Elsevier

Toxicon

Volume 56, Issue 8, December 2010, Pages 1372-1380
Toxicon

Latrotoxin-induced exocytosis in mast cells transfected with latrophilin

https://doi.org/10.1016/j.toxicon.2010.08.002Get rights and content

Abstract

α-Latrotoxin (α-LTX) is known to cause massive exocytosis from presynaptic nerve terminals. We investigated the effects of α-LTX on exocytotic release from mast cells, typical non-neuronal secretory cells. When we transfected mast cells with latrophilin, a specific receptor for α-LTX, α-LTX caused intracellular Ca2+ to increase and led to exocytosis in the presence of extracellular Ca2+. On the other hand, neither Ca2+ increase nor exocytosis was observed in the absence of extracellular Ca2+. These results indicate that α-LTX, together with latrophilin, works as a Ca2+ ionophore. However, α-LTX had additional effects on signal transduction in mast cells. We found that inhibitors of protein kinase C (PKC) partially suppressed exocytosis. Furthermore, several soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including SNAP-23, were phosphorylated by α-LTX. These results suggest that exocytosis induced by α-LTX can be explained by (1) elevation of intracellular Ca2+, (2) phosphorylation of SNARE proteins including SNAP-23, syntaxin-4 and VAMP-8 through PKC-dependent and -independent pathways. Our study may provide a new system to investigate the action of α-LTX and the mechanism of exocytosis in mast cells.

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.).

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