Slp4-a/granuphilin-a regulates dense-core vesicle exocytosis in PC12 cells.
Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic beta-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542-28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitro and in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218). How Slp4-a.Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a.Rab8 and Slp4-a.Rab27A complexes, but not Slp4-a.Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
Kanno
Eiko
E
Saegusa
Chika
C
Ogata
Yukie
Y
Kuroda
Taruho S
TS
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology*; D018076:DNA, Complementary / Q000378:metabolism; D005089:Exocytosis; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D016716:PC12 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C512779:SYTL4 protein, human; C122015:Sytl4 protein, mouse; C512778:Sytl4 protein, rat; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D005982:Glutathione Transferase; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; C513177:Rab8b protein, rat; D020691:rab GTP-Binding Proteins; D020826:rab3A GTP-Binding Protein