High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion

Details

Serval ID
serval:BIB_06E5ED2601FB
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion
Journal
Journal of Cell Science
Author(s)
Joberty  G., Stabila  P. F., Coppola  T., Macara  I. G., Regazzi  R.
ISSN
0021-9533 (Print)
Publication state
Published
Issued date
10/1999
Volume
112 ( Pt 20)
Pages
3579-87
Notes
Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S. --- Old month value: Oct
Abstract
Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca(2+)-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic alpha-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca(2+)-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion. High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca(2+)-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.
Keywords
Adaptor Proteins, Signal Transducing Amino Acid Sequence Animals Binding Sites Cell Line Cytoplasmic Granules/metabolism GTP Phosphohydrolases/metabolism Intracellular Membranes/metabolism Molecular Sequence Data Mutagenesis, Site-Directed Nerve Tissue Proteins/chemistry/genetics/*metabolism PC12 Cells Point Mutation Rats Recombinant Fusion Proteins/metabolism Recombinant Proteins/chemistry/metabolism Sequence Alignment Sequence Homology, Amino Acid Transfection Vesicular Transport Proteins rab GTP-Binding Proteins/chemistry/genetics/*metabolism rab3 GTP-Binding Proteins/chemistry/*metabolism
Pubmed
Web of science
Create date
24/01/2008 14:30
Last modification date
20/08/2019 12:29
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