RRC ID 32583
Author Fukuda M.
Title Slp4-a/granuphilin-a inhibits dense-core vesicle exocytosis through interaction with the GDP-bound form of Rab27A in PC12 cells.
Journal J Biol Chem
Abstract 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.
Volume 278(17)
Pages 15390-6
Published 2003-4-25
DOI 10.1074/jbc.M213090200
PII M213090200
PMID 12590134
MeSH Amino Acid Substitution Animals Base Sequence Carrier Proteins / metabolism Carrier Proteins / physiology* DNA Mutational Analysis Exocytosis* / genetics Guanosine Diphosphate / metabolism Mice Molecular Sequence Data Munc18 Proteins Nerve Tissue Proteins / metabolism Neuropeptide Y / metabolism PC12 Cells / metabolism* Protein Binding / genetics Protein Binding / physiology Protein Structure, Tertiary Rats Secretory Vesicles* Vesicular Transport Proteins / metabolism rab GTP-Binding Proteins / genetics rab GTP-Binding Proteins / metabolism* rab27 GTP-Binding Proteins
IF 4.238
Times Cited 52
DNA material Slp4-a (RDB03421) pEF-T7-mouse Slac2-b (RDB15095) pEF-FLAG-mouse Munc18-1 (RDB15177).