Slp4-a/granuphilin-a inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells.
Slp4-a (synaptotagmin-like protein 4-a)/granuphilin-a is specifically localized on dense-core vesicles in PC12 cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A via the N-terminal Slp homology domain (SHD) (Fukuda, M., Kanno, E., Saegusa, C., Ogata, Y., and Kuroda, T. S. (2002) J. Biol. Chem. 277, 39673-39678). However, the mechanism of the inhibition by Slp4-a has never been elucidated at the molecular level and is still a matter of controversy. In this study, I discovered an unexpected biochemical property of Slp4-a, that Slp4-a, but not other Rab27 effectors reported thus far, is capable of interacting with both Rab27A(T23N), a dominant negative form that mimics the GDP-bound form, and Rab27A(Q78L), a dominant active form that mimics the GTP-bound form, whereas Slp4-a specifically recognizes the GTP-bound form of Rab3A and Rab8A and does not recognize their GDP-bound form. I show by deletion and mutation analyses that the TGDWFY sequence in SHD2 is essential for Rab27A(T23N) binding, whereas SHD1 is involved in Rab27A(Q78L) binding. I further show by immunoprecipitation and cotransfection assays that Munc18-1, but not syntaxin IA, directly interacts with the C-terminal domain of Slp4-a in a Rab27A-independent manner. Expression of Slp4-a mutants that lack Rab27A(T23N) binding activity (i.e. specific binding to Rab27A(Q78L)) completely reverses the inhibitory effect of the wild-type Slp4-a on high KCl-dependent neuropeptide Y secretion in PC12 cells. The results strongly indicate that interaction of Slp4-a with the GDP-bound form of Rab27A, not with syntaxin IA or Munc18-1, is the primary reason that Slp4-a expression inhibits dense core vesicle exocytosis in PC12 cells.
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
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D004252:DNA Mutational Analysis; D005089:Exocytosis / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism; D016716:PC12 Cells / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D022142:Secretory Vesicles; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000075745:rab27 GTP-Binding Proteins
D002352:Carrier Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009478:Neuropeptide Y; C512779:SYTL4 protein, human; C497938:Stxbp1 protein, mouse; C497939:Stxbp1 protein, rat; C122015:Sytl4 protein, mouse; C512778:Sytl4 protein, rat; D033921:Vesicular Transport Proteins; D000075745:rab27 GTP-Binding Proteins; D006153:Guanosine Diphosphate; C512962:Rab27a protein, mouse; C512961:Rab27a protein, rat; D020691:rab GTP-Binding Proteins