
Introduction
α-Latrotoxin (α-LTX), a neurotoxin obtained from the black-widow spider (Latrodectus tredecimguttatus), causes massive exocytosis from presynaptic nerve terminals (Longenecker et al., 1970, Ceccarelli et al., 1979, Südhof, 2001). Three specific receptors for α-LTX, neurexin (Ushkaryov et al., 1992, Geppert et al., 1998, Sugita et al., 1999), latrophilin (Davletov et al., 1996, Krasnoperov et al., 1997, Sugita et al., 1998), and protein tyrosine phosphatases (PTPs) (Krasnoperov et al., 2002), have been reported. Neurexin binds to α-LTX only in the presence of extracellular Ca2+, while latrophilin and PTPs bind to α-LTX even in the absence of extracellular Ca2+. Because the contribution of PTPs is small (Ushkaryov et al., 2004), latrophilin is a key receptor of Ca2+-independent secretion. It is well-known that α-LTX works as a Ca2+ ionophore (Chanturiya and Nikoloshina, 1994, Van Renterghem et al., 2000). However, Ca2+-independent secretion by α-LTX mediated by latrophilin has not been elucidated clearly.
It has been reported that the binding of α-LTX to latrophilin activates phospholipase C (PLC) and generates IP3, resulting in Ca2+ release from the intracellular Ca2+ stores (Vicentini and Meldolesi, 1984, Rahman et al., 1999, Ashton et al., 2001). At the nerve terminal, synaptic vesicle fusion is mediated by core membrane fusion machinery comprised of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, synaptobrevin/VAMP2, SNAP-25, and syntaxin-1 (Südhof et al., 1993, Chen and Scheller, 2001). Several lines of evidence indicate that the Ca2+-independent release mechanism by α-LTX requires the synaptic SNARE proteins synaptobrevin/VAMP, SNAP-25, and Munc13-1 (Südhof et al., 1993, Deák et al., 2009).
Mast cell is a specialized cell that is involved in allergic responses. Mast cells have been intensively studied to clarify their mechanism of exocytosis as non-neuronal secretory cells. Cross-linking of high-affinity receptor for IgE (FcεRI) by multivalent antigen causes IP3 production and increase in intracellular Ca2+ concentrations, resulting in exocytotic release (Holgate, 1999, Turner and Kinet, 1999). As in the case of nerve terminal, several groups, including ours, have shown that SNARE proteins regulate exocytosis in mast cells (Guo et al., 1998, Hibi et al., 2000, Paumet et al., 2000; Puri, 2003). Furthermore, it has been shown that SNARE accessory proteins such as Munc18-2 and complexin II regulates exocytosis in mast cells (Martin-Verdeaux et al., 2003, Tadokoro et al., 2005, Tadokoro et al., 2007). We also found that the active-zone protein Munc13-1 and ELKS positively regulates exocytosis in mast cells (Miura et al., 2008, Nomura et al., 2009). These results caused us to examine whether or not α-LTX induces exocytosis by itself without antigen stimulation. In this study, we investigated the effects of α-LTX on RBL-2H3 cells transfected with latrophilin to understand the mechanism of exocytosis in mast cells and the action mechanism of α-LTX.
Section snippets
Chemicals
U73122, U73343, Gö6983, Gö6976, and phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem.Ionomycin and Fura/2AM were purchased from Sigma and Dojindo (Kumamoto, Japan), respectively. α-LTX was obtained from Alomone labs (Israel).
Cell culture
Rat basophilic leukemia cells (RBL-2H3) were cultured in Eagle’s minimal essential medium from Nissui (Tokyo, Japan) with 10% fetal calf serum (Boehringer Mannheim) at 37 °C in an atmosphere of 5% CO2.
Plasmid construction and transfection
Poly(A)+ RNA was obtained using the QuickPrep Micro
Expression of latrophilin in RBL-2H3 cells
Because mast cells do not express the receptor for α-LTX, latrophilin, which is involved in the Ca2+-independent release of neurotransmitter, we first established cell lines that express latrophilin. To investigate the localization of latrophilin, we expressed latrophilin with a YFP-tag at the C-terminus. In neuronal cells, latrophilin is expressed on the plasma membrane and works as a receptor for α-LTX. Thus, we first examined the intracellular localization of latrophilin in mast cells. Fig. 1
Discussion
We investigated the effects of α-LTX on the exocytotic process in mast cells that were transfected with the receptor of α-LTX, latrophilin. We found that exogenously introduced latrophilin acted as a receptor for α-LTX and was able to induce Ca2+ mobilization and exocytosis. In the absence of extracellular Ca2+, neither Ca2+ increase nor exocytosis was observed, suggesting that α-LTX was bound to latrophilin and acted as a Ca2+ ionophore. However, this does not mean that α-LTX works solely as a
Acknowledgement
This work was supported in part by the Ministry of Education, Culture, Sports, and Technology of Japan (Grants 18370064 to N.H.).
References (40)
- et al.
α-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles
J. Biol. Chem.
(2001) - et al.
Isolation and biochemical characterization of a Ca2+-independent α latrotoxin-binding protein
J. Biol. Chem.
(1996) - et al.
Neurexin I is a major α-latrotoxin receptor that cooperates in α-latrotoxin action
J. Biol. Chem.
(1998) - et al.
Characterization of αlatrotoxin interaction with rat brain synaptosomes and PC12 cells
Toxicon
(1982) - et al.
Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells
Cell
(1998) - et al.
Phosphorylation of SNAP-23 regulates exocytosis from mast cells
J. Biol. Chem.
(2005) - et al.
Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes
Biochem. Biophys. Res. Commun.
(2000) - et al.
α-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor
Neuron
(1997) - et al.
Protein-tyrosine phosphatase-sigma is a novel member of the functional family of α-latrotoxin receptors
J. Biol. Chem.
(2002) - et al.
α-Latrotoxin induces exocytosis by inhibition of voltage dependent K+ channels and by stimulation of L-type Ca2+ channels via Latrophilin in β-cells
J. Biol. Chem.
(2006)