N-terminal insertion and C-terminal ankyrin-like repeats of alpha-latrotoxin are critical for Ca2+-dependent exocytosis.
Alpha-latrotoxin, a potent stimulator of exocytosis from neurons and neuroendocrine cells, has been studied intensively, but the mechanisms of its actions are poorly understood. Here, we developed a new method to generate active recombinant alpha-latrotoxin and conducted a structure/function analysis of the toxin in stimulating Ca2+-dependent exocytosis. alpha-Latrotoxin consists of a conserved N-terminal domain and C-terminal ankyrin-like repeats. After cleavage of an N-terminally fused purification tag of glutathione S-transferase (GST), the recombinant toxin strongly stimulated exocytosis, whereas the GST-fused toxin was much less potent. The GST-fused toxin bound to the receptors [neurexin 1alpha; CL1 (CIRL/latrophilin 1)] as efficiently as did the GST-cleaved toxin but was much less effective in inserting into the plasma membrane and inducing cation conductance. The toxin with deletion of the last two ankyrin-like repeats still bound the receptors but could neither stimulate exocytosis nor induce cation conductance efficiently. The abilities of the mutated toxins to stimulate exocytosis correlated well with their abilities to induce cation conductance, but not their binding to the receptors. Our results indicate that (1) C-terminal ankyrin-like repeats and a free (unfused) N terminus are both required for the toxin to form pores, which is essential for Ca2+-dependent exocytosis, and (2) receptor binding alone is not sufficient to stimulate Ca2+-dependent exocytosis.
Li
Gang
G
Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, M5T 2S8, Canada.
Lee
David
D
Wang
Li
L
Khvotchev
Mikhail
M
Chiew
Soon Kwang
SK
Arunachalam
Lakshmanan
L
Collins
Tony
T
Feng
Zhong-Ping
ZP
Sugita
Shuzo
S
D000818:Animals; D017487:Ankyrins / Q000235:genetics* / Q000378:metabolism*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002522:Chlorocebus aethiops; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D020449:Repetitive Sequences, Amino Acid; D013111:Spider Venoms / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017487:Ankyrins; D013111:Spider Venoms; C020763:alpha-latrotoxin; D002118:Calcium