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Peter A. Friedman - One of the best experts on this subject based on the ideXlab platform.
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Site-specific polyubiquitination differentially regulates Parathyroid Hormone Receptor–initiated MAPK signaling and cell proliferation
The Journal of biological chemistry, 2018Co-Authors: Qiangmin Zhang, W. Bruce Sneddon, Alessandro Bisello, Kunhong Xiao, Jennifer Mcgarvey, Hongda Liu, Lei Song, Peter A. FriedmanAbstract:G protein-coupled Receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR Parathyroid Hormone Receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.
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site specific polyubiquitination differentially regulates Parathyroid Hormone Receptor initiated mapk signaling and cell proliferation
Journal of Biological Chemistry, 2018Co-Authors: Qiangmin Zhang, Alessandro Bisello, Kunhong Xiao, Jennifer Mcgarvey, Hongda Liu, Lei Song, Bruce W Sneddon, Peter A. FriedmanAbstract:G protein-coupled Receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR Parathyroid Hormone Receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.
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Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor
Biochemistry, 2017Co-Authors: Tatyana Mamonova, Alessandro Bisello, Qiangmin Zhang, Mintu Chandra, Brett M. Collins, Edward Sarfo, Kunhong Xiao, Peter A. FriedmanAbstract:Na+/H+ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the Parathyroid Hormone Receptor (PTHR), a family B G-protein-coupled Receptor. PTHR and NHERF1 bind through a PDZ–ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1–34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ–ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ–PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions −3, −5, and −6 upstream of the ...
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Structural Basis for NHERF1 PDZ Domain Binding
Biochemistry, 2012Co-Authors: Tatyana Mamonova, Maria Kurnikova, Peter A. FriedmanAbstract:The Na+/H+ exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The Parathyroid Hormone Receptor (...
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dynamic na h exchanger regulatory factor 1 association and dissociation regulate Parathyroid Hormone Receptor trafficking at membrane microdomains
Journal of Biological Chemistry, 2011Co-Authors: Juan A. Ardura, Bin Wang, Jean-pierre Vilardaga, Simon C. Watkins, Peter A. FriedmanAbstract:Abstract Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic Postsynaptic density 95/Disc large/Zona occludens (PDZ) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled Receptors (GPCR) and other membrane-delimited proteins. The Parathyroid Hormone Receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a GPCR harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live-cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon Receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the Receptor and induces Receptor aggregation in long-lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the Receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates Receptor trafficking and signaling in a spatially and temporally coordinated manner.
Jean-pierre Vilardaga - One of the best experts on this subject based on the ideXlab platform.
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Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism
Journal of the American Chemical Society, 2019Co-Authors: Shi Liu, Frederic Jean-alphonse, Alex D. White, Denise Wootten, Patrick M. Sexton, Thomas J. Gardella, Jean-pierre Vilardaga, Samuel H. GellmanAbstract:The type-1 Parathyroid Hormone Receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, Parathyroid Hormone (PTH) and PTH-related protein (PTHrP). PTH ...
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Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.
The Journal of biological chemistry, 2016Co-Authors: Jennifer Mcgarvey, W. Bruce Sneddon, Alessandro Bisello, Frederic Jean-alphonse, Tatyana Mamonova, Qiangmin Zhang, Kunhong Xiao, Shanna L. Bowman, Juan A. Ardura, Jean-pierre VilardagaAbstract:The G protein-coupled Parathyroid Hormone Receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon Parathyroid Hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface Receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.
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International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors
Pharmacological reviews, 2015Co-Authors: Thomas J. Gardella, Jean-pierre VilardagaAbstract:The type-1 Parathyroid Hormone Receptor (PTHR1) is a family B G protein–coupled Receptor (GPCR) that mediates the actions of two polypeptide ligands; Parathyroid Hormone (PTH), an endocrine Hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 Parathyroid Hormone Receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these Receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic “two-site” mode of ligand binding now thought to be used by each of the family B peptide Hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall Receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gαs/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH Receptors.
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dynamic na h exchanger regulatory factor 1 association and dissociation regulate Parathyroid Hormone Receptor trafficking at membrane microdomains
Journal of Biological Chemistry, 2011Co-Authors: Juan A. Ardura, Bin Wang, Jean-pierre Vilardaga, Simon C. Watkins, Peter A. FriedmanAbstract:Abstract Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic Postsynaptic density 95/Disc large/Zona occludens (PDZ) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled Receptors (GPCR) and other membrane-delimited proteins. The Parathyroid Hormone Receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a GPCR harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live-cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon Receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the Receptor and induces Receptor aggregation in long-lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the Receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates Receptor trafficking and signaling in a spatially and temporally coordinated manner.
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Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains
The Journal of biological chemistry, 2011Co-Authors: Juan A. Ardura, Bin Wang, Jean-pierre Vilardaga, Simon C. Watkins, Peter A. FriedmanAbstract:Abstract Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic Postsynaptic density 95/Disc large/Zona occludens (PDZ) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled Receptors (GPCR) and other membrane-delimited proteins. The Parathyroid Hormone Receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a GPCR harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live-cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon Receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the Receptor and induces Receptor aggregation in long-lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the Receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates Receptor trafficking and signaling in a spatially and temporally coordinated manner.
Thomas J. Gardella - One of the best experts on this subject based on the ideXlab platform.
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The Parathyroid Hormone Receptor Type 1
Osteoporosis, 2020Co-Authors: Thomas J. GardellaAbstract:This chapter describes the molecular and structural properties of the Parathyroid Hormone Receptor type 1 (PTHR1), with emphasis on mechanisms of ligand binding and signal activation, as well as therapeutic applications. The basic protein architecture and mechanistic actions of the PTHR1 can now be viewed in light of the recently reported X-ray crystal and cryo-EM structures obtained for this Receptor, which provide insights at an atomic level of resolution. These new structures confirm that the PTHR1 adopts an overall protein topology similar to that seen in peptide Hormone-binding G protein-coupled Receptors that comprise the class B GPCR subgroup and that it furthermore utilizes a similar basic mechanism of ligand binding and activation. Pharmacologic studies on the PTHR1 have led to the concept that structurally distinct PTH and PTHrP ligands and analogs can bind with altered affinities to different PTHR1 conformations to thus induce different types of signaling responses, including prolonged cAMP responses from endosomes. These distinct modes of action suggest potential new paths to explore for therapeutic ligand development. A number of diseases are linked to mutations in the genes for the PTHR1 or its ligands, PTH and PTHrP, and the evolving approaches for developing new PTHR1 ligands, including small molecules, may lead to new modes of treatment for such diseases.
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Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism
Journal of the American Chemical Society, 2019Co-Authors: Shi Liu, Frederic Jean-alphonse, Alex D. White, Denise Wootten, Patrick M. Sexton, Thomas J. Gardella, Jean-pierre Vilardaga, Samuel H. GellmanAbstract:The type-1 Parathyroid Hormone Receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, Parathyroid Hormone (PTH) and PTH-related protein (PTHrP). PTH ...
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Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution
Journal of medicinal chemistry, 2017Co-Authors: Ross W. Cheloha, Thomas J. Gardella, Tomoyuki Watanabe, Bingming Chen, Niyanta N. Kumar, Robert G. Thorne, Samuel H. GellmanAbstract:The Parathyroid Hormone Receptor 1 (PTHR1) is a member of the B-family of GPCRs; these Receptors are activated by long polypeptide Hormones and constitute targets of drug development efforts. Parathyroid Hormone (PTH, 84 residues) and PTH-related protein (PTHrP, 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1–34), is used clinically to treat osteoporosis. Conventional peptides in the 20–40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1–ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with β-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α → β replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis, in cases remaining active after extended exposure to simulated gastric fluid. The...
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G-Protein-coupled Receptors: Decoding mixed signals.
Nature chemical biology, 2017Co-Authors: Thomas J. GardellaAbstract:A new mechanism of functional crosstalk between two distinct G-protein-coupled Receptors (GPCRs)—the Parathyroid Hormone Receptor (PTHR) and β2-adrenergic Receptor (β2 Ar)—that occurs at the level of G protein βγ subunits and a specific adenylyl cyclase isoform is identified. This crosstalk augments cAMP signaling by the PTHR from endosomes, and thus promotes the actions of PTH ligands in bone target cells.
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Backbone Modification of a Parathyroid Hormone Receptor-1 Antagonist/Inverse Agonist.
ACS chemical biology, 2016Co-Authors: Ross W. Cheloha, Samuel H. Gellman, Tomoyuki Watanabe, Thomas R. Dean, Thomas J. GardellaAbstract:A backbone-modified peptide derived from Parathyroid Hormone (PTH) is shown to function as an inhibitor and inverse agonist of Parathyroid Hormone Receptor-1 (PTHR1) signaling. This Receptor acts to regulate calcium and phosphate homeostasis, as well as bone turnover and development. PTH is a natural agonist of PTHR1, and PTH(1–34) displays full activity relative to the natural 84-residue Hormone. PTH(1–34) is used clinically to treat osteoporosis. N-terminally truncated derivatives of PTH(1–34), such as PTH(7–34), are known to bind to PTHR1 without initiating intracellular signaling and can thus act as competitive antagonists of PTH-induced signaling at PTHR1. In some cases, N-terminally truncated PTH derivatives also act as inverse agonists of PTHR1 variants that display pathologically high levels of signaling in the absence of PTH. Many analogues of PTH, however, are rapidly degraded by proteases, which may limit biomedical application. We show that backbone modification via periodic replacement of α-a...
Alessandro Bisello - One of the best experts on this subject based on the ideXlab platform.
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Site-specific polyubiquitination differentially regulates Parathyroid Hormone Receptor–initiated MAPK signaling and cell proliferation
The Journal of biological chemistry, 2018Co-Authors: Qiangmin Zhang, W. Bruce Sneddon, Alessandro Bisello, Kunhong Xiao, Jennifer Mcgarvey, Hongda Liu, Lei Song, Peter A. FriedmanAbstract:G protein-coupled Receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR Parathyroid Hormone Receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.
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site specific polyubiquitination differentially regulates Parathyroid Hormone Receptor initiated mapk signaling and cell proliferation
Journal of Biological Chemistry, 2018Co-Authors: Qiangmin Zhang, Alessandro Bisello, Kunhong Xiao, Jennifer Mcgarvey, Hongda Liu, Lei Song, Bruce W Sneddon, Peter A. FriedmanAbstract:G protein-coupled Receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR Parathyroid Hormone Receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.
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Origins of PDZ Binding Specificity. A Computational and Experimental Study Using NHERF1 and the Parathyroid Hormone Receptor
Biochemistry, 2017Co-Authors: Tatyana Mamonova, Alessandro Bisello, Qiangmin Zhang, Mintu Chandra, Brett M. Collins, Edward Sarfo, Kunhong Xiao, Peter A. FriedmanAbstract:Na+/H+ exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the Parathyroid Hormone Receptor (PTHR), a family B G-protein-coupled Receptor. PTHR and NHERF1 bind through a PDZ–ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1–34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ–ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ–PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions −3, −5, and −6 upstream of the ...
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Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.
The Journal of biological chemistry, 2016Co-Authors: Jennifer Mcgarvey, W. Bruce Sneddon, Alessandro Bisello, Frederic Jean-alphonse, Tatyana Mamonova, Qiangmin Zhang, Kunhong Xiao, Shanna L. Bowman, Juan A. Ardura, Jean-pierre VilardagaAbstract:The G protein-coupled Parathyroid Hormone Receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon Parathyroid Hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface Receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.
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nherf1 regulates Parathyroid Hormone Receptor membrane retention without affecting recycling
Journal of Biological Chemistry, 2007Co-Authors: Bin Wang, Alessandro Bisello, Yanmei Yang, Guillermo Romero, Peter A. FriedmanAbstract:Na/H exchange regulatory factor-1 (NHERF1) is a PDZ protein that regulates trafficking of several G protein-coupled Receptors. The phenotype of NHERF1-null mice suggests that the Parathyroid Hormone (PTH) Receptor (PTH1R) is the principal GPCR interacting with NHERF1. The effect of NHERF1 on Receptor recycling is unknown. Here, we characterized NHERF1 effects on PTH1R membrane tethering and recycling by radio-ligand binding and recovery after maximal Receptor endocytosis. Using Chinese hamster ovary cells expressing the PTH1R, where NHERF1 expression could be induced by tetracycline, NHERF1 inhibited PTH1R endocytosis and delayed PTH1R recycling. NHERF1 also inhibited PTH-induced Receptor internalization in MC4 osteoblast cells. Reducing constitutive NHERF1 levels in HEK-293 cells with short hairpin RNA directed against NHERF1 augmented PTH1R endocytosis in response to PTH. Mutagenesis of the PDZ-binding domains or deletion of the MERM domain of NHERF1 demonstrated that both are required for inhibition of endocytosis and recycling. Likewise, an intact COOH-terminal PDZ recognition motif in PTH1R is needed. The effect of NHERF1 on Receptor internalization and recycling was not associated with altered Receptor expression or binding, activation, or phosphorylation but involved beta-arrestin and dynamin. We conclude that NHERF1 inhibits endocytosis without affecting PTH1R recycling in MC4 and PTH1R-expressing HEK-293 cells. Such an effect may protect against PTH resistance or PTH1R down-regulation in certain cells harboring NHERF1.
Bin Wang - One of the best experts on this subject based on the ideXlab platform.
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dynamic na h exchanger regulatory factor 1 association and dissociation regulate Parathyroid Hormone Receptor trafficking at membrane microdomains
Journal of Biological Chemistry, 2011Co-Authors: Juan A. Ardura, Bin Wang, Jean-pierre Vilardaga, Simon C. Watkins, Peter A. FriedmanAbstract:Abstract Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic Postsynaptic density 95/Disc large/Zona occludens (PDZ) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled Receptors (GPCR) and other membrane-delimited proteins. The Parathyroid Hormone Receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a GPCR harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live-cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon Receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the Receptor and induces Receptor aggregation in long-lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the Receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates Receptor trafficking and signaling in a spatially and temporally coordinated manner.
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Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains
The Journal of biological chemistry, 2011Co-Authors: Juan A. Ardura, Bin Wang, Jean-pierre Vilardaga, Simon C. Watkins, Peter A. FriedmanAbstract:Abstract Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic Postsynaptic density 95/Disc large/Zona occludens (PDZ) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled Receptors (GPCR) and other membrane-delimited proteins. The Parathyroid Hormone Receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a GPCR harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live-cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon Receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the Receptor and induces Receptor aggregation in long-lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the Receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates Receptor trafficking and signaling in a spatially and temporally coordinated manner.
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Formation of a Ternary Complex among NHERF1, β-Arrestin, and Parathyroid Hormone Receptor
The Journal of biological chemistry, 2010Co-Authors: Christoph Klenk, Peter A. Friedman, Jean-pierre Vilardaga, Bin Wang, Thorsten Vetter, Alexander Zürn, Martin J. LohseAbstract:β-Arrestins are crucial regulators of G-protein coupled Receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted Receptor phosphorylation is required for β-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in β-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate Receptor signaling and trafficking. Na+/H+ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, β-arrestin2, and the Parathyroid Hormone Receptor type 1 (PTHR). We show that NHERF1 and β-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, β-arrestin2 binding is promoted by Receptor activation. NHERF1 interacts directly with β-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and β-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing β-arrestin2 into close proximity to the PTHR, thereby facilitating β-arrestin2 recruitment after Receptor activation.
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A naturally occurring isoform inhibits Parathyroid Hormone Receptor trafficking and signaling
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2010Co-Authors: Verónica Alonso, W. Bruce Sneddon, Bin Wang, Juan A. Ardura, Peter A. FriedmanAbstract:Parathyroid Hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive Receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both Receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional Receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.
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Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits
The Journal of biological chemistry, 2010Co-Authors: Bin Wang, Guillermo Romero, Juan A. Ardura, Yanmei S. Yang, Randy A. Hall, Peter A. FriedmanAbstract:The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The Parathyroid Hormone Receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [35S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance Receptor-mediated stimulation of Gαq but have no effect on stimulation of Gαi or Gαs. In contrast, PTHR associations with NHERF2 enhance Receptor-mediated stimulation of both Gαq and Gαi but decrease stimulation of Gαs. Consistent with these functional data, NHERF2 formed cellular complexes with both Gαq and Gαi, whereas NHERF1 was found to interact only with Gαq. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.
