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First published online September 3, 2008
doi: 10.1242/10.1242/jcs.030544

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The GDP-dependent Rab27a effector coronin 3 controls endocytosis of secretory membrane in insulin-secreting cell lines

¹ Department of Pharmacology, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hasama, Yufu, Oita 879-5593, Japan
² Division of Molecular Metabolism and Diabetes, Tohoku University Graduate School of Medicine, 2-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan
³ Department of Anatomy I, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
⁴ Protein Research Network, Inc., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

View larger version (62K): [in this window] [in a new window]   Fig. 1. Identification of coronin 3 as a GDP-Rab27a-interacting protein in pancreatic β cells. (A,B) Eluates from affinity columns were analyzed by silver staining (A) and immunoblotting with anti-coronin 3 antibody (B). (C) Immunoprecipitation of MIN6 extracts using anti-Rab27a antibody and immunoblotting using anti-Rab27a and anti-coronin 3 antibodies. 0.2% of the input protein was co-immunoprecipitated. (D,E) Mouse pancreata were double-stained using anti-insulin antibody and anti-Rab27a antibody (D) or anti-coronin 3 antibody (E). Scale bars, 20 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Coronin 3 binds glucose-induced GDP-Rab27a through the β-propeller structure. (A) Immunoprecipitation of COS-7 extracts that express FLAG-Rab27a mutants and GFP-coronin 3 (upper panel) or GFP-SHD (lower panel) using anti-GFP antibody, and immunoblotting using anti-GFP and anti-FLAG antibodies. 0.2% of the input protein was co-immunoprecipitated. (B) In-vitro-binding assay of purified FLAG-coronin 3 and Rab27a mutants and immunoblotting using anti-FLAG and anti-GST antibodies. (C) Immunoprecipitation of COS-7 extracts that express FLAG-Rab27a-T23N and GFP-coronin 3 deletion mutants using anti-GFP antibody, and immunoblotting using anti-FLAG antibody. 2.0% of the input protein was co-immunoprecipitated except for WT (0.2%). (D) The indicated concentrations of MBP–coronin-3-C were incubated with beads conjugated to GST-Rab27a-T23N; the Kd value was calculated using Scatchard analysis. (E) COS7 lysates that express T7-Rab27a mutants were incubated with beads conjugated to GST-SHD or GST-coronin-3-C. The bound proteins were analyzed by immunoblotting using anti-T7 antibody. (F) MIN6 cells stimulated with 20 mM glucose were extracted and incubated with beads conjugated to GST-SHD or GST-coronin-3-C. The bound proteins were analyzed by immunoblotting using anti-Rab27a antibody.

View larger version (57K): [in this window] [in a new window]   Fig. 3. The interaction between GDP-Rab27a and coronin 3 is essential for endocytosis. (A) Coronin-3-silenced MIN6 cells were analyzed by immunoblotting with anti-coronin 3 and anti-Rab27a antibodies. (B) Coronin-3-silenced MIN6 cells that express GFP (green in overlay) as a transfection marker were labeled with FM4-64 (top panel, and red in overlay). Fluorescence intensity of FM4-64 on the line is shown in lower panels. Scale bar, 10 µm. (C) GFP-coronin three mutants expressing MIN6 cells were labeled with FM4-64. For rescue experiments, T7-Rab27a-T23N was co-transfected. Dashed outlines indicate transfected cells. Scale bar, 10 µm. (D) Fluorescence intensity of FM4-64 was analyzed. Among the transfected cells, the rate of cells with cytoplasmic distribution of fluorescence is given presented as a percentage. More than 40 randomly selected cells (more than ten cells per experiment) were examined. Data are expressed as the mean ± s.d. from four independent experiments. All the experiments were carried out in the presence of 25 mM glucose.

View larger version (50K): [in this window] [in a new window]   Fig. 4. The interaction between GDP-Rab27a and coronin 3 is essential for internalization of phogrin. (A) Coronin 3 siRNA was co-transfected with phogrin-GFP into MIN6 cells. GFP-positive cells were regarded as coronin-3-silenced cells. Arrowheads denote phogrin-GFP localized in the cell periphery. Fluorescence intensity of phogrin-GFP on the line is shown in lower panels. Scale bar, 10 µm. (B) MIN6 cells expressing DsRed-coronin 3 mutant and phogrin-GFP were evaluated. For rescue experiments, T7-Rab27a-T23N was co-transfected. Dashed outlines indicate transfected cells. Scale bar, 10 µm. (C) Fluorescence intensity was analyzed in the same way as in Fig. 3D. All experiments were carried out in the presence of 25 mM glucose.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Schematic model for Rab27a function at membrane traffic. GTP-Rab27a functions at pre-exocytotic stages through interaction with its GTP-dependent effectors under basal conditions. Glucose triggers exocytosis via closure of the ATP-sensitive K+ channel, opening of the voltage-dependent Ca2+ channel and an eventual rise in cytosolic Ca2+. Glucose also causes a shift from GTP-Rab27a to GDP-Rab27a, which regulates endocytosis through interaction with coronin 3. Both GTP- and GDP-bound forms of Rab27a control a series of the insulin granule traffic at the distinct stages.

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