title: Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner.
abstract: Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic beta-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereas Munc18-2 itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4-a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a.syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane.
authors.affiliation: Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
authors.forename: Akane
authors.initials: A
authors.lastname: Imai
authors.forename: Tomoko
authors.initials: T
authors.lastname: Nashida
authors.forename: Hiromi
authors.initials: H
authors.lastname: Shimomura
mesh_terms: D000681:Amylases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D051379:Mice; D046912:Multiprotein Complexes; D051938:Munc18 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010306:Parotid Gland / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050827:Syntaxin 1; C122015:Sytl4 protein, mouse; C512778:Sytl4 protein, rat; D033921:Vesicular Transport Proteins; D000681:Amylases