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Involvement of Phospholipase C‐related but catalytically inactive protein in regulated exocytosis through the pleckstrin homology and the C2 domains
Author(s) -
Takeuchi Hiroshi,
Zhang Zhao,
Gao Jing,
Wang DaGuang,
Hirata Masato
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.591.7
Subject(s) - pleckstrin homology domain , exocytosis , syntaxin , microbiology and biotechnology , biology , mutant , secretion , phospholipase d , c2 domain , munc 18 , vesicle , biochemistry , signal transduction , synaptic vesicle , membrane , gene
Phospholipase C‐related but catalytically inactive protein (PRIP) is a scaffolding protein identified in our laboratory. We have recently found that the secretion of various peptide hormones from multiple organs were upregulated in PRIP knock‐out mice, indicating that PRIP regulates the exocytic event common to dense‐core vesicles in a negative manner. In this study, we investigated the molecular mechanism underlying the inhibitory role of PRIP in exocytosis. We examined the effect of exogenously expressed various mutants of PRIP on noradrenaline (NA) secretion using rat pheochromocytoma cell line, PC12. Expression of the wild‐type PRIP suppressed High‐K + induced NA release from PC12 cells, while the mutant of which Arg‐134 in the pleckstrin homology (PH) domain was replaced with Glu (R134Q) to loose the binding to membrane phospholipid PtdIns(4,5)P 2 , little suppressed NA release. The mutant lacking the C2 domain was also less effective than the wild‐type PRIP to suppress exocytosis. We found that the C2 domain of PRIP interacts with the components of SNARE complex including syntaxin 1A and synaptosomal‐associated protein of 25 kDa (SNAP‐25) and inhibited the formation of stable SDS‐resistant SNARE complexes. The results suggest that the direct bindings of the PH domain to PtdIns(4,5)P 2 and the C2 domain to SNARE proteins are both required for PRIP to negatively regulate exocytosis.