Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B–Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B–Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B–Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.

Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic ␤-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-a⅐syntaxin-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.Small GTPase Rab27A is expressed in a wide variety of secretory cells (1) and has been suggested to control secretion by these cells through interaction with cell type-or tissue-specific Rab27A effector(s) (for review, see Refs. 2-4). To date three distinct groups of Rab27A-binding proteins have been reported in humans and mice (4). The first group includes the members of the synaptotagmin-like protein (Slp) 2 family (Slp1-5) and rabphilin (5-12), all of which contain an N-terminal Rab27A binding domain (also called Slp homology domain (SHD)) and C-terminal tandem C2 domains that potentially bind phospholipids (5,13,14). The second group of Rab27A-binding proteins includes the members of the Slac2 family (Slac2-a-c) and Noc2, all of which contain an N-terminal Rab27A-binding domain but lack tandem C2 domains (6,7,11,(15)(16)(17)(18). Both Slac2-a/melanophilin and Slac2-c/MyRIP contain a myosin binding domain at the middle of the molecule (10, 19 -23) and an actin binding domain at the C terminus (17, 22). The last Rab27A-binding protein identified is Munc13-4, a putative priming factor for exocytosis (24,25). Although Rab27A has been shown to be involved in the control of hormone secretion by endocrine cells through interaction with Slp4-a ...

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We investigated the expression of the genes encoding Rab proteins, low molecular weight GTP-binding proteins, in the rat parotid gland by the use of reverse transcription-polymerase chain reaction, and detected cDNAs of Rab3D, Rab4, and Rab26. We further examined the characteristics and localization of Rab26 by western blotting and light and electron microscopic immunocytochemistry. Western blotting using an antibody against the Rab26-specific, C-terminal peptide detected the His-tagged Rab26 protein as a single 27-kDa band. This band also displayed binding to [alpha-32P]GTP. The fraction containing secretory granule membranes in an acinar cell homogenate was immunostained with the antibody. Supporting this, the immunocytochemical reaction for Rab26 was localized immediately around secretory granules in the acinar cells. The immunostaining disappeared from the acinar cells after treatment of rats with isoproterenol. These findings suggest that Rab26 participates in the regulated secretion of granules and functionally belongs to the Rab3 group.

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.