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Nigel W. Bunnett - One of the best experts on this subject based on the ideXlab platform.

  • Endothelin Converting Enzyme 1 actions determine differential trafficking and signaling of corticotropin releasing factor receptor 1 at high agonist concentrations
    Molecular Endocrinology, 2012
    Co-Authors: Burcu Hasdemir, Nigel W. Bunnett, Shilpi Mahajan, Min Liao, Aditi Bhargava
    Abstract:

    CRF receptor 1 (CRF1), a key neuroendocrine mediator of the stress response, has two known agonists corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1). Here we report that Endothelin-Converting Enzyme-1 (ECE-1) differentially degrades CRF and Ucn1; ECE-1 cleaves Ucn1, but not CRF, at critical residue Arginine-34/35′, which is essential for ligand-receptor binding. At near KD agonist concentration (30 nm), both Ucn1- and CRF-mediated Ca2+ mobilization are ECE-1 dependent. Interestingly, at high agonist concentration (100 nm), Ucn1-mediated Ca2+ mobilization remains ECE-1 dependent, whereas CRF-mediated mobilization becomes independent of ECE-1 activity. At high agonist concentration, ECE-1 inhibition disrupted Ucn1-, but not CRF-induced CRF1 recycling and resensitization, but did not prolong the association of CRF1 with β-arrestins. RNA interference-mediated knockdown of Rab suggests that both Ucn1- and CRF-induced CRF1 resensitization is dependent on activity of Rab11, but not of Rab4. CRF1 behav...

  • endosomal Endothelin Converting Enzyme 1 a regulator of β arrestin dependent erk signaling
    Journal of Biological Chemistry, 2009
    Co-Authors: Graeme S. Cottrell, Dirk Roosterman, Benjamin E. Padilla, Martin Steinhoff, Silvia Amadesi, Daniel P Poole, Jane E Murphy, Markus Hardt, Nigel W. Bunnett
    Abstract:

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that Endothelin-Converting Enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

  • Endothelin Converting Enzyme 1 promotes re sensitization of neurokinin 1 receptor dependent neurogenic inflammation
    British Journal of Pharmacology, 2009
    Co-Authors: Fiore Cattaruzza, Graeme S. Cottrell, Natalya Vaksman, Nigel W. Bunnett
    Abstract:

    Background and purpose:  The metalloendopeptidase Endothelin-Converting Enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big Endothelin to Endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK1) receptor. We investigated whether ECE-1 regulates NK1 receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium. Experimental approach:  We examined ECE-1 expression, SP trafficking and NK1 receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats. Key results:  HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK1 receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization. Conclusions and implications:  By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK1 receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.

  • Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
    Endocrinology, 2008
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Nigel W. Bunnett, Cordula Kempkes, Christoph W. Turck, Martin Steinhoff
    Abstract:

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), Endothelin-Converting Enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 Converting Enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

Martin Steinhoff - One of the best experts on this subject based on the ideXlab platform.

  • endosomal Endothelin Converting Enzyme 1 a regulator of β arrestin dependent erk signaling
    Journal of Biological Chemistry, 2009
    Co-Authors: Graeme S. Cottrell, Dirk Roosterman, Benjamin E. Padilla, Martin Steinhoff, Silvia Amadesi, Daniel P Poole, Jane E Murphy, Markus Hardt, Nigel W. Bunnett
    Abstract:

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that Endothelin-Converting Enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

  • Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
    Endocrinology, 2008
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Nigel W. Bunnett, Cordula Kempkes, Christoph W. Turck, Martin Steinhoff
    Abstract:

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), Endothelin-Converting Enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 Converting Enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and beta arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

  • Endothelin-Converting Enzyme 1 degrades neuropeptides in endosomes to control receptor recycling
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Laurent Muller, Christopher B. Eckman, Nigel W. Bunnett, Martin Steinhoff
    Abstract:

    Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase Endothelin-Converting Enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK1R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK1R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal Enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Graeme S. Cottrell - One of the best experts on this subject based on the ideXlab platform.

  • endosomal Endothelin Converting Enzyme 1 a regulator of β arrestin dependent erk signaling
    Journal of Biological Chemistry, 2009
    Co-Authors: Graeme S. Cottrell, Dirk Roosterman, Benjamin E. Padilla, Martin Steinhoff, Silvia Amadesi, Daniel P Poole, Jane E Murphy, Markus Hardt, Nigel W. Bunnett
    Abstract:

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that Endothelin-Converting Enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

  • Endothelin Converting Enzyme 1 promotes re sensitization of neurokinin 1 receptor dependent neurogenic inflammation
    British Journal of Pharmacology, 2009
    Co-Authors: Fiore Cattaruzza, Graeme S. Cottrell, Natalya Vaksman, Nigel W. Bunnett
    Abstract:

    Background and purpose:  The metalloendopeptidase Endothelin-Converting Enzyme 1 (ECE-1) is prominently expressed in the endothelium where it converts big Endothelin to Endothelin-1, a vasoconstrictor peptide. Although ECE-1 is found in endosomes in endothelial cells, the role of endosomal ECE-1 is unclear. ECE-1 degrades the pro-inflammatory neuropeptide substance P (SP) in endosomes to promote recycling and re-sensitization of its neurokinin 1 (NK1) receptor. We investigated whether ECE-1 regulates NK1 receptor re-sensitization and the pro-inflammatory effects of SP in the endothelium. Experimental approach:  We examined ECE-1 expression, SP trafficking and NK1 receptor re-sensitization in human microvascular endothelial cells (HMEC-1), and investigated re-sensitization of SP-induced plasma extravasation in rats. Key results:  HMEC-1 expressed all four ECE-1 isoforms (a-d), and fluorescent SP trafficked to early endosomes containing ECE-1b/d. The ECE-1 inhibitor SM-19712 prevented re-sensitization of SP-induced Ca2+ signals in HMEC-1 cells. Immunoreactive ECE-1 and NK1 receptors co-localized in microvascular endothelial cells in the rat. SP-induced extravasation of Evans blue in the urinary bladder, skin and ears of the rat desensitized when the interval between two SP injections was 10 min, and re-sensitized after 480 min. SM-19712 inhibited this re-sensitization. Conclusions and implications:  By degrading endocytosed SP, ECE-1 promotes the recycling and re-sensitization of NK1 receptors in endothelial cells, and thereby induces re-sensitization of the pro-inflammatory effects of SP. Thus, ECE-1 inhibitors may ameliorate the pro-inflammatory actions of SP.

  • Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
    Endocrinology, 2008
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Nigel W. Bunnett, Cordula Kempkes, Christoph W. Turck, Martin Steinhoff
    Abstract:

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), Endothelin-Converting Enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 Converting Enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and beta arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

Benjamin E. Padilla - One of the best experts on this subject based on the ideXlab platform.

  • endosomal Endothelin Converting Enzyme 1 a regulator of β arrestin dependent erk signaling
    Journal of Biological Chemistry, 2009
    Co-Authors: Graeme S. Cottrell, Dirk Roosterman, Benjamin E. Padilla, Martin Steinhoff, Silvia Amadesi, Daniel P Poole, Jane E Murphy, Markus Hardt, Nigel W. Bunnett
    Abstract:

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that Endothelin-Converting Enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

  • Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
    Endocrinology, 2008
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Nigel W. Bunnett, Cordula Kempkes, Christoph W. Turck, Martin Steinhoff
    Abstract:

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), Endothelin-Converting Enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 Converting Enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and beta arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

  • Endothelin-Converting Enzyme 1 degrades neuropeptides in endosomes to control receptor recycling
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Laurent Muller, Christopher B. Eckman, Nigel W. Bunnett, Martin Steinhoff
    Abstract:

    Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase Endothelin-Converting Enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK1R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK1R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal Enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Dirk Roosterman - One of the best experts on this subject based on the ideXlab platform.

  • endosomal Endothelin Converting Enzyme 1 a regulator of β arrestin dependent erk signaling
    Journal of Biological Chemistry, 2009
    Co-Authors: Graeme S. Cottrell, Dirk Roosterman, Benjamin E. Padilla, Martin Steinhoff, Silvia Amadesi, Daniel P Poole, Jane E Murphy, Markus Hardt, Nigel W. Bunnett
    Abstract:

    Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and β-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. β-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that Endothelin-Converting Enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, β-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls β-arrestin-dependent signaling of endocytosed GPCRs.

  • Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
    Endocrinology, 2008
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Nigel W. Bunnett, Cordula Kempkes, Christoph W. Turck, Martin Steinhoff
    Abstract:

    Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with β-arrestin1-enhanced green fluorescence protein (EGFP), Endothelin-Converting Enzyme-1 (ECE-1), and rab5a. Internalized 125I-Tyr11-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized 125I-Tyr1-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A1. ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 Converting Enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and β arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.

  • Endothelin Converting Enzyme 1 regulates endosomal sorting of calcitonin receptor like receptor and beta arrestins
    Journal of Cell Biology, 2007
    Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W. Bunnett
    Abstract:

    Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of Endothelin-Converting Enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

  • Endothelin-Converting Enzyme 1 degrades neuropeptides in endosomes to control receptor recycling
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Dirk Roosterman, Graeme S. Cottrell, Benjamin E. Padilla, Laurent Muller, Christopher B. Eckman, Nigel W. Bunnett, Martin Steinhoff
    Abstract:

    Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with β-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide–receptor–arrestin complex is sequestered into endosomes. Although dissociation of β-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase Endothelin-Converting Enzyme 1 (ECE-1), determines stability of the peptide–receptor–arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of β-arrestins followed by trafficking of the SP–NK1R–β-arrestin complex to early endosomes containing ECE-1a–d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; β-arrestins returned to the cytosol, and the NK1R, freed from β-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide–receptor–arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control β-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal Enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.