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Find video protocols related to scientific articles indexed in Pubmed.
Allosteric Modulation of ?-Arrestin-biased Angiotensin II Type 1 Receptor Signaling by Membrane Stretch.
J. Biol. Chem.
PUBLISHED: 08-28-2014
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It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, ?-arrestin, in a manner consistent with previously identified ?-arrestin-biased ligands. However, the ligand-independent mechanism by which mechanical stretch promotes ?-arrestin-biased signaling remains unknown. Implicit in the concept of biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct ?-arrestin-activating conformations of the AT1R by using ?-arrestin2-biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to ?-arrestin (AT1R-?-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the ?-arrestin-biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-?-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET ?-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize ?-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific ?-arrestin-biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.
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??Zr-huJ591 immuno-PET imaging in patients with advanced metastatic prostate cancer.
Eur. J. Nucl. Med. Mol. Imaging
PUBLISHED: 08-21-2014
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Given the bone tropism of prostate cancer, conventional imaging modalities poorly identify or quantify metastatic disease. (89)Zr-huJ591 positron emission tomography (PET) imaging was performed in patients with metastatic prostate cancer to analyze and validate this as an imaging biomarker for metastatic disease. The purpose of this initial study was to assess safety, biodistribution, normal organ dosimetry, and optimal imaging time post-injection for lesion detection.
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Recent developments in biased agonism.
Curr. Opin. Cell Biol.
PUBLISHED: 04-01-2014
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The classic paradigm of G protein-coupled receptor (GPCR) activation was based on the understanding that agonist binding to a receptor induces or stabilizes a conformational change to an 'active' conformation. In the past decade, however, it has been appreciated that ligands can induce distinct 'active' receptor conformations with unique downstream functional signaling profiles. Building on the initial recognition of the existence of such 'biased ligands', recent years have witnessed significant developments in several areas of GPCR biology. These include increased understanding of structural and biophysical mechanisms underlying biased agonism, improvements in characterization and quantification of ligand efficacy, as well as clinical development of these novel ligands. Here we review recent major developments in these areas over the past several years.
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Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR).
J. Biol. Chem.
PUBLISHED: 03-25-2014
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The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and ?-arrestin (?arr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-?arr2 fusion proteins with their respective molecular efficacies for activating Gq and ?arr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and ?arr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.
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Visualization of arrestin recruitment by a G-protein-coupled receptor.
Nature
PUBLISHED: 01-13-2014
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G-protein-coupled receptors (GPCRs) are critically regulated by ?-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ?2 adrenergic receptor (?2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ?-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-?-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ?2AR-?-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ?2AR and ?-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ?-arrestin 1 to the ?2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ?-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ?-arrestin 1 when coupled to the ?2AR. A molecular model of the ?2AR-?-arrestin signalling complex was made by docking activated ?-arrestin 1 and ?2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.
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Regulation of Beta-2-Adrenergic Receptor Function by Conformationally Selective Single-domain Intrabodies.
Mol. Pharmacol.
PUBLISHED: 12-06-2013
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The biological activity induced by ligand binding to orthosteric or allosteric sites on a GPCR is mediated by stabilization of specific receptor conformations. In the case of the ?2 adrenergic receptor, these ligands are generally small molecule agonists or antagonists. However, recently a monomeric single domain antibody (nanobody) from the Camelid family was found to allosterically bind and stabilize an active conformation of the ?2 adrenergic receptor (?2AR). Here we set out to study the functional interaction of 18 related nanobodies with the ?2 adrenergic receptor to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence related nanobody families with preferences for active (agonist occupied) or inactive (antagonist occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface, therefore we transiently expressed them intracellularly (intrabodies) to test their effects on ?2AR-dependent signaling Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist or antagonist-occupied receptors. When expressed as intrabodies inhibited G-protein activation (cyclic AMP accumulation), GRK-mediated receptor phosphorylation, ?-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, ?-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.
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Biopsy of suspicious bone lesions in patients with a single known malignancy: prevalence of a second malignancy.
AJR Am J Roentgenol
PUBLISHED: 11-23-2013
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OBJECTIVE. The probability that a suspicious bone lesion in a patient with one known malignancy is actually due to a second, previously unknown primary malignancy has been reported to be 2-8%. We sought to determine this prevalence as well as that of benign diagnoses in a larger number of patients in a tertiary cancer center. MATERIALS AND METHODS. The medical records of 482 consecutive patients (254 women and 228 men) with only one known primary malignancy each (excluding nonmelanoma skin cancer) and who underwent biopsy of a suspicious bone lesion were retrospectively reviewed. The results of bone biopsy were classified as benign, metastasis of the known primary malignancy, due to a second primary malignancy, or nondiagnostic or indeterminate. RESULTS. In 103 of 482 (21%) patients, bone biopsy results were benign, 316 (66%) were due to metastases of the known malignancy, 15 (3%) were due to a second malignancy, and 48 (10%) were nondiagnostic or indeterminate. Second malignancies included osteosarcoma (n = 4); soft-tissue sarcoma (n = 2); lymphoma (n = 2); plasma cell malignancy (n = 2); and lung cancer, thyroid cancer, renal cancer, chondrosarcoma, and carcinoma of unknown primary (n = 1 each). CONCLUSION. In 3% of patients with one known malignancy and a suspicious bone lesion, the lesion was due to a previously unknown second malignancy; in 21% of patients, the lesion was benign. Bone biopsy is recommended in the management of patients with one known cancer and a suspicious bone lesion only if the presence of a second malignancy would alter clinical management.
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Arrestins come of age: a personal historical perspective.
Prog Mol Biol Transl Sci
PUBLISHED: 06-15-2013
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Visual arrestin and the two ?-arrestins (1 and 2) were originally discovered 25-30 years ago in the context of their ability to desensitize phosphorylated G protein-coupled receptors (rhodopsin and the ?2-adrenergic receptor, respectively). A fourth retinal-specific member of the family (X-arrestin) was discovered later. Over the past 10-15 years, however, it has become clear that these versatile molecules subserve a host of other roles in modulating and mediating the function of most GPCRs as well as other types of receptors. Functioning as multifunctional adaptor proteins, the ?-arrestins also play prominent roles in receptor endocytosis, signaling, trafficking, and ubiquitination among others. Here, I provide a brief personal perspective on how the field has evolved since its inception and speculate on future directions.
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Conformation guides molecular efficacy in docking screens of activated ?-2 adrenergic G protein coupled receptor.
ACS Chem. Biol.
PUBLISHED: 03-21-2013
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A prospective, large library virtual screen against an activated ?2-adrenergic receptor (?2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated ?2AR structure returned few hits of only marginal potency.
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Musculoskeletal tumors and tumor-like conditions: common and avoidable pitfalls at imaging in patients with known or suspected cancer: Part A: benign conditions that may mimic malignancy.
Int Orthop
PUBLISHED: 01-31-2013
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A wide range of musculoskeletal tumors and tumor-like conditions may be encountered when patients undergo radiological examinations. The imaging features of certain normal, reactive, benign neoplastic, inflammatory, traumatic, and degenerative processes in the musculoskeletal system may mimic malignant tumor; misinterpretation of the imaging findings can lead to inappropriate clinical management of the patient. This review describes and illustrates a number of such mimics that we have commonly encountered in our oncological imaging practice, and provides suggestions for avoiding each of these pitfalls. Because many orthopaedic surgeons interpret radiological images themselves, they need to be as aware as radiologists about these issues.
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Musculoskeletal tumours and tumour-like conditions: common and avoidable pitfalls at imaging in patients with known or suspected cancer: Part B: malignant mimics of benign tumours.
Int Orthop
PUBLISHED: 01-31-2013
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A wide range of musculoskeletal tumours and tumour-like conditions may be encountered when patients undergo radiological examinations. Some malignant musculoskeletal lesions may mimic benign tumours at imaging, being confused with benign cystic lesions or haematomas. Also, inappropriately selected magnetic resonance (MR) image sequences or computed tomography (CT) display windows can lead to misdiagnosis. Many orthopaedic surgeons interpret radiological images themselves, and therefore need to be as aware of these issues as radiologists are. This review describes and illustrates a number of such errors that commonly occur, and provides suggestions for avoiding these pitfalls.
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Myxofibrosarcoma: prevalence and diagnostic value of the "tail sign" on magnetic resonance imaging.
Skeletal Radiol.
PUBLISHED: 01-15-2013
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Myxofibrosarcoma frequently shows curvilinear extensions of high T2 signal that also enhance on magnetic resonance imaging; these "tails" represent fascial extension of tumor at histopathological examination. This study was performed to determine whether the tail sign is helpful in distinguishing myxofibrosarcoma from other myxoid-containing neoplasms.
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Structure of active ?-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.
Nature
PUBLISHED: 01-07-2013
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The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ?-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ?-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ?-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ?-arrestin-1. The structure of the ?-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ?-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the lariat loop implicated in maintaining the inactive state of ?-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ?-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.
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?-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration.
PLoS ONE
PUBLISHED: 01-01-2013
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Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds ?-arrestin-1 and ?-arrestin-2. Previous studies have implicated ?-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the ?-arrestins and found that ?-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that ?-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, ?-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either ?-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the ?-arrestin biased ligand [Sar(1),Ile(4),Ile(8)]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a ?-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by ?-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.
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Percutaneous CT-guided bone biopsy: diagnosis of malignancy in lesions with initially indeterminate biopsy results and CT features associated with diagnostic or indeterminate results.
AJR Am J Roentgenol
PUBLISHED: 11-24-2011
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The purpose of our study was to determine the proportion of bone lesions with indeterminate results after initial percutaneous CT-guided bone biopsy that ultimately are found to be malignant and whether CT features are associated with diagnostic outcomes.
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Molecular mechanism of ?-arrestin-biased agonism at seven-transmembrane receptors.
Annu. Rev. Pharmacol. Toxicol.
PUBLISHED: 09-19-2011
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The concept of biased agonism has recently come to the fore with the realization that seven-transmembrane receptors (7TMRs, also known as G protein-coupled receptors, or GPCRs) activate complex signaling networks and can adopt multiple active conformations upon agonist binding. As a consequence, the "efficacy" of receptors, which was classically considered linear, is now recognized as pluridimensional. Biased agonists selectively stabilize only a subset of receptor conformations induced by the natural "unbiased" ligand, thus preferentially activating certain signaling mechanisms. Such agonists thus reveal the intriguing possibility that one can direct cellular signaling with unprecedented precision and specificity and support the notion that biased agonists may identify new classes of therapeutic agents that have fewer side effects. This review focuses on one particular class of biased ligands that has the ability to alter the balance between G protein-dependent and ?-arrestin-dependent signal transduction.
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Distinct phosphorylation sites on the ?(2)-adrenergic receptor establish a barcode that encodes differential functions of ?-arrestin.
Sci Signal
PUBLISHED: 08-27-2011
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Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with ?-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through ?-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of ?-arrestin engagement may aid in the discovery of therapeutic agents targeting individual ?-arrestin functions. The ?(2)-adrenergic receptor (?(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the ?(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and ?-arrestin-mediated pathways or with a biased ligand that promotes signaling only through ?-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of ?-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited ?-arrestin, thus regulating its functional activities.
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A stress response pathway regulates DNA damage through ?2-adrenoreceptors and ?-arrestin-1.
Nature
PUBLISHED: 07-18-2011
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The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the fight-or-flight response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates ?(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated ?(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of ?-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which ?-adrenergic catecholamines, acting through both Gs-PKA and ?-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, ?-arrestin-1 (ARRB1), activated via ?(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offsprings genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.
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Variability of lung tumor measurements on repeat computed tomography scans taken within 15 minutes.
J. Clin. Oncol.
PUBLISHED: 07-05-2011
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We use changes in tumor measurements to assess response and progression, both in routine care and as the primary objective of clinical trials. However, the variability of computed tomography (CT) -based tumor measurement has not been comprehensively evaluated. In this study, we assess the variability of lung tumor measurement using repeat CT scans performed within 15 minutes of each other and discuss the implications of this variability in a clinical context.
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Quantifying ligand bias at seven-transmembrane receptors.
Mol. Pharmacol.
PUBLISHED: 05-24-2011
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Seven transmembrane receptors (7TMRs), commonly referred to as G protein-coupled receptors, form a large part of the "druggable" genome. 7TMRs can signal through parallel pathways simultaneously, such as through heterotrimeric G proteins from different families, or, as more recently appreciated, through the multifunctional adapters, ?-arrestins. Biased agonists, which signal with different efficacies to a receptors multiple downstream pathways, are useful tools for deconvoluting this signaling complexity. These compounds may also be of therapeutic use because they have distinct functional and therapeutic profiles from "balanced agonists." Although some methods have been proposed to identify biased ligands, no comparison of these methods applied to the same set of data has been performed. Therefore, at this time, there are no generally accepted methods to quantify the relative bias of different ligands, making studies of biased signaling difficult. Here, we use complementary computational approaches for the quantification of ligand bias and demonstrate their application to two well known drug targets, the ?2 adrenergic and angiotensin II type 1A receptors. The strategy outlined here allows a quantification of ligand bias and the identification of weakly biased compounds. This general method should aid in deciphering complex signaling pathways and may be useful for the development of novel biased therapeutic ligands as drugs.
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Multiple ligand-specific conformations of the ?2-adrenergic receptor.
Nat. Chem. Biol.
PUBLISHED: 04-22-2011
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Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins ?-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this biased agonism remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the ?(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.
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Activity of Sorafenib against desmoid tumor/deep fibromatosis.
Clin. Cancer Res.
PUBLISHED: 03-29-2011
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Desmoid tumors (deep fibromatoses) are clonal connective tissue malignancies that do not metastasize, but have a significant risk of local recurrence, and are associated with morbidity and occasionally mortality. Responses of desmoid patients to sorafenib on an expanded access program led us to review our experience.
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?-Arrestin-mediated receptor trafficking and signal transduction.
Trends Pharmacol. Sci.
PUBLISHED: 03-23-2011
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?-Arrestins function as endocytic adaptors and mediate trafficking of a variety of cell-surface receptors, including seven-transmembrane receptors (7TMRs). In the case of 7TMRs, ?-arrestins carry out these tasks while simultaneously inhibiting upstream G-protein-dependent signaling and promoting alternate downstream signaling pathways. The mechanisms by which ?-arrestins interact with a continuously expanding ensemble of protein partners and perform their multiple functions including trafficking and signaling are currently being uncovered. Molecular changes at the level of protein conformation as well as post-translational modifications of ?-arrestins probably form the basis for their dynamic interactions during receptor trafficking and signaling. It is becoming increasingly evident that ?-arrestins, originally discovered as 7TMR adaptor proteins, indeed have much broader and more versatile roles in maintaining cellular homeostasis. In this review paper, we assess the traditional and novel functions of ?-arrestins and discuss the molecular attributes that might facilitate multiple interactions in regulating cell signaling and receptor trafficking.
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Emerging paradigms of ?-arrestin-dependent seven transmembrane receptor signaling.
Trends Biochem. Sci.
PUBLISHED: 03-21-2011
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?-Arrestins, originally discovered to desensitize activated seven transmembrane receptors (7TMRs; also known as G-protein-coupled receptors, GPCRs), are now well established mediators of receptor endocytosis, ubiquitylation and G protein-independent signaling. Recent global analyses of ?-arrestin interactions and ?-arrestin-dependent phosphorylation events have uncovered several previously unanticipated roles of ?-arrestins in a range of cellular signaling events. These findings strongly suggest that the functional roles of ?-arrestins are much broader than currently understood. Biophysical studies aimed at understanding multiple active conformations of the 7TMRs and the ?-arrestins have begun to unravel the mechanistic basis for the diverse functional capabilities of ?-arrestins in cellular signaling.
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?-arrestin deficiency protects against pulmonary fibrosis in mice and prevents fibroblast invasion of extracellular matrix.
Sci Transl Med
PUBLISHED: 03-18-2011
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Idiopathic pulmonary fibrosis is a progressive disease that causes unremitting extracellular matrix deposition with resulting distortion of pulmonary architecture and impaired gas exchange. ?-Arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Here, we examine the role of ?-arrestin1 and ?-arrestin2 in the pathobiology of pulmonary fibrosis. In the bleomycin-induced mouse lung fibrosis model, loss of either ?-arrestin1 or ?-arrestin2 resulted in protection from mortality, inhibition of matrix deposition, and protected lung function. Fibrosis was prevented despite preserved recruitment of inflammatory cells and fibroblast chemotaxis. However, isolated lung fibroblasts from bleomycin-treated ?-arrestin-null mice failed to invade extracellular matrix and displayed altered expression of genes involved in matrix production and degradation. Furthermore, knockdown of ?-arrestin2 in fibroblasts from patients with idiopathic pulmonary fibrosis attenuated the invasive phenotype. These data implicate ?-arrestins as mediators of fibroblast invasion and the development of pulmonary fibrosis, and as a potential target for therapeutic intervention in patients with idiopathic pulmonary fibrosis.
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Phase II study of modified docetaxel, cisplatin, and fluorouracil with bevacizumab in patients with metastatic gastroesophageal adenocarcinoma.
J. Clin. Oncol.
PUBLISHED: 12-28-2010
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To evaluate the safety and efficacy of a modified administration schedule of docetaxel, cisplatin, and fluorouracil (mDCF) with bevacizumab in patients with advanced gastroesophageal malignancies.
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Imaging characteristics of primary osteosarcoma: nonconventional subtypes.
Radiographics
PUBLISHED: 11-13-2010
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Osteosarcoma (OS) is a common primary malignant tumor of bone that produces osteoid matrix. According to the World Health Organization, OS of bone is classified into eight subtypes with distinct biologic behaviors and clinical outcomes: conventional, telangiectatic, small cell, low-grade central, secondary, parosteal, periosteal, and high-grade surface. Imaging plays a crucial role in the diagnosis of each subtype of OS and ultimately in patients survival because the diagnosis is based on a combination of histopathologic and imaging features. Conventional OS is the most common subtype of OS and is readily identified at radiography as an intramedullary mass with immature cloudlike bone formation in the metaphyses of long bones. The imaging features of less common subtypes of primary OS are variable and frequently overlap with those of multiple benign and malignant entities, creating substantial diagnostic challenges. For accurate diagnosis, it is important to be aware of radiographic and cross-sectional imaging features that allow differentiation of each nonconventional subtype of OS from its mimics.
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Therapeutic potential of ?-arrestin- and G protein-biased agonists.
Trends Mol Med
PUBLISHED: 09-27-2010
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Members of the seven-transmembrane receptor (7TMR), or G protein-coupled receptor (GPCR), superfamily represent some of the most successful targets of modern drug therapy, with proven efficacy in the treatment of a broad range of human conditions and disease processes. It is now appreciated that ?-arrestins, once viewed simply as negative regulators of traditional 7TMR-stimulated G protein signaling, act as multifunctional adapter proteins that regulate 7TMR desensitization and trafficking and promote distinct intracellular signals in their own right. Moreover, several 7TMR biased agonists, which selectively activate these divergent signaling pathways, have been identified. Here we highlight the diversity of G protein- and ?-arrestin-mediated functions and the therapeutic potential of selective targeting of these in disease states.
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International Workshop at the Nobel Forum, Karolinska Institutet on G protein-coupled receptors: finding the words to describe monomers, oligomers, and their molecular mechanisms and defining their meaning. Can a consensus be reached?
J. Recept. Signal Transduct. Res.
PUBLISHED: 09-23-2010
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A meeting was held May 19, 2010 at the Karolinski Institute on Nomenclature in Pharmacology. This meeting occurred in conjunction with the Symposium The Changing World of G Protein Coupled Receptors: From Monomers to Dimers and Receptor Mosaics (Higher-order Oligomers) held the previous day at the Royal Swedish Academy of Science. Two broad topics of nomenclature were discussed; ligand nomenclature and the definition of receptor-receptor interactions. This paper summarizes discussions on these topics along with a consensus definition of the term receptor-receptor interaction.
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Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-04-2010
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beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.
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Arresting a transient receptor potential (TRP) channel: beta-arrestin 1 mediates ubiquitination and functional down-regulation of TRPV4.
J. Biol. Chem.
PUBLISHED: 07-22-2010
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?-Arrestins, originally discovered to desensitize activated G protein-coupled receptors, (aka seven-transmembrane receptors, 7TMRs) also mediate 7TMR internalization and G protein-independent signaling via these receptors. More recently, several regulatory roles of ?-arrestins for atypical 7TMRs and non-7TM receptors have emerged. Here, we uncover an entirely novel regulatory role of ?-arrestins in cross-talk between the angiotensin receptor (AT1aR) and a member of the transient receptor potential (TRP) ion channel family, TRPV4. AT1aR and TRPV4 form a constitutive complex in the plasma membrane, and angiotensin stimulation leads to recruitment of ?-arrestin 1 to this complex. Surprisingly, angiotensin stimulation results in ubiquitination of TRPV4, a process that requires ?-arrestin 1, and subsequently to internalization and functional down-regulation of TRPV4. ?-Arrestin 1 interacts with, and acts as an adaptor for AIP4, an E3 ubiquitin ligase responsible for TRPV4 ubiquitination. Thus, our data provide the first evidence of a functional link between ?-arrestins and TRPV4 and uncovers an entirely novel mechanism to maintain appropriate intracellular Ca(2+) concentration to avoid excessive Ca(2+) signaling.
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Teaching old receptors new tricks: biasing seven-transmembrane receptors.
Nat Rev Drug Discov
PUBLISHED: 05-01-2010
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Seven-transmembrane receptors (7TMRs; also known as G protein-coupled receptors) are the largest class of receptors in the human genome and are common targets for therapeutics. Originally identified as mediators of 7TMR desensitization, beta-arrestins (arrestin 2 and arrestin 3) are now recognized as true adaptor proteins that transduce signals to multiple effector pathways. Signalling that is mediated by beta-arrestins has distinct biochemical and functional consequences from those mediated by G proteins, and several biased ligands and receptors have been identified that preferentially signal through either G protein- or beta-arrestin-mediated pathways. These ligands are not only useful tools for investigating the biochemistry of 7TMR signalling, they also have the potential to be developed into new classes of therapeutics.
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A phase I pharmacokinetic study of pulse-dose vorinostat with flavopiridol in solid tumors.
Invest New Drugs
PUBLISHED: 04-05-2010
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Vorinostat (V) at levels >2.5 µM enhances chemotherapy in vitro. Yet the approved oral dose of 400 mg inconsistently achieves this level in patients. We developed an intermittent oral pulse-dose schedule of V to increase serum levels. We combined V with the cyclin dependent kinase inhibitor flavopiridol (F) which increases V-induced apoptosis.
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A phase I clinical trial of FOLFIRI in combination with the pan-cyclin-dependent kinase (CDK) inhibitor flavopiridol.
Cancer Chemother. Pharmacol.
PUBLISHED: 02-02-2010
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The cyclin-dependent kinase inhibitor flavopiridol increases irinotecan- and fluorouracil-induced apoptosis. We conducted a phase I trial of FOLFIRI + flavopiridol in patients with advanced solid tumors.
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Beta-arrestin- but not G protein-mediated signaling by the "decoy" receptor CXCR7.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-17-2009
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Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through beta-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both beta-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling "decoys" because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through beta-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or beta-arrestin depletion by siRNA. This example of an endogenous "beta-arrestin-biased" 7TMR that signals through beta-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through beta-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.
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Phase I study of flavopiridol with oxaliplatin and fluorouracil/leucovorin in advanced solid tumors.
Clin. Cancer Res.
PUBLISHED: 11-24-2009
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Flavopiridol, a cyclin-dependent kinase inhibitor, has promising clinical activity when combined with chemotherapy. Preclinical data indicate that flavopiridol enhances oxaliplatin- and fluorouracil (5FU)-induced apoptosis in a sequence-dependent manner.
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Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways.
Dev. Cell
PUBLISHED: 10-27-2009
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Arrestins were identified as mediators of G protein-coupled receptor (GPCR) desensitization and endocytosis. However, it is now clear that they scaffold many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors--including those relevant to the Hedgehog, Wnt, Notch, and TGFbeta pathways--and downstream kinases such as the MAPK and Akt/PI3K cascades. The involvement of arrestins in many discrete developmental signaling events suggests an indispensable role for these multifaceted molecular scaffolds.
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G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.
J. Biol. Chem.
PUBLISHED: 10-02-2009
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Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3beta, a cytosolic kinase for phosphorylation of a downstream effector beta-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of beta-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished beta-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.
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A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation.
Sci Transl Med
PUBLISHED: 08-04-2009
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About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through beta-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp(12),Tyr(34))-PTH(7-34) (PTH-betaarr), which activates beta-arrestin but not classic G protein signaling. In mice, PTH-betaarr induces anabolic bone formation, as does the nonselective agonist PTH(1-34), which activates both mechanisms. In beta-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-betaarr is ablated. The beta-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the beta-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.
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Imaging findings, prevalence and outcome of de novo and secondary malignant fibrous histiocytoma of bone.
Skeletal Radiol.
PUBLISHED: 07-18-2009
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To evaluate the radiographic and magnetic resonance (MR) imaging features of primary and secondary malignant fibrous histiocytoma in bone and determine the demographics, prevalence and outcome of patients with this tumor.
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Oxygen-regulated beta(2)-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL.
Sci Signal
PUBLISHED: 07-09-2009
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Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although beta-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the beta(2)-adrenergic receptor (beta(2)AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the beta(2)AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with beta(2)AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the beta(2)AR to serve as an endogenous beta(2)AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous beta(2)AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.
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Evaluating variability in tumor measurements from same-day repeat CT scans of patients with non-small cell lung cancer.
Radiology
PUBLISHED: 06-30-2009
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To evaluate the variability of tumor unidimensional, bidimensional, and volumetric measurements on same-day repeat computed tomographic (CT) scans in patients with non-small cell lung cancer.
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Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-02-2009
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CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ss-arrestin system. CCL19 leads to robust CCR7 phosphorylation and beta-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to beta-arrestin2 recruitment, only CCL19 leads to redistribution of beta-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and beta-arrestin2 to ERK kinases. Thus, this mechanism for "ligand bias" whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of beta-arrestin.
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A phase II study of flavopiridol (Alvocidib) in combination with docetaxel in refractory, metastatic pancreatic cancer.
Pancreatology
PUBLISHED: 05-19-2009
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Pancreatic adenocarcinoma (PC) harbors frequent alterations in p16, resulting in cell cycle dysregulation. A phase I study of docetaxel and flavopiridol, a pan-cyclin-dependent kinase inhibitor, demonstrated encouraging clinical activity in PC. This phase II study was designed to further define the efficacy and toxicity of this regimen in patients with previously treated PC.
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Beta-arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-10-2009
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Beta-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seven-transmembrane receptors (7TMRs). Agonist-stimulated ubiquitination of beta-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid beta(2)-adrenergic receptor (beta(2)AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds beta-arrestin2 and leads to the deubiquitination of beta-arrestins. USP33 and Mdm2 function reciprocally and favor respectively the stability or lability of the receptor beta-arrestin complex, thus regulating the longevity and subcellular localization of receptor signalosomes. Receptors such as the beta(2)AR, previously shown to form loose complexes with beta-arrestin ("class A") promote a beta-arrestin conformation conducive for binding to the deubiquitinase, whereas the vasopressin V2R, which forms tight beta-arrestin complexes ("class B"), promotes a distinct beta-arrestin conformation that favors dissociation of the enzyme. Thus, USP33-beta-arrestin interaction is a key regulatory step in 7TMR trafficking and signal transmission from the activated receptors to downstream effectors.
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Local changes in bone marrow at MRI after treatment of extremity soft tissue sarcoma.
Skeletal Radiol.
PUBLISHED: 04-01-2009
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To determine the prevalence and appearance of magnetic resonance imaging (MRI) signal changes that occur in local bone marrow after radiation therapy (RT) and/or chemotherapy for extremity soft tissue sarcoma (STS).
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Independent beta-arrestin2 and Gq/protein kinase Czeta pathways for ERK stimulated by angiotensin type 1A receptors in vascular smooth muscle cells converge on transactivation of the epidermal growth factor receptor.
J. Biol. Chem.
PUBLISHED: 03-02-2009
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Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and beta-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the beta-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated beta-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of beta-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Czeta. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. beta-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and beta-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.
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beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.
J. Clin. Invest.
PUBLISHED: 02-18-2009
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Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.
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{beta}-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation.
J. Biol. Chem.
PUBLISHED: 01-26-2009
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beta-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that beta-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate beta-arrestin but not G protein-dependent signaling, and are abrogated by beta-arrestin-2 small interfering RNA. These findings establish a key role for beta-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved.
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Pharmacological blockade of a ?(2)AR-?-arrestin-1 signaling cascade prevents the accumulation of DNA damage in a behavioral stress model.
Cell Cycle
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Chronic stress is known to have a profound negative impact on human health and has been suggested to influence a number of disease states. However, the mechanisms underlying the deleterious effects of stress remain largely unknown. Stress is known to promote the release of epinephrine, a catecholamine stress hormone that binds to ?(2)-adrenergic receptors (?(2)ARs) with high affinity. Our previous work has demonstrated that chronic stimulation of a ?(2)AR-?-arrestin-1-mediated signaling pathway by infusion of isoproterenol suppresses p53 levels and impairs genomic integrity. In this pathway, ?-arrestin-1, which is activated via ?(2)ARs, facilitates the AKT-mediated activation of Mdm2 and functions as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, Mdm2. Here, we show that chronic restraint stress in mice recapitulates the effects of isoproterenol infusion to reduce p53 levels and results in the accumulation of DNA damage in the frontal cortex of the brain, two effects that are abrogated by the ?-blocker, propranolol and by genetic deletion of ?-arrestin-1. These data suggest that the ?(2)AR-?-arrestin-1 signaling pathway may represent an attractive therapeutic target to prevent some of the negative consequences of stress in the treatment of stress-related disorders.
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Renal cortical tumors: use of multiphasic contrast-enhanced MR imaging to differentiate benign and malignant histologic subtypes.
Radiology
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To investigate the use of quantitative multiphasic contrast material-enhanced magnetic resonance (MR) imaging in differentiating between common benign and malignant histologic subtypes of renal cortical tumors.
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?-Arrestin2 mediates the initiation and progression of myeloid leukemia.
Proc. Natl. Acad. Sci. U.S.A.
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?-Arrestins were initially discovered as negative regulators of G protein-coupled receptor signaling. Although ?-arrestins have more recently been implicated as scaffold proteins that interact with various mitogenic and developmental signals, the genetic role of ?-arrestins in driving oncogenesis is not known. Here we have investigated the role of ?-arrestin in hematologic malignancies and have found that although both ?-arrestin1 and -2 are expressed in the hematopoietic system, loss of ?-arrestin2 preferentially leads to a severe impairment in the establishment and propagation of the chronic and blast crisis phases of chronic myelogenous leukemia (CML). These defects are linked to a reduced frequency, as well as defective self-renewal capacity of the cancer stem-cell population, in mouse models and in human CML patient samples. At a molecular level, the loss of ?-arrestin2 leads to a significant inhibition of ?-catenin stabilization, and ectopic activation of Wnt signaling reverses the defects observed in the ?-arrestin2 mutant cells. These data cumulatively show that ?-arrestin2 is essential for CML disease propagation and indicate that ?-arrestins and the Wnt/?-catenin pathway lie in a signaling hierarchy in the context of CML cancer stem cell maintenance.
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Competing G protein-coupled receptor kinases balance G protein and ?-arrestin signaling.
Mol. Syst. Biol.
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Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through ?-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on ?-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.