Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation of Grn knockout mice. (A) Targeted disruption of the granuphilin gene on mouse chromosome X. The targeting vector contains a neomycin resistance gene driven by the pgk promoter (pgk-neo) and a diphtheria toxin A fragment gene driven by the MC1 promoter (DTA) as positive and negative selection markers, respectively. Exon structures are vertically lined and partially shown from the second (Ex2) to sixth exon (Ex6). Homologous recombination results in replacement of the genomic region from the third to fifth exon with pgk-neo. A, ApaI; V, EcoRV; H, HindIII restriction sites. (B) Genomic Southern blot analysis of the backcrossed progenies from a cross of F1 female heterozygotes (Grn+/−) with wild-type (Grn+/Y) C3H/He male. The locations of the 5′ external probe and the 3′ external probe are shown with horizontal closed boxes in A. The 5′ probe hybridizes to a 13.4-kb ApaI fragment from the wild-type locus and to a 16.7-kb fragment from the mutated locus, and the 3′ probe hybridizes to an 8.3-kb EcoRV fragment from the wild-type locus and to an 8.7-kb fragment from the mutated locus, respectively. (C) The protein extracts from pancreatic islets were electrophoresed for immunoblotting with antigranuphilin (αGrp-N) and α-tubulin antibodies. (D) PCR genotyping of the backcrossed progenies. PCR with Grn/Fow1, Grn/Rev1, and Neo/Fow2 mixed primers produces 364- and 760-bp fragments for wild-type and mutated alleles, respectively, as shown in boxes in A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171228&req=5

fig1: Generation of Grn knockout mice. (A) Targeted disruption of the granuphilin gene on mouse chromosome X. The targeting vector contains a neomycin resistance gene driven by the pgk promoter (pgk-neo) and a diphtheria toxin A fragment gene driven by the MC1 promoter (DTA) as positive and negative selection markers, respectively. Exon structures are vertically lined and partially shown from the second (Ex2) to sixth exon (Ex6). Homologous recombination results in replacement of the genomic region from the third to fifth exon with pgk-neo. A, ApaI; V, EcoRV; H, HindIII restriction sites. (B) Genomic Southern blot analysis of the backcrossed progenies from a cross of F1 female heterozygotes (Grn+/−) with wild-type (Grn+/Y) C3H/He male. The locations of the 5′ external probe and the 3′ external probe are shown with horizontal closed boxes in A. The 5′ probe hybridizes to a 13.4-kb ApaI fragment from the wild-type locus and to a 16.7-kb fragment from the mutated locus, and the 3′ probe hybridizes to an 8.3-kb EcoRV fragment from the wild-type locus and to an 8.7-kb fragment from the mutated locus, respectively. (C) The protein extracts from pancreatic islets were electrophoresed for immunoblotting with antigranuphilin (αGrp-N) and α-tubulin antibodies. (D) PCR genotyping of the backcrossed progenies. PCR with Grn/Fow1, Grn/Rev1, and Neo/Fow2 mixed primers produces 364- and 760-bp fragments for wild-type and mutated alleles, respectively, as shown in boxes in A.

Mentions: To study the cellular function of granuphilin, we generated a mouse line lacking granuphilin using the gene knockout approach (Fig. 1). The granuphilin gene (Grn) is mapped on the X chromosome and therefore is present as a single copy in male mice. Mice heterozygous for the targeted allele in the female (Grn+/−) were obtained by crossing the male chimeras with female C3H/He mice (Grn+/+). Mutant (Grn−/Y) and control wild-type (Grn+/Y) male mice for the experiments were obtained by backcrossing the female Grn+/− mice with male C3H/He mice. In the wild-type mouse islets, granuphilin-a was predominantly expressed relative to the granuphilin-b isoform (Fig. 1 C), in contrast to the roughly equal expression of the two isoforms in the cultured β cell line MIN6 (Yi et al., 2002). Immunoblot and immunofluorescent analyses confirmed the absence of granuphilin-a and -b in the pancreatic β cells of the Grn−/Y mice (Fig. 1 C and not depicted).


Granuphilin molecularly docks insulin granules to the fusion machinery.

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

Generation of Grn knockout mice. (A) Targeted disruption of the granuphilin gene on mouse chromosome X. The targeting vector contains a neomycin resistance gene driven by the pgk promoter (pgk-neo) and a diphtheria toxin A fragment gene driven by the MC1 promoter (DTA) as positive and negative selection markers, respectively. Exon structures are vertically lined and partially shown from the second (Ex2) to sixth exon (Ex6). Homologous recombination results in replacement of the genomic region from the third to fifth exon with pgk-neo. A, ApaI; V, EcoRV; H, HindIII restriction sites. (B) Genomic Southern blot analysis of the backcrossed progenies from a cross of F1 female heterozygotes (Grn+/−) with wild-type (Grn+/Y) C3H/He male. The locations of the 5′ external probe and the 3′ external probe are shown with horizontal closed boxes in A. The 5′ probe hybridizes to a 13.4-kb ApaI fragment from the wild-type locus and to a 16.7-kb fragment from the mutated locus, and the 3′ probe hybridizes to an 8.3-kb EcoRV fragment from the wild-type locus and to an 8.7-kb fragment from the mutated locus, respectively. (C) The protein extracts from pancreatic islets were electrophoresed for immunoblotting with antigranuphilin (αGrp-N) and α-tubulin antibodies. (D) PCR genotyping of the backcrossed progenies. PCR with Grn/Fow1, Grn/Rev1, and Neo/Fow2 mixed primers produces 364- and 760-bp fragments for wild-type and mutated alleles, respectively, as shown in boxes in A.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Generation of Grn knockout mice. (A) Targeted disruption of the granuphilin gene on mouse chromosome X. The targeting vector contains a neomycin resistance gene driven by the pgk promoter (pgk-neo) and a diphtheria toxin A fragment gene driven by the MC1 promoter (DTA) as positive and negative selection markers, respectively. Exon structures are vertically lined and partially shown from the second (Ex2) to sixth exon (Ex6). Homologous recombination results in replacement of the genomic region from the third to fifth exon with pgk-neo. A, ApaI; V, EcoRV; H, HindIII restriction sites. (B) Genomic Southern blot analysis of the backcrossed progenies from a cross of F1 female heterozygotes (Grn+/−) with wild-type (Grn+/Y) C3H/He male. The locations of the 5′ external probe and the 3′ external probe are shown with horizontal closed boxes in A. The 5′ probe hybridizes to a 13.4-kb ApaI fragment from the wild-type locus and to a 16.7-kb fragment from the mutated locus, and the 3′ probe hybridizes to an 8.3-kb EcoRV fragment from the wild-type locus and to an 8.7-kb fragment from the mutated locus, respectively. (C) The protein extracts from pancreatic islets were electrophoresed for immunoblotting with antigranuphilin (αGrp-N) and α-tubulin antibodies. (D) PCR genotyping of the backcrossed progenies. PCR with Grn/Fow1, Grn/Rev1, and Neo/Fow2 mixed primers produces 364- and 760-bp fragments for wild-type and mutated alleles, respectively, as shown in boxes in A.
Mentions: To study the cellular function of granuphilin, we generated a mouse line lacking granuphilin using the gene knockout approach (Fig. 1). The granuphilin gene (Grn) is mapped on the X chromosome and therefore is present as a single copy in male mice. Mice heterozygous for the targeted allele in the female (Grn+/−) were obtained by crossing the male chimeras with female C3H/He mice (Grn+/+). Mutant (Grn−/Y) and control wild-type (Grn+/Y) male mice for the experiments were obtained by backcrossing the female Grn+/− mice with male C3H/He mice. In the wild-type mouse islets, granuphilin-a was predominantly expressed relative to the granuphilin-b isoform (Fig. 1 C), in contrast to the roughly equal expression of the two isoforms in the cultured β cell line MIN6 (Yi et al., 2002). Immunoblot and immunofluorescent analyses confirmed the absence of granuphilin-a and -b in the pancreatic β cells of the Grn−/Y mice (Fig. 1 C and not depicted).

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