The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Masayuki Komada - One of the best experts on this subject based on the ideXlab platform.
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Regulation of epidermal growth factor Receptor down Regulation by ubpy mediated deubiquitination at endosomes
Molecular Biology of the Cell, 2005Co-Authors: Emi Mizuno, Takanobu Iura, Akiko Mukai, Tamotsu Yoshimori, Naomi Kitamura, Masayuki KomadaAbstract:Ligand-activated Receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “Receptor down-Regulation” process is crucial to terminate the cell proliferation signals produced by activated Receptors. During the process, ubiquitination of the Receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-Regulation of epidermal growth factor (EGF) Receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated Receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-Regulation by deubiquitinating EGFR on endosomes.
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Regulation of epidermal growth factor Receptor down-Regulation by UBPY-mediated deubiquitination at endosomes.
Molecular biology of the cell, 2005Co-Authors: Emi Mizuno, Takanobu Iura, Akiko Mukai, Tamotsu Yoshimori, Naomi Kitamura, Masayuki KomadaAbstract:Ligand-activated Receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “Receptor down-Regulation” process is crucial to terminate the cell proliferation signals produced by activated Receptors. During the process, ubiquitination of the Receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-Regulation of epidermal growth factor (EGF) Receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated Receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-Regulation by deubiquitinating EGFR on endosomes.
John A. Williams - One of the best experts on this subject based on the ideXlab platform.
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down-Regulation of the G-proteins G(q)α and G11α by transfected human M3 muscarinic acetylcholine Receptors in Chinese hamster ovary cells is independent of Receptor down-Regulation
Biochemical Journal, 1995Co-Authors: E. M. A. Van De Westerlo, Jun Yang, C. Logsdon, John A. WilliamsAbstract:Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine Receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 Receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This Receptor, however, induces Gq alpha/G11 alpha down-Regulation similarly to wild-type M3 Receptors, indicating that G-protein down-Regulation is not directly coupled to Receptor down-Regulation. Thus down-Regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.
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Human m3 muscarinic acetylcholine Receptor carboxyl-terminal threonine residues are required for agonist-induced Receptor down-Regulation
Molecular pharmacology, 1993Co-Authors: Jun Yang, Craig D. Logsdon, Teit E. Johansen, John A. WilliamsAbstract:The mechanisms involved in agonist-induced down-Regulation of the human m3 muscarinic acetylcholine Receptor were investigated by site-directed mutagenesis of the Receptor cytoplasmic carboxyl terminus. Threonine residues (Thr550,553,554) were converted into alanines collectively and individually. The mutated and wild-type Receptor cDNAs stably expressed in Chinese hamster ovary cells displayed similar antagonist- and agonist-binding properties. Furthermore, mutant Receptors showed the same efficacy and potency for carbachol-induced activation of phosphoinositide hydrolysis as did the wild-type clone. In all cases the maximal increase in phosphoinositide hydrolysis was 8-9-fold. In contrast to normal intracellular signaling, however, the mutant Receptor with all three threonines changed to alanines (Ala550,553,554) failed to undergo normal down-Regulation in response to carbachol. After a 24-hr incubation in the presence of 1 mM carbachol, subsequent N-[3H]methylscopolamine binding was reduced by 66% for the wild-type clone but by only 12% for the mutant Receptor. The Ala553,554 mutant also showed a profound reduction in Receptor down-Regulation. Subsequent studies showed that a small but significant blockage of Receptor down-Regulation also could be produced by converting a single threonine residue (Thr553) to alanine. The fact that these effects were not due to nonspecific conformational changes was suggested by the lack of effects on binding, signal transduction, and down-Regulation of converting Thr550 to alanine or converting two cysteine residues (Cys561,563) to glycines in an adjacent region. A similar reduction in Receptor number also was observed in binding studies using the membrane-permeant ligand [3H]scopolamine. These results show that threonine residues in the carboxyl-terminal domain of the human m3 muscarinic acetylcholine Receptor are important in agonist-induced Receptor down-Regulation.
Jun Yang - One of the best experts on this subject based on the ideXlab platform.
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muscarinic acetylcholine Receptor down Regulation limits the extent of inhibition of cell cycle progression in chinese hamster ovary cells
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Katharina Detjen, Jun Yang, Craig D. LogsdonAbstract:Abstract Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-Regulation-resistant mutant m3 muscarinic Receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-Regulation of m3 muscarinic acetylcholine Receptors was the principal mechanism of desensitization of Receptor-activated inositol phosphate phospholipid hydrolysis in these cells. Activation of wild-type and mutant Receptors inhibited anchorage-independent growth as assayed by colony formation in agar. However, the potency for inhibition of anchorage-independent growth was greater for cells expressing the mutant Receptor. Activation of either Receptor also initially inhibited anchorage-dependent cell proliferation in randomly growing populations. Rates of DNA synthesis and cell division were profoundly reduced by carbachol in cells expressing either Receptor at early time points. Analysis of cell cycle parameters indicated that cell cycle progression was inhibited at transitions from G1 to S and G2/M to G1 phases. However, mutant Receptor effects on anchorage-dependent growth were sustained, whereas wild-type Receptor effects were transient. Thus, Receptor down-Regulation restored cell cycle progression. In contrast, activation of either Receptor blocked entry into the cell cycle from quiescence, and this response was not reduced by Receptor down-Regulation. Therefore, activation of m3 muscarinic acetylcholine Receptors inhibited CHO cell anchorage-dependent and -independent growth. In anchored cells carbachol inhibited the cell cycle at three distinct points. Inhibitions at two of these points were eliminated by wild-type Receptor down-Regulation while the other was not. These results directly demonstrate that desensitization mechanisms can act as principal determinants of cellular growth responses.
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down-Regulation of the G-proteins G(q)α and G11α by transfected human M3 muscarinic acetylcholine Receptors in Chinese hamster ovary cells is independent of Receptor down-Regulation
Biochemical Journal, 1995Co-Authors: E. M. A. Van De Westerlo, Jun Yang, C. Logsdon, John A. WilliamsAbstract:Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine Receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 Receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This Receptor, however, induces Gq alpha/G11 alpha down-Regulation similarly to wild-type M3 Receptors, indicating that G-protein down-Regulation is not directly coupled to Receptor down-Regulation. Thus down-Regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure.
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Human m3 muscarinic acetylcholine Receptor carboxyl-terminal threonine residues are required for agonist-induced Receptor down-Regulation
Molecular pharmacology, 1993Co-Authors: Jun Yang, Craig D. Logsdon, Teit E. Johansen, John A. WilliamsAbstract:The mechanisms involved in agonist-induced down-Regulation of the human m3 muscarinic acetylcholine Receptor were investigated by site-directed mutagenesis of the Receptor cytoplasmic carboxyl terminus. Threonine residues (Thr550,553,554) were converted into alanines collectively and individually. The mutated and wild-type Receptor cDNAs stably expressed in Chinese hamster ovary cells displayed similar antagonist- and agonist-binding properties. Furthermore, mutant Receptors showed the same efficacy and potency for carbachol-induced activation of phosphoinositide hydrolysis as did the wild-type clone. In all cases the maximal increase in phosphoinositide hydrolysis was 8-9-fold. In contrast to normal intracellular signaling, however, the mutant Receptor with all three threonines changed to alanines (Ala550,553,554) failed to undergo normal down-Regulation in response to carbachol. After a 24-hr incubation in the presence of 1 mM carbachol, subsequent N-[3H]methylscopolamine binding was reduced by 66% for the wild-type clone but by only 12% for the mutant Receptor. The Ala553,554 mutant also showed a profound reduction in Receptor down-Regulation. Subsequent studies showed that a small but significant blockage of Receptor down-Regulation also could be produced by converting a single threonine residue (Thr553) to alanine. The fact that these effects were not due to nonspecific conformational changes was suggested by the lack of effects on binding, signal transduction, and down-Regulation of converting Thr550 to alanine or converting two cysteine residues (Cys561,563) to glycines in an adjacent region. A similar reduction in Receptor number also was observed in binding studies using the membrane-permeant ligand [3H]scopolamine. These results show that threonine residues in the carboxyl-terminal domain of the human m3 muscarinic acetylcholine Receptor are important in agonist-induced Receptor down-Regulation.
Emi Mizuno - One of the best experts on this subject based on the ideXlab platform.
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Regulation of epidermal growth factor Receptor down Regulation by ubpy mediated deubiquitination at endosomes
Molecular Biology of the Cell, 2005Co-Authors: Emi Mizuno, Takanobu Iura, Akiko Mukai, Tamotsu Yoshimori, Naomi Kitamura, Masayuki KomadaAbstract:Ligand-activated Receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “Receptor down-Regulation” process is crucial to terminate the cell proliferation signals produced by activated Receptors. During the process, ubiquitination of the Receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-Regulation of epidermal growth factor (EGF) Receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated Receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-Regulation by deubiquitinating EGFR on endosomes.
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Regulation of epidermal growth factor Receptor down-Regulation by UBPY-mediated deubiquitination at endosomes.
Molecular biology of the cell, 2005Co-Authors: Emi Mizuno, Takanobu Iura, Akiko Mukai, Tamotsu Yoshimori, Naomi Kitamura, Masayuki KomadaAbstract:Ligand-activated Receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This “Receptor down-Regulation” process is crucial to terminate the cell proliferation signals produced by activated Receptors. During the process, ubiquitination of the Receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-Regulation of epidermal growth factor (EGF) Receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated Receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-Regulation by deubiquitinating EGFR on endosomes.
Lakshmi A Devi - One of the best experts on this subject based on the ideXlab platform.
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thr located within the cooh terminal tail of the opiate Receptor is involved in Receptor down Regulation
Journal of Biological Chemistry, 1996Co-Authors: Svetlana Cvejic, Nino Trapaidze, Curt Cyr, Lakshmi A DeviAbstract:Abstract Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of Receptors, or down-Regulation. The molecular mechanism of the opiate Receptor down-Regulation is not known. In order to address this, we generated a number of mutants of the opiate Receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the Receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated Receptor does not show down-Regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type Receptor and the Receptor with the deletion of only 15 COOH-terminal residues show substantial down-Regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the Receptor down-Regulation. In order to identify residues that play a key role in down-Regulation, point mutations of residues within this region were examined for their ability to modulate Receptor down-Regulation. The Receptor with a mutation of Thr to Ala does not down-regulate, whereas the Receptor with a mutation of Ser to Gly down-regulates with a time course similar to that of the wild type Receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-Regulation and that Thr is critical for the agonist-mediated down-Regulation of the opiate Receptor.
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Thr353, Located within the COOH-terminal Tail of the ´ Opiate Receptor, Is Involved in Receptor down-Regulation
The Journal of biological chemistry, 1996Co-Authors: Svetlana Cvejic, Nino Trapaidze, Curt Cyr, Lakshmi A DeviAbstract:Abstract Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of Receptors, or down-Regulation. The molecular mechanism of the opiate Receptor down-Regulation is not known. In order to address this, we generated a number of mutants of the opiate Receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the Receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated Receptor does not show down-Regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type Receptor and the Receptor with the deletion of only 15 COOH-terminal residues show substantial down-Regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the Receptor down-Regulation. In order to identify residues that play a key role in down-Regulation, point mutations of residues within this region were examined for their ability to modulate Receptor down-Regulation. The Receptor with a mutation of Thr to Ala does not down-regulate, whereas the Receptor with a mutation of Ser to Gly down-regulates with a time course similar to that of the wild type Receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-Regulation and that Thr is critical for the agonist-mediated down-Regulation of the opiate Receptor.
