RalA and RalB function as the critical GTP sensors for GTP-dependent exocytosis.
Although it has been established that the activation of GTPases by non-hydrolyzable GTP stimulates neurotransmitter release from many different secretory cell types, the underlying mechanisms remain unclear. In the present study we aimed to elucidate the functional role(s) for endogenous Ras-like protein A (RalA) and RalB GTPases in GTP-dependent exocytosis. For this purpose stable neuroendocrine pheochromocytoma 12 (PC12) cell lines were generated in which the expressions of both RalA and RalB were strongly downregulated. In these double knock-down cells GTP-dependent exocytosis was reduced severely and was restored after the expression of RalA or RalB was reintroduced by transfection. In contrast, Ca2+-dependent exocytosis and the docking of dense core vesicles analyzed by electron microscopy remained unchanged in the double knock-down cells. Furthermore, the transfected RalA and RalB appeared to be localized primarily on the dense core vesicles in undifferentiated and nerve growth factor-differentiated PC12 cells. Our results indicate that endogenous RalA and RalB function specifically as GTP sensors for the GTP-dependent exocytosis of dense core vesicles, but they are not required for the general secretory pathways, including tethering of vesicles to the plasma membrane and Ca2+-dependent exocytosis.
Li
Gang
G
Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada M5T 2S8.
Han
Liping
L
Chou
Ting-Chieh
TC
Fujita
Yoshihito
Y
Arunachalam
Lakshmanan
L
Xu
Ainan
A
Wong
Aaron
A
Chiew
Soon-Kwang
SK
Wan
Qi
Q
Wang
Li
L
Sugita
Shuzo
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D020725:ral GTP-Binding Proteins / Q000378:metabolism*
C082190:Ralb protein, rat; D006160:Guanosine Triphosphate; C492197:Rala protein, rat; D020725:ral GTP-Binding Proteins; D002118:Calcium