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

Authors :
Joberty, Gérard
Stabila, Paul F.
Coppola, Thierry
Macara, Ian G.
Regazzi, Romano
Source :
Journal of Cell Science; October 1999, Vol. 112 Issue: 20 p3579-3587, 9p
Publication Year :
1999

Abstract

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca2+-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 α-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 Ca2+-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 Ca2+-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.

Details

Language :
English
ISSN :
00219533 and 14779137
Volume :
112
Issue :
20
Database :
Supplemental Index
Journal :
Journal of Cell Science
Publication Type :
Periodical
Accession number :
ejs58996061
Full Text :
https://doi.org/10.1242/jcs.112.20.3579