The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Narin Osman - One of the best experts on this subject based on the ideXlab platform.
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Suramin inhibits PDGF-stimulated Receptor Phosphorylation, proteoglycan synthesis and glycosaminoglycan hyperelongation in human vascular smooth muscle cells
The Journal of pharmacy and pharmacology, 2013Co-Authors: Peter J. Little, Muhamad Ashraf Rostam, Terrence J. Piva, Robel Getachew, Danielle Kamato, Daniel Guidone, Mandy L. Ballinger, Wenhua Zheng, Narin OsmanAbstract:Objectives: Suramin is a polysulfonated naphthylurea with antiparasitic and potential antineoplastic activity. Suramin's pharmacological actions, which have not yet been fully elucidated, include antagonism of the action of platelet-derived growth factor (PDGF) at its Receptor. We investigated the effects of suramin on PDGF-stimulated proteoglycan synthesis. Methods: Human vascular smooth muscle cells (VSMCs) were incubated in the presence and absence of PDGF and suramin with [H]thymidine or SO as radiolabels. Mitogenic response was determined by [H]thymidine incorporation. PDGFβ Receptor Phosphorylation was assessed by western blotting. Proteoglycan size and glycosaminoglycan chain synthesis and size were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Alphascreen phosphotyrosine assay kit was used to investigate PDGFβ Receptor tyrosine kinase inhibition by suramin. Key findings: Suramin decreased PDGF-stimulated proliferation, proteoglycan synthesis and GAG chain hyperelongation. Suramin also directly inhibited PDGFβ Receptor kinase activity as well as PDGFβ Receptor Phosphorylation in intact VSMCs. Conclusions: These data show that inhibition of PDGFβ Receptor Phosphorylation in intact cells is necessary to define a fully active PDGF antagonist. They also confirm that PDGFβ Receptor kinase activity is necessary for PDGF-mediated atherogenic changes in proteoglycan synthesis and support efforts to develop PDGFβ Receptor antagonists as potential anti-atherosclerotic agents.
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Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFβ Receptor Phosphorylation.
Archives of biochemistry and biophysics, 2012Co-Authors: Peter J. Little, Robel Getachew, Mandy L. Ballinger, Wenhua Zheng, Hossein Babaahmadi Rezaei, Estella Sanchez-guerrero, Levon M. Khachigian, Haitao Wang, Sufen Liao, Narin OsmanAbstract:The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated Receptor Phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block Phosphorylation of the activation site of the PDGF Receptor at Tyr857 and two other downstream sites Tyr 751 and Tyr1021. Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF Receptor Phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF Receptor and that PDGF mediates GAG elongation via the PDGF Receptor.
Horace H. Loh - One of the best experts on this subject based on the ideXlab platform.
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FK506-Binding Protein 12 Modulates μ-Opioid Receptor Phosphorylation and Protein Kinase Cε–Dependent Signaling by Its Direct Interaction with the Receptor
Molecular pharmacology, 2013Co-Authors: Yu Qiu, Horace H. Loh, Wei Zhao, Yan Wang, Eddie Huie, Shan Jiang, Ying Hui Yan, Hong Zhuan Chen, Ping-yee LawAbstract:Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid Receptor (OPRM1) desensitization and tolerance development. It was recently shown that Receptor Phosphorylation by G protein–coupled Receptor kinase regulates agonist-dependent selective signaling and that inefficient Phosphorylation of OPRM1 leads to PKCe activation and subsequent responses. Here, we demonstrate that such Receptor Phosphorylation and PKCe activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro353 residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced Receptor Phosphorylation at Ser375. The morphine-induced PKCe activation and the recruitment of PKCe to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKCe. Further analysis of the Receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the Receptor. The blockade of the FKBP12 association with the Receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro353 to Ala resulted in a reduction in PKCe and calcineurin recruitment to the Receptor signaling complex. The Receptor-associated calcineurin modulates OPRM1 Phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the Receptor Phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the Phosphorylation of the Receptor and triggers the recruitment and activation of PKCe.
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Modulating μ-Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKCϵ Activation and Dendritic Spine Stability
The Journal of biological chemistry, 2011Co-Authors: Hui Zheng, Ji Chu, Horace H. Loh, Yuhan Zhang, Ping-yee LawAbstract:A new role of G protein-coupled Receptor (GPCR) Phosphorylation was demonstrated in the current studies by using the μ-opioid Receptor (OPRM1) as a model. Morphine induces a low level of Receptor Phosphorylation and uses the PKCϵ pathway to induce ERK Phosphorylation and Receptor desensitization, whereas etorphine, fentanyl, and [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive Receptor Phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 Phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCϵ pathway. This was not due to the decreased recruitment of β-arrestin2 to the Receptor signaling complex, because these agonists were unable to use the PKCϵ pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled Receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCϵ, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCϵ. Furthermore, by overexpressing wild-type OPRM1 and a Phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that Receptor Phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent Phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.
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Morphine-induced μ-opioid Receptor rapid desensitization is independent of Receptor Phosphorylation and β-arrestins
Cellular signalling, 2008Co-Authors: Ji Chu, Horace H. Loh, Hui Zheng, Ping-yee LawAbstract:Abstract Receptor desensitization involving Receptor Phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid Receptor (OPRM1). However, the roles of Receptor Phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca 2+ ([Ca 2+ ] i )release to monitor Receptor activation, as predicted, [D-Ala 2 , N -Me-Phe 4 , Gly 5 -ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a Receptor Phosphorylation- and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when Phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca 2+ ] i release to a greater extent than that of DAMGO and could induce the Phosphorylation of Ser 375 residue, morphine-induced desensitization was not influenced by mutating the Phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr.
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μ-Opioid Receptor Desensitization ROLE OF Receptor Phosphorylation, INTERNALIZATION, AND RESENSITIZATION
The Journal of biological chemistry, 2003Co-Authors: Yu Qiu, Ping-yee Law, Horace H. LohAbstract:Abstract It is generally accepted that the internalization and desensitization of μ-opioid Receptor (MOR) involves Receptor Phosphorylation and β-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo Phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced Phosphorylation sites, did not exhibit detectable agonist-induced Phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of β-arrestin 2 when compared with wild type MOR, but the level of Receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type Receptors recycled back to membrane after agonist-induced internalization, which contributed to the Receptor resensitization and thus partially reversed the Receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the Receptor internalization and the recycling of internalized Receptor to cell surface in an active state. Taken together, our data indicated that Receptor Phosphorylation is not absolutely required in the internalization, but Receptor Phosphorylation and subsequent β-arrestin recruitment play important roles in the resensitization of internalized Receptors.
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The Absence of a Direct Correlation between the Loss of [d-Ala2,MePhe4,Gly5-ol]Enkephalin Inhibition of Adenylyl Cyclase Activity and Agonist-induced μ-Opioid Receptor Phosphorylation
The Journal of biological chemistry, 1999Co-Authors: Rachid El Kouhen, Ping-yee Law, Odile Maestri El Kouhen, Horace H. LohAbstract:Chronic activation of the mu-opioid Receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the Receptor. It has been suggested that opioid Receptor Phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent Phosphorylation of the Receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the Receptor, but is not involved in DAMGO-induced MOR1TAG Phosphorylation. The rapid rate of Receptor Phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between Receptor Phosphorylation and the loss of response was further demonstrated when Receptor Phosphorylation was increased by either calyculin A or overexpression of the G-protein Receptor kinases. Calyculin A increased the magnitude of MOR1TAG Phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) Receptors were expressed in the same system, overexpression of beta-adrenergic Receptor kinase 2 elevated agonist-induced Phosphorylation for both Receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic Receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced Receptor desensitization. Thus, these data show that Phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the Receptor.
Ping-yee Law - One of the best experts on this subject based on the ideXlab platform.
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FK506-Binding Protein 12 Modulates μ-Opioid Receptor Phosphorylation and Protein Kinase Cε–Dependent Signaling by Its Direct Interaction with the Receptor
Molecular pharmacology, 2013Co-Authors: Yu Qiu, Horace H. Loh, Wei Zhao, Yan Wang, Eddie Huie, Shan Jiang, Ying Hui Yan, Hong Zhuan Chen, Ping-yee LawAbstract:Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid Receptor (OPRM1) desensitization and tolerance development. It was recently shown that Receptor Phosphorylation by G protein–coupled Receptor kinase regulates agonist-dependent selective signaling and that inefficient Phosphorylation of OPRM1 leads to PKCe activation and subsequent responses. Here, we demonstrate that such Receptor Phosphorylation and PKCe activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro353 residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced Receptor Phosphorylation at Ser375. The morphine-induced PKCe activation and the recruitment of PKCe to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKCe. Further analysis of the Receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the Receptor. The blockade of the FKBP12 association with the Receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro353 to Ala resulted in a reduction in PKCe and calcineurin recruitment to the Receptor signaling complex. The Receptor-associated calcineurin modulates OPRM1 Phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the Receptor Phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the Phosphorylation of the Receptor and triggers the recruitment and activation of PKCe.
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Modulating μ-Opioid Receptor Phosphorylation Switches Agonist-dependent Signaling as Reflected in PKCϵ Activation and Dendritic Spine Stability
The Journal of biological chemistry, 2011Co-Authors: Hui Zheng, Ji Chu, Horace H. Loh, Yuhan Zhang, Ping-yee LawAbstract:A new role of G protein-coupled Receptor (GPCR) Phosphorylation was demonstrated in the current studies by using the μ-opioid Receptor (OPRM1) as a model. Morphine induces a low level of Receptor Phosphorylation and uses the PKCϵ pathway to induce ERK Phosphorylation and Receptor desensitization, whereas etorphine, fentanyl, and [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive Receptor Phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 Phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCϵ pathway. This was not due to the decreased recruitment of β-arrestin2 to the Receptor signaling complex, because these agonists were unable to use the PKCϵ pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled Receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCϵ, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCϵ. Furthermore, by overexpressing wild-type OPRM1 and a Phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that Receptor Phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent Phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.
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Morphine-induced μ-opioid Receptor rapid desensitization is independent of Receptor Phosphorylation and β-arrestins
Cellular signalling, 2008Co-Authors: Ji Chu, Horace H. Loh, Hui Zheng, Ping-yee LawAbstract:Abstract Receptor desensitization involving Receptor Phosphorylation and subsequent βArrestin (βArr) recruitment has been implicated in the tolerance development mediated by μ-opioid Receptor (OPRM1). However, the roles of Receptor Phosphorylation and βArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca 2+ ([Ca 2+ ] i )release to monitor Receptor activation, as predicted, [D-Ala 2 , N -Me-Phe 4 , Gly 5 -ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a Receptor Phosphorylation- and βArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when Phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from βArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from βArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca 2+ ] i release to a greater extent than that of DAMGO and could induce the Phosphorylation of Ser 375 residue, morphine-induced desensitization was not influenced by mutating the Phosphorylation sites or in MEF cells lacking βArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of βArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of βArr.
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μ-Opioid Receptor Desensitization ROLE OF Receptor Phosphorylation, INTERNALIZATION, AND RESENSITIZATION
The Journal of biological chemistry, 2003Co-Authors: Yu Qiu, Ping-yee Law, Horace H. LohAbstract:Abstract It is generally accepted that the internalization and desensitization of μ-opioid Receptor (MOR) involves Receptor Phosphorylation and β-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo Phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced Phosphorylation sites, did not exhibit detectable agonist-induced Phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of β-arrestin 2 when compared with wild type MOR, but the level of Receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type Receptors recycled back to membrane after agonist-induced internalization, which contributed to the Receptor resensitization and thus partially reversed the Receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the Receptor internalization and the recycling of internalized Receptor to cell surface in an active state. Taken together, our data indicated that Receptor Phosphorylation is not absolutely required in the internalization, but Receptor Phosphorylation and subsequent β-arrestin recruitment play important roles in the resensitization of internalized Receptors.
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The Absence of a Direct Correlation between the Loss of [d-Ala2,MePhe4,Gly5-ol]Enkephalin Inhibition of Adenylyl Cyclase Activity and Agonist-induced μ-Opioid Receptor Phosphorylation
The Journal of biological chemistry, 1999Co-Authors: Rachid El Kouhen, Ping-yee Law, Odile Maestri El Kouhen, Horace H. LohAbstract:Chronic activation of the mu-opioid Receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the Receptor. It has been suggested that opioid Receptor Phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO) induced a time- and concentration-dependent Phosphorylation of the Receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the Receptor, but is not involved in DAMGO-induced MOR1TAG Phosphorylation. The rapid rate of Receptor Phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between Receptor Phosphorylation and the loss of response was further demonstrated when Receptor Phosphorylation was increased by either calyculin A or overexpression of the G-protein Receptor kinases. Calyculin A increased the magnitude of MOR1TAG Phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when mu- and delta-opioid (DOR1TAG) Receptors were expressed in the same system, overexpression of beta-adrenergic Receptor kinase 2 elevated agonist-induced Phosphorylation for both Receptors. However, in the same cell lines under the same conditions, overexpression of beta-adrenergic Receptor kinase 2 and beta-arrestin 2 accelerated the rate of DPDPE- but not DAMGO-induced Receptor desensitization. Thus, these data show that Phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the Receptor.
Abdul B. Abou-samra - One of the best experts on this subject based on the ideXlab platform.
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Disruption of parathyroid hormone and parathyroid hormone-related peptide Receptor Phosphorylation prolongs ERK1/2 MAPK activation and enhances c-fos expression.
American journal of physiology. Endocrinology and metabolism, 2012Co-Authors: Hesham A. Tawfeek, Abdul B. Abou-samraAbstract:Previous studies have demonstrated that parathyroid hormone (PTH) binding to the PTH/PTH-related peptide Receptor (PPR) stimulates G protein coupling, Receptor Phosphorylation, β-arrestin transloca...
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Negative regulation of parathyroid hormone (PTH)-activated phospholipase C by PTH/PTH-related peptide Receptor Phosphorylation and protein kinase A.
Endocrinology, 2008Co-Authors: Hesham A. Tawfeek, Abdul B. Abou-samraAbstract:PTH binding to the PTH/PTHrP Receptor activates adenylate cyclase/protein kinase A (PKA) and phospholipase C (PLC) pathways and increases Receptor Phosphorylation. The mechanisms regulating PTH activation of PLC signaling are poorly understood. In the current study, we explored the role of PTH/PTHrP Receptor Phosphorylation and PKA in PTH activation of PLC. When treated with PTH, LLCPK-1 cells stably expressing a green fluorescent protein (GFP)-tagged wild-type (WT) PTH/PTHrP Receptor show a small dose-dependent increase in PLC signaling as measured by inositol trisphosphate accumulation assay. In contrast, PTH treatment of LLCPK-1 cells stably expressing a GFP-tagged Receptor mutated in its carboxyl-terminal tail so that it cannot be phosphorylated (PD-GFP) results in significantly higher PLC activation (P < 0.001). The effects of PTH on PLC activation are dose dependent and reach maximum at the 100 nm PTH dose. When WT Receptor-expressing cells are pretreated with H89, a specific inhibitor of PKA, PTH activation of PLC signaling is enhanced in a dose-dependent manner. H89 pretreatment in PD-GFP cells causes a further increase in PLC activation in response to PTH treatment. Interestingly, PTH and forskolin (adenylate cyclase/PKA pathway activator) treatment causes an increase in PLCβ3 Phosphorylation at the Ser1105 inhibitory site and that increase is blocked by the PKA inhibitor, H89. Expression of a mutant PLCβ3 in which Ser1105 was mutated to alanine (PLCβ3-SA), in WT or PD cells increases PTH stimulation of inositol 1,4,5-trisphosphate formation. Altogether, these data suggest that PTH signaling to PLC is negatively regulated by PTH/PTHrP Receptor Phosphorylation and PKA. Furthermore, Phosphorylation at Ser1105 is demonstrated as a regulatory mechanism of PLCβ3 by PKA.
Trudy A Kohout - One of the best experts on this subject based on the ideXlab platform.
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differential desensitization Receptor Phosphorylation β arrestin recruitment and erk1 2 activation by the two endogenous ligands for the cc chemokine Receptor 7
Journal of Biological Chemistry, 2004Co-Authors: Trudy A Kohout, Stephen J. Perry, Greg J. Reinhart, Sachiko Junger, Shelby L Nicholas, Scott R StruthersAbstract:Many members of the chemokine Receptor family of G protein-coupled Receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single Receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of Receptor Phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced Phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase Phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated Phosphorylation of the Receptor, whereas residues serine 356, 357, 364, and 365 are important for basal Receptor Phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 Phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the Phosphorylation sites in the tail of the Receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the Receptor and activation of ERK1/2, and another that is impaired in these functions.
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differential desensitization Receptor Phosphorylation beta arrestin recruitment and erk1 2 activation by the two endogenous ligands for the cc chemokine Receptor 7
Journal of Biological Chemistry, 2004Co-Authors: Trudy A Kohout, Shelby Nicholas, Stephen J. Perry, Greg J. Reinhart, Sachiko Junger, Scott R StruthersAbstract:Many members of the chemokine Receptor family of G protein-coupled Receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single Receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of Receptor Phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced Phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase Phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated Phosphorylation of the Receptor, whereas residues serine 356, 357, 364, and 365 are important for basal Receptor Phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 Phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the Phosphorylation sites in the tail of the Receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the Receptor and activation of ERK1/2, and another that is impaired in these functions.
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Differential desensitization, Receptor Phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine Receptor 7
The Journal of biological chemistry, 2004Co-Authors: Trudy A Kohout, Shelby Nicholas, Stephen J. Perry, Greg J. Reinhart, Sachiko Junger, R. Scott StruthersAbstract:Many members of the chemokine Receptor family of G protein-coupled Receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single Receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of Receptor Phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced Phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase Phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated Phosphorylation of the Receptor, whereas residues serine 356, 357, 364, and 365 are important for basal Receptor Phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 Phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the Phosphorylation sites in the tail of the Receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the Receptor and activation of ERK1/2, and another that is impaired in these functions.
