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Nigel P Birch - One of the best experts on this subject based on the ideXlab platform.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and Cellular Endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-ΔTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-ΔTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.
    Biochemistry, 2003
    Co-Authors: Irina Arnaoutova, Angela M Smith, Leigh C Coates, Nigel P Birch, Juanita C. Sharpe, Savita Dhanvantari, Chris R. Snell, Y Peng Loh
    Abstract:

    The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Y Peng Loh - One of the best experts on this subject based on the ideXlab platform.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and Cellular Endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-ΔTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-ΔTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.
    Biochemistry, 2003
    Co-Authors: Irina Arnaoutova, Angela M Smith, Leigh C Coates, Nigel P Birch, Juanita C. Sharpe, Savita Dhanvantari, Chris R. Snell, Y Peng Loh
    Abstract:

    The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Angela M Smith - One of the best experts on this subject based on the ideXlab platform.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and Cellular Endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-ΔTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-ΔTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.
    Biochemistry, 2003
    Co-Authors: Irina Arnaoutova, Angela M Smith, Leigh C Coates, Nigel P Birch, Juanita C. Sharpe, Savita Dhanvantari, Chris R. Snell, Y Peng Loh
    Abstract:

    The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Leigh C Coates - One of the best experts on this subject based on the ideXlab platform.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and Cellular Endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-ΔTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-ΔTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The prohormone processing enzyme PC3 is a lipid raft-associated transmembrane protein.
    Biochemistry, 2003
    Co-Authors: Irina Arnaoutova, Angela M Smith, Leigh C Coates, Nigel P Birch, Juanita C. Sharpe, Savita Dhanvantari, Chris R. Snell, Y Peng Loh
    Abstract:

    The biosynthesis of most biologically active peptides involves the action of prohomone convertases, including PC3 (also known as PC1), that catalyze limited proteolysis of precursor proteins. Proteolysis of prohormones occurs mainly in the granules of the regulated secretory pathway. It has been proposed that the targeting of these processing enzymes to secretory granules involves their association with lipid rafts in granule membranes. We now provide evidence for the interaction of the 86 and 64 kDa forms of PC3 with secretory granule membranes. Furthermore, both forms of PC3 were resistant to extraction with TX-100, were floated to low-density fractions in sucrose gradients, and were partially extracted upon cholesterol depletion by methyl-beta-cyclodextrin, indicating that they were associated with lipid rafts in the membranes. Protease protection assays, immunolabeling, and biotinylation of proteins in intact secretory granules identified an approximately 115-residue cytoplasmic tail for 86 kDa PC3. Using two-dimensional gel electrophoresis and a specific antibody, a novel, raft-associated form of 64 kDa PC3 that contains a transmembrane domain consisting of residues 619-638 was identified. This form was designated as 64 kDa PC3-TM, and differs from the 64 kDa mature form of PC3. We present a model of the membrane topology of PC3, where it is anchored to lipid rafts in secretory granule membranes via the transmembrane domain. We demonstrate that the transmembrane domain of PC3 alone was sufficient to target the extracellular domain of the IL2 receptor alpha-subunit (Tac) to secretory granules.

Hong Lou - One of the best experts on this subject based on the ideXlab platform.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and cellular endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

  • The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.
    Molecular and Cellular Endocrinology, 2006
    Co-Authors: Hong Lou, Angela M Smith, Leigh C Coates, Niamh X Cawley, Y Peng Loh, Nigel P Birch
    Abstract:

    The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617–638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC31–638 (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC31–616 (PC3-616) and PC3-ΔTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-ΔTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.