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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 complex formation of granuphilin-interacting syntaxin-1a and Munc18-1 in islets. (A) Expression levels of syntaxin-1a and Munc18-1 in islets. Islet protein was extracted by homogenization with 1× SDS sample buffer and the equivalent amount from 16 to 20 islets was loaded per lane. Protein-transferred membranes were separated into upper and lower parts at the region representing a molecular mass of ∼45 kD and then reacted with anti–Munc18-1/anti–α-tubulin (upper) and anti–syntaxin-1a/anti-Rab27a (lower) antibody mixtures. Signals with short- and long-term exposure in immunoblots (IB) reveal the decreased levels of syntaxin-1a and Munc18-1 in Grn−/Y islets, respectively (left). Protein expression levels in Grn+/Y (open bars) and Grn−/Y islets (closed bars) were quantified from seven experimental preparations (right). *, P = 0.018. (B) Immunofluorescent analysis of syntaxin-1a (green) and glucagon (red) in frozen pancreatic tissue sections. The syntaxin-1a immunosignal is specifically reduced in the glucagon-negative cells mainly located in the center area of the islet. Bars, 50 μm (left). The cell area (μm2) and the immunofluorescent intensity of syntaxin-1a were quantified for glucagon-positive (+) and -negative (−) islet cells (n = 15) of Grn+/Y (open bars) and Grn−/Y mice (closed bars; right). **, P < 0.0001. (C) The complex formation between syntaxin-1a and Munc18-1. The sample lysates for immunoprecipitation were prepared with 1% Triton X-100–containing buffer, and the amounts of Munc18-1 and syntaxin-1a were adjusted equally between Grn+/Y and Grn−/Y islets (left). These and MIN6 cell lysates were immunoprecipitated with anti–Munc18-1 serum. In the control lane, MIN6 cell extracts were immunoprecipitated with normal rabbit serum. The immunoprecipitates (IP) were analyzed by immunoblotting with anti–Munc18-1 and anti–syntaxin-1a mAb (middle). Relative intensities of signals in Grn−/Y islets (closed bars) to those in Grn+/Y islets (open bars) were calculated from six independent experiments (right). *, P = 0.027. Results are provided as mean ± SEM.
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fig7: Expression and complex formation of granuphilin-interacting syntaxin-1a and Munc18-1 in islets. (A) Expression levels of syntaxin-1a and Munc18-1 in islets. Islet protein was extracted by homogenization with 1× SDS sample buffer and the equivalent amount from 16 to 20 islets was loaded per lane. Protein-transferred membranes were separated into upper and lower parts at the region representing a molecular mass of ∼45 kD and then reacted with anti–Munc18-1/anti–α-tubulin (upper) and anti–syntaxin-1a/anti-Rab27a (lower) antibody mixtures. Signals with short- and long-term exposure in immunoblots (IB) reveal the decreased levels of syntaxin-1a and Munc18-1 in Grn−/Y islets, respectively (left). Protein expression levels in Grn+/Y (open bars) and Grn−/Y islets (closed bars) were quantified from seven experimental preparations (right). *, P = 0.018. (B) Immunofluorescent analysis of syntaxin-1a (green) and glucagon (red) in frozen pancreatic tissue sections. The syntaxin-1a immunosignal is specifically reduced in the glucagon-negative cells mainly located in the center area of the islet. Bars, 50 μm (left). The cell area (μm2) and the immunofluorescent intensity of syntaxin-1a were quantified for glucagon-positive (+) and -negative (−) islet cells (n = 15) of Grn+/Y (open bars) and Grn−/Y mice (closed bars; right). **, P < 0.0001. (C) The complex formation between syntaxin-1a and Munc18-1. The sample lysates for immunoprecipitation were prepared with 1% Triton X-100–containing buffer, and the amounts of Munc18-1 and syntaxin-1a were adjusted equally between Grn+/Y and Grn−/Y islets (left). These and MIN6 cell lysates were immunoprecipitated with anti–Munc18-1 serum. In the control lane, MIN6 cell extracts were immunoprecipitated with normal rabbit serum. The immunoprecipitates (IP) were analyzed by immunoblotting with anti–Munc18-1 and anti–syntaxin-1a mAb (middle). Relative intensities of signals in Grn−/Y islets (closed bars) to those in Grn+/Y islets (open bars) were calculated from six independent experiments (right). *, P = 0.027. Results are provided as mean ± SEM.

Mentions: Besides Rab27a, granuphilin directly binds to the plasma membrane–anchored SNARE protein syntaxin-1a, and this interaction is positively regulated by Rab27a (Torii et al., 2002). Granuphilin has also been shown to bind to Munc18-1 in vitro and in a mammalian two-hybrid assay (Coppola et al., 2002). It was, however, very difficult to detect the endogenous protein complex between granuphilin and Munc18-1 in the islets using coimmunoprecipitation (unpublished data). Further, the amount of Munc18-1 in the granuphilin immunoprecipitates is highly correlated with the amount of coprecipitated syntaxin-1a in MIN6 cells, as previously reported (Torii et al., 2002). Because Munc18-1 itself interacts with syntaxin-1a (Dulubova et al., 1999; Misura et al., 2000; Yang et al., 2000), it is currently unknown whether granuphilin forms a complex with Munc18-1 directly or through syntaxin-1a. The expression levels of syntaxin-1a and Munc18-1 were profoundly diminished by ∼50% in the mutant islets (49.4 ± 8.7% for syntaxin-1a and 49.5 ± 3.6% for Munc18-1; P = 0.018), although those of α-tubulin and Rab27a were not significantly different (94.5 ± 5.7% for α-tubulin and 119.6 ± 13.7% for Rab27a; Fig. 7 A and Fig. S1). Remarkably, immunofluorescent analysis demonstrated that the decrease of syntaxin-1a expression was specific to the glucagon-negative, deducible β cells (Fig. 7 B), where granuphilin is specifically expressed in normal islets (Wang et al., 1999), indicating an even more severe reduction in β cells. We then examined the complex formation between Munc18-1 and syntaxin-1a. Because the expression levels of both proteins are reduced in mutant islets, we adjusted the amount of lysate proteins equally between the control and mutant samples for the coimmunoprecipitation experiments (Fig. 7 C). Although the amount of Munc18-1 in its immunoprecipitate was similar, the amount of coprecipitated syntaxin-1a was significantly reduced in the mutant islets (112.0 ± 10.5% for Munc18-1 and 61.0 ± 6.2% for syntaxin-1a of the control; P = 0.027). The impairment of the complex formation in physiological cells should be more severe without the adjustment of protein levels. These results indicate that granuphilin is essential both for the preservation of expression levels of syntaxin-1a and Munc18-1 and for the maintenance of the syntaxin-1a–Munc18-1 complex formation.


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 complex formation of granuphilin-interacting syntaxin-1a and Munc18-1 in islets. (A) Expression levels of syntaxin-1a and Munc18-1 in islets. Islet protein was extracted by homogenization with 1× SDS sample buffer and the equivalent amount from 16 to 20 islets was loaded per lane. Protein-transferred membranes were separated into upper and lower parts at the region representing a molecular mass of ∼45 kD and then reacted with anti–Munc18-1/anti–α-tubulin (upper) and anti–syntaxin-1a/anti-Rab27a (lower) antibody mixtures. Signals with short- and long-term exposure in immunoblots (IB) reveal the decreased levels of syntaxin-1a and Munc18-1 in Grn−/Y islets, respectively (left). Protein expression levels in Grn+/Y (open bars) and Grn−/Y islets (closed bars) were quantified from seven experimental preparations (right). *, P = 0.018. (B) Immunofluorescent analysis of syntaxin-1a (green) and glucagon (red) in frozen pancreatic tissue sections. The syntaxin-1a immunosignal is specifically reduced in the glucagon-negative cells mainly located in the center area of the islet. Bars, 50 μm (left). The cell area (μm2) and the immunofluorescent intensity of syntaxin-1a were quantified for glucagon-positive (+) and -negative (−) islet cells (n = 15) of Grn+/Y (open bars) and Grn−/Y mice (closed bars; right). **, P < 0.0001. (C) The complex formation between syntaxin-1a and Munc18-1. The sample lysates for immunoprecipitation were prepared with 1% Triton X-100–containing buffer, and the amounts of Munc18-1 and syntaxin-1a were adjusted equally between Grn+/Y and Grn−/Y islets (left). These and MIN6 cell lysates were immunoprecipitated with anti–Munc18-1 serum. In the control lane, MIN6 cell extracts were immunoprecipitated with normal rabbit serum. The immunoprecipitates (IP) were analyzed by immunoblotting with anti–Munc18-1 and anti–syntaxin-1a mAb (middle). Relative intensities of signals in Grn−/Y islets (closed bars) to those in Grn+/Y islets (open bars) were calculated from six independent experiments (right). *, P = 0.027. Results are provided as mean ± SEM.
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fig7: Expression and complex formation of granuphilin-interacting syntaxin-1a and Munc18-1 in islets. (A) Expression levels of syntaxin-1a and Munc18-1 in islets. Islet protein was extracted by homogenization with 1× SDS sample buffer and the equivalent amount from 16 to 20 islets was loaded per lane. Protein-transferred membranes were separated into upper and lower parts at the region representing a molecular mass of ∼45 kD and then reacted with anti–Munc18-1/anti–α-tubulin (upper) and anti–syntaxin-1a/anti-Rab27a (lower) antibody mixtures. Signals with short- and long-term exposure in immunoblots (IB) reveal the decreased levels of syntaxin-1a and Munc18-1 in Grn−/Y islets, respectively (left). Protein expression levels in Grn+/Y (open bars) and Grn−/Y islets (closed bars) were quantified from seven experimental preparations (right). *, P = 0.018. (B) Immunofluorescent analysis of syntaxin-1a (green) and glucagon (red) in frozen pancreatic tissue sections. The syntaxin-1a immunosignal is specifically reduced in the glucagon-negative cells mainly located in the center area of the islet. Bars, 50 μm (left). The cell area (μm2) and the immunofluorescent intensity of syntaxin-1a were quantified for glucagon-positive (+) and -negative (−) islet cells (n = 15) of Grn+/Y (open bars) and Grn−/Y mice (closed bars; right). **, P < 0.0001. (C) The complex formation between syntaxin-1a and Munc18-1. The sample lysates for immunoprecipitation were prepared with 1% Triton X-100–containing buffer, and the amounts of Munc18-1 and syntaxin-1a were adjusted equally between Grn+/Y and Grn−/Y islets (left). These and MIN6 cell lysates were immunoprecipitated with anti–Munc18-1 serum. In the control lane, MIN6 cell extracts were immunoprecipitated with normal rabbit serum. The immunoprecipitates (IP) were analyzed by immunoblotting with anti–Munc18-1 and anti–syntaxin-1a mAb (middle). Relative intensities of signals in Grn−/Y islets (closed bars) to those in Grn+/Y islets (open bars) were calculated from six independent experiments (right). *, P = 0.027. Results are provided as mean ± SEM.
Mentions: Besides Rab27a, granuphilin directly binds to the plasma membrane–anchored SNARE protein syntaxin-1a, and this interaction is positively regulated by Rab27a (Torii et al., 2002). Granuphilin has also been shown to bind to Munc18-1 in vitro and in a mammalian two-hybrid assay (Coppola et al., 2002). It was, however, very difficult to detect the endogenous protein complex between granuphilin and Munc18-1 in the islets using coimmunoprecipitation (unpublished data). Further, the amount of Munc18-1 in the granuphilin immunoprecipitates is highly correlated with the amount of coprecipitated syntaxin-1a in MIN6 cells, as previously reported (Torii et al., 2002). Because Munc18-1 itself interacts with syntaxin-1a (Dulubova et al., 1999; Misura et al., 2000; Yang et al., 2000), it is currently unknown whether granuphilin forms a complex with Munc18-1 directly or through syntaxin-1a. The expression levels of syntaxin-1a and Munc18-1 were profoundly diminished by ∼50% in the mutant islets (49.4 ± 8.7% for syntaxin-1a and 49.5 ± 3.6% for Munc18-1; P = 0.018), although those of α-tubulin and Rab27a were not significantly different (94.5 ± 5.7% for α-tubulin and 119.6 ± 13.7% for Rab27a; Fig. 7 A and Fig. S1). Remarkably, immunofluorescent analysis demonstrated that the decrease of syntaxin-1a expression was specific to the glucagon-negative, deducible β cells (Fig. 7 B), where granuphilin is specifically expressed in normal islets (Wang et al., 1999), indicating an even more severe reduction in β cells. We then examined the complex formation between Munc18-1 and syntaxin-1a. Because the expression levels of both proteins are reduced in mutant islets, we adjusted the amount of lysate proteins equally between the control and mutant samples for the coimmunoprecipitation experiments (Fig. 7 C). Although the amount of Munc18-1 in its immunoprecipitate was similar, the amount of coprecipitated syntaxin-1a was significantly reduced in the mutant islets (112.0 ± 10.5% for Munc18-1 and 61.0 ± 6.2% for syntaxin-1a of the control; P = 0.027). The impairment of the complex formation in physiological cells should be more severe without the adjustment of protein levels. These results indicate that granuphilin is essential both for the preservation of expression levels of syntaxin-1a and Munc18-1 and for the maintenance of the syntaxin-1a–Munc18-1 complex formation.

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