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Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

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Expression and intracellular distribution of granuphilin-binding Rab3a and -27a. (A) Immunoblot analysis of Rab3a, Rab27a, and α-tubulin in total lysates from Grn+/Y and Grn−/Y islets. (B) Coimmunoprecipitation of granuphilin in the Rab3a or -27a immunoprecipitates. Protein from 400–600 islets was extracted with 1% Triton X-100–containing lysis buffer and immunoprecipitated (IP) with anti-Rab3a or anti-Rab27a antibodies followed by immunoblotting (IB) with antigranuphilin αGrp-N and anti-Rabs antibodies. (C) Immunofluorescent labeling of Rab3a, Rab27a, and insulin in the islet monolayer culture. Rab27a is diffusely dispersed of in Grn−/Y cells in contrast to the marginal distribution in Grn+/Y cells (arrowheads). Bars, 25 μm.
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fig6: Expression and intracellular distribution of granuphilin-binding Rab3a and -27a. (A) Immunoblot analysis of Rab3a, Rab27a, and α-tubulin in total lysates from Grn+/Y and Grn−/Y islets. (B) Coimmunoprecipitation of granuphilin in the Rab3a or -27a immunoprecipitates. Protein from 400–600 islets was extracted with 1% Triton X-100–containing lysis buffer and immunoprecipitated (IP) with anti-Rab3a or anti-Rab27a antibodies followed by immunoblotting (IB) with antigranuphilin αGrp-N and anti-Rabs antibodies. (C) Immunofluorescent labeling of Rab3a, Rab27a, and insulin in the islet monolayer culture. Rab27a is diffusely dispersed of in Grn−/Y cells in contrast to the marginal distribution in Grn+/Y cells (arrowheads). Bars, 25 μm.

Mentions: To explore the molecular mechanism that underlies these phenotypes, we investigated the granuphilin-interacting proteins. Because granuphilin can bind to both Rab27a and -3a in vitro (Coppola et al., 2002; Yi et al., 2002), we examined the effect of granuphilin deletion on the expression and cellular localization of these Rab proteins. The expression level of either Rab27a or -3a was not affected by loss of granuphilin (Fig. 6 A and see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200505179/DC1, for the entire images of immunoblots). Consistent with our previous findings in MIN6 cells (Torii et al., 2002; Yi et al., 2002), coimmunoprecipitation assays demonstrated the preferential interaction of granuphilin with Rab27a in wild-type islets, whereas the interaction with Rab3a was barely detectable (Fig. 6 B). Although we could not detect changes in the distribution pattern in the pancreatic islet sections (unpublished data), we observed a marked redistribution of Rab27a, but not of Rab3a, in the monolayer β cells (Fig. 6 C). Compared with the marginal distribution to the plasma membrane in control cells, Rab27a was diffusely dispersed in the cytoplasm of the granuphilin-deficient cells. These results strongly support our previous proposals that Rab27a is a physiologically dominant granuphilin-binding Rab protein in vivo (Torii et al., 2002; Yi et al., 2002; Kasai et al., 2005).


Granuphilin molecularly docks insulin granules to the fusion machinery.

Gomi H, Mizutani S, Kasai K, Itohara S, Izumi T - J. Cell Biol. (2005)

Expression and intracellular distribution of granuphilin-binding Rab3a and -27a. (A) Immunoblot analysis of Rab3a, Rab27a, and α-tubulin in total lysates from Grn+/Y and Grn−/Y islets. (B) Coimmunoprecipitation of granuphilin in the Rab3a or -27a immunoprecipitates. Protein from 400–600 islets was extracted with 1% Triton X-100–containing lysis buffer and immunoprecipitated (IP) with anti-Rab3a or anti-Rab27a antibodies followed by immunoblotting (IB) with antigranuphilin αGrp-N and anti-Rabs antibodies. (C) Immunofluorescent labeling of Rab3a, Rab27a, and insulin in the islet monolayer culture. Rab27a is diffusely dispersed of in Grn−/Y cells in contrast to the marginal distribution in Grn+/Y cells (arrowheads). Bars, 25 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171228&req=5

fig6: Expression and intracellular distribution of granuphilin-binding Rab3a and -27a. (A) Immunoblot analysis of Rab3a, Rab27a, and α-tubulin in total lysates from Grn+/Y and Grn−/Y islets. (B) Coimmunoprecipitation of granuphilin in the Rab3a or -27a immunoprecipitates. Protein from 400–600 islets was extracted with 1% Triton X-100–containing lysis buffer and immunoprecipitated (IP) with anti-Rab3a or anti-Rab27a antibodies followed by immunoblotting (IB) with antigranuphilin αGrp-N and anti-Rabs antibodies. (C) Immunofluorescent labeling of Rab3a, Rab27a, and insulin in the islet monolayer culture. Rab27a is diffusely dispersed of in Grn−/Y cells in contrast to the marginal distribution in Grn+/Y cells (arrowheads). Bars, 25 μm.
Mentions: To explore the molecular mechanism that underlies these phenotypes, we investigated the granuphilin-interacting proteins. Because granuphilin can bind to both Rab27a and -3a in vitro (Coppola et al., 2002; Yi et al., 2002), we examined the effect of granuphilin deletion on the expression and cellular localization of these Rab proteins. The expression level of either Rab27a or -3a was not affected by loss of granuphilin (Fig. 6 A and see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200505179/DC1, for the entire images of immunoblots). Consistent with our previous findings in MIN6 cells (Torii et al., 2002; Yi et al., 2002), coimmunoprecipitation assays demonstrated the preferential interaction of granuphilin with Rab27a in wild-type islets, whereas the interaction with Rab3a was barely detectable (Fig. 6 B). Although we could not detect changes in the distribution pattern in the pancreatic islet sections (unpublished data), we observed a marked redistribution of Rab27a, but not of Rab3a, in the monolayer β cells (Fig. 6 C). Compared with the marginal distribution to the plasma membrane in control cells, Rab27a was diffusely dispersed in the cytoplasm of the granuphilin-deficient cells. These results strongly support our previous proposals that Rab27a is a physiologically dominant granuphilin-binding Rab protein in vivo (Torii et al., 2002; Yi et al., 2002; Kasai et al., 2005).

Bottom Line: The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis.Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells.The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Endocrinology and Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan.

ABSTRACT
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin- mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin- beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

Show MeSH
Related in: MedlinePlus