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First published online 23 March 2004
doi: 10.1242/jcs.01048

Journal of Cell Science 117, 1945-1953 (2004)
Published by The Company of Biologists 2004
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The small GTPase Rab27B regulates amylase release from rat parotid acinar cells

Akane Imai1, Sumio Yoshie2, Tomoko Nashida1, Hiromi Shimomura1 and Mitsunori Fukuda3,*

1 Department of Biochemistry, The Nippon Dental University, School of Dentistry at Niigata, 1-8, Hamaura-cho, Niigata 951-8580, Japan
2 Histology, The Nippon Dental University, School of Dentistry at Niigata, 1-8, Hamaura-cho, Niigata 951-8580, Japan
3 Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan



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  		Fig. 1. Expression of small GTPase Rab27 and its effectors (Slp and Slac2) in rat parotid glands. (A) Expression of the Slp and Slac2 family members in rat parotid glands. Similar amounts of recombinant T7-tagged Slp1-5 and Slac2-a/b/c expressed in COS-7 cells (lane 1; see B) and total homogenates of rat parotid glands (50 µg; lane 2) were loaded on 7.5% SDS-PAGE and immunoblotted with anti-Slp1, anti-Slp2-a, anti-Slp3-a, anti-Slp4-a, anti-Slp5, anti-Slac2-a, anti-Slac2-b, or anti-Slac2-c specific antibody. Note that the Slp4-a and Slac2-c proteins, but not the other Rab27 effectors, were easily detected in rat parotid glands (arrowheads). The asterisk indicates non-specific interaction of the anti-Slac2-a antibody. (B) Recombinant T7-Slp1-5 and T7-Slac2-a/b/c were used as positive controls in A. Similar amounts of the T7-tagged proteins except for T7-Slac2-b were loaded into each lane. The positions of the molecular mass markers (in kDa) are shown on the left. (C) Expression of Rab27A and Rab27B in rat parotid glands. The same amounts of FLAG-tagged Rab27A (lane 1) and Rab27B (lane 2), and total homogenates of rat parotid glands (50 µg; lane 3) were loaded on 12.5% SDS-PAGE and immunoblotted with anti-Rab27A (top panel), anti-Rab27B (middle panel), or anti-FLAG tag antibody (bottom panel). Judging from the intensity of the Rab27A and Rab27B bands calibrated with the FLAG-tagged recombinant proteins, expression of Rab27B was dominant in parotid glands. Under our experimental conditions, Rab27A, Rab27B, Slp4-a, and Slac2-c were detected as single immunoreactive bands.

 


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  		Fig. 2. Localization of Rab27–Slac2-c and Rab27–Slp4-a complexes on secretory granules in rat parotid glands. (A) In vivo formation of Rab27–Slac2-c and Rab27–Slp4-a complexes in rat parotid glands. Immunoprecipitation with anti-Slp4-a, anti-Slac2-c, or anti-Syt I IgG was performed as described in the Materials and Methods. Co-immunoprecipitated Rab27A/B was detected by immunoblotting with anti-Rab27A/B specific antibody (12.5% non-reduced gels; solid arrowheads in the top and middle panels). Immunoprecipitated Slp4-a, Slac2-c and Syt I were separately visualized by immunoblotting with specific antibodies (10% reduced gels; open arrowheads in bottom panel). The asterisks in lane 3 indicate the degradation products of Slac2-c. Note that both anti-Slp4-a and anti-Slac2-c IgGs, but not Syt I IgG, immunoprecipitated Rab27A and Rab27B (lanes 2 and 3 in the top and middle panels). The positions of the molecular mass markers (in kDa) are shown on the left. (B) Localization of Rab27A, Rab27B, Slac2-c and Slp4-a proteins in secretory-granule-enriched fractions. Subcellular fractionation of the rat parotid glands was performed as described previously (Imai et al., 2003Go). A 25 µg amount of total homogenate (lane 1), apical plasma membrane (lane 2), and secretory granule membrane (lane 3) from rat parotid glands was loaded on 7.5% (for Slp4-a and Slac2-c) or 12.5% SDS-PAGE (for Rab27A/B) and immunoblotted with anti-Rab27A (top panel), anti-Rab27B (second panel), anti-Slp4-a (third panel), or anti-Slac2-c antibody (bottom panel). Note that both the Rab27A and Rab27B proteins were highly enriched on the secretory granule membranes, whereas the Slp4-a and Slac2-c proteins were localized on both the apical plasma membrane and the secretory granule membrane. The positions of the molecular mass markers (in kDa) are shown on the right.

 


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  		Fig. 3. Colocalization of Rab27B, Slp4-a and Slac2-c with VAMP-2, a secretory granule marker, in rat parotid acinar cells. Each section was immunostained for Rab27B (A,D), Slp4-a (B,G), or Slac2-c (C,J) and double-stained for VAMP-2 (E,H,K) to confirm the presence of secretory granules in the apical regions of the acinar cells. The merged images are shown in F, I and L. Note that Rab27B, Slp4-a and Slac2-c were mainly colocalized with VAMP-2 on secretory granules that are close to the apical plasma membrane of the acinar cells (F,I,L, respectively). The cells are outlined in white; ap, apical plasma membrane. Scale bars: 5 µm.

 


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  		Fig. 4. Functional involvement of the Rab27B–Slac2-c complex in amylase release from SLO-permeabilized parotid acinar cells. (A) Inhibition of amylase release by SHD, a specific Rab27 binding domain, in SLO-permeabilized parotid acinar cells. GST-SHD, but not GST alone, inhibited amylase release in a dose-dependent manner. (B) Inhibition of amylase release by ABD, an actin binding domain of Slac2-c, in SLO-permeabilized parotid acinar cells. GST-ABD, but not GST-ABD(RA) lacking actin binding capacity, inhibited amylase release in a dose-dependent manner. (C) Inhibition of amylase release by anti-Rab27B and anti-Slac2-c specific antibodies. Both anti-Rab27B and Slac2-c IgGs inhibited amylase release in a dose-dependent manner. By contrast, control IgG (up to 100 µg/ml) had no significant effect on amylase release. IPR-stimulated amylase release from SLO-permeabilized parotid acinar cells was measured as described in the Materials and Methods. The released amylase activity is expressed as a percentage of the IPR-stimulated release without rabbit IgG or GST fusion proteins. Bars indicate the mean±s.e.m. of 3-5 independent experiments, performed in triplicate. *P<0.01, Student's t-test.

 


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  		Fig. 5. Effect of anti-Rab27B and anti-Slac2-c-SHD antibodies on the interaction between Rab27B and Slac2-c in vitro. (A) FLAG-Rab27B binding activity of T7-Slac2-c and T7-Slp4-a in the presence or absence of anti-Rab27B IgG. The FLAG-Rab27B beads (bottom panel) were incubated with T7-Slac2-c (lanes 1 and 2) or T7-Slp4-a (lanes 3 and 4) in the presence or absence of the anti-Rab27B IgG as described in the Materials and Methods, and the T7-tagged proteins trapped by the beads were analyzed by immunoblotting with HRP-conjugated anti-T7 tag antibody (1/10,000 dilution) as described previously (Fukuda et al., 1999Go). Note that the anti-Rab27B IgG specifically disrupted the Rab27B–Slac2-c interaction (lane 2, middle panel) but had no effect on the Rab27B–Slp4-a interaction (lane 4, middle panel). (B) Inhibition of the Rab27B–Slac2-c interaction by the anti-Slac2-c antibody. The T7-Slac2-c beads (bottom panel) were incubated with either the anti-Slac2-c IgG or a control rabbit IgG, and the FLAG-Rab27B trapped by the beads was analyzed by immunoblotting with HRP-conjugated anti-FLAG tag antibody as described previously (Fukuda et al., 1999Go; Fukuda et al., 2002bGo). Note that the anti-Slac2-c IgG, but not control IgG, inhibited the interaction between Rab27B and Slac2-c (compare lanes 2 and 3 in the middle panel). Input means 1/80 volume of the reaction mixtures used for the immunoprecipitation studies (top panels in A and B). The positions of the molecular mass markers (in kDa) are shown on the left.

 

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© The Company of Biologists Ltd 2004