title: PRIP (phospholipase C-related but catalytically inactive protein) inhibits exocytosis by direct interactions with syntaxin 1 and SNAP-25 through its C2 domain.
abstract: Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K(+)-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.
authors: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan; Stomatological Hospital of Hebei Medical University, Shijiazhuang 050017, China.
authors: Zhao
authors: Z
authors: Zhang
authors: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan; Division of Applied Pharmacology, Kyushu Dental College, Kitakyushu 803-8580, Japan. Electronic address: r12takeuchi@fa.kyu-dent.ac.jp.
authors: Hiroshi
authors: H
authors: Takeuchi
authors: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors: Jing
authors: J
authors: Gao
authors: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors: DaGuang
authors: D
authors: Wang
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors: Declan J
authors: DJ
authors: James
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors: Thomas F J
authors: TFJ
authors: Martin
authors: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan. Electronic address: hirata1@dent.kyushu-u.ac.jp.
authors: Masato
authors: M
authors: Hirata
mesh_terms: D000818:Animals; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D005089:Exocytosis; D008081:Liposomes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D009638:Norepinephrine / Q000737:chemistry; D056918:Nuclear Receptor Coactivators / Q000737:chemistry / Q000502:physiology*; D016716:PC12 Cells; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D050600:SNARE Proteins / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050863:Synaptotagmin I / Q000737:chemistry; D050827:Syntaxin 1 / Q000737:chemistry*
chemical_list: D008081:Liposomes; C486560:Ncoa6 protein, rat; D056918:Nuclear Receptor Coactivators; D011994:Recombinant Proteins; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; D004247:DNA; D011188:Potassium; D009638:Norepinephrine