Slp4-a/Granuphilin-a Interacts with Syntaxin-2/3 in a Munc18-2-dependent Manner

Akane Imai,Hiromi Shimomura,Mitsunori Fukuda,Tomoko Nashida

doi:10.1074/jbc.m505759200

Akane Imai, Hiromi Shimomura + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m505759200

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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.

Highlights

(Slp1–5) and rabphilin [5,6,7,8,9,10,11,12], all of which contain an N-terminal Rab27A binding domain ( called synaptotagmin-like protein (Slp) homology domain (SHD)) and C-terminal tandem C2 domains that potentially bind phospholipids [5, 13, 14]
We found that Slp4a1⁄7syntaxin-2 complex is present in rat parotid glands and that introduction of the recombinant linker domain of Slp4-a or the antibody against the Slp4-a linker domain into streptolysin O (SLO)-permeabilized parotid acinar cells severely attenuates isoproterenol (IPR)-stimulated amylase release
Slp4-a Interacts with Syntaxin-2/3 in a Munc18-2-dependent Manner—In our previous studies we showed that Slp4-a directly interacts with Munc18-1 in PC12 cells [29] and that Slp4-a is expressed in exocrine parotid acinar cells [32], which do not endogenously express either Munc18-1 or syntaxin-1a [34, 35]

Summary

Introduction

(Slp1–5) and rabphilin [5,6,7,8,9,10,11,12], all of which contain an N-terminal Rab27A binding domain ( called Slp homology domain (SHD)) and C-terminal tandem C2 domains that potentially bind phospholipids [5, 13, 14]. The results showed that Munc18-1 interacted with the Slp4-a deletion mutants that contained the linker domain of Slp4-a (amino acid residues 144 –354), a previously uncharacterized region between the SHD and the C2A domain (open arrowhead, lanes 1–3 in the middle panel of Fig. 4B) and that the C2 domains (putative protein interaction sites) were unnecessary for Munc18-1 binding.

Results

Conclusion