R J Lefkowitz

Duke University, Durham, North Carolina, United States

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Publications (869)7630.41 Total impact

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    ABSTRACT: Angiotensin II type 1a receptor (AT1aR)-mediated activation of mitogen-activated protein kinases (MAPKs) contributes to thoracic aortic aneurysm (TAA) development in Marfan Syndrome (MFS). β-arrestin2 (βarr2) is known to mediate AT1aR-dependent MAPK activation as well as pro-proliferative and pro-fibrotic signaling in aortic vascular smooth muscle cells. We, therefore, investigated whether βarr2-dependent signaling contributes to TAA formation in MFS. We utilized a murine model of MFS (Fbn(C1039G/+)) to generate a MFS murine model in combination with genetic βarr2 deletion (Fbn(C1039G/+)/βarr2(-/-)). Fbn(C1039G/+)/βarr2(-/-) mice displayed delayed aortic root dilation compared to Fbn(C1039G/+) mice. The mRNA and protein expression of several mediators of TAA formation including matrix metalloproteinase (MMP) -2 and -9 were reduced in the aorta of Fbn(C1039G/+)/βarr2(-/-) mice relative to Fbn(C1039G/+) mice. Activation of extracellular-regulated kinase 1/2 (ERK 1/2) was also decreased in the aortas of Fbn(C1039G/+)/βarr2(-/-) mice compared to Fbn(C1039G/+) animals. siRNA targeting βarr2 inhibited angiotensin-stimulated expression of pro-aneurysmal signaling mediators in primary aortic root smooth muscle cells. Angiotensin-stimulated expression of the pro-aneurysmal signaling mediators MMP-2 and -9 was inhibited by blockade of ERK 1/2 or the epidermal growth factor receptor, whereas blockade of the transforming growth factor-β receptor had no effect. These results suggest that βarr2 contributes to TAA formation in MFS by regulating ERK 1/2-dependent expression of pro-aneurysmal genes and proteins downstream of the AT1aR. Importantly, this demonstration of the unique signaling mechanism by which βarr2 contributes to aneurysm formation identifies multiple novel potential therapeutic targets in MFS.
    Preview · Article · Sep 2015 · AJP Heart and Circulatory Physiology
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    Robert J Lefkowitz
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    ABSTRACT: As academic physician-scientists, one of the most important things we do is mentor young trainee-scientists. There obviously is no one right way to mentor or a set of rules one can follow; it's a very personal matter, and very much depends on one's personality. For much of my career, I gave very little thought as to how I mentored my trainees or to whether I was any good at it. Like many investigators, perhaps, I was just too busy with the daily activities of research to consider how I was guiding my students. Here, I take a look back and reflect on my experiences as a mentor and the factors that I believe contribute to the success of trainees as independent scientists.
    Preview · Article · Aug 2015 · The Journal of clinical investigation
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    Wei Tang · Ryan T Strachan · Robert J Lefkowitz · Howard A Rockman
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    ABSTRACT: It has recently been appreciated that the angiotensin-II type-1 receptor (AT1R), a prototypic member of the G protein-coupled receptor (GPCR) superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-biased ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-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 if mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-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-β-arrestin2 fusion compared to the wild type AT1R, and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations which 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 β-arrestin-biased active conformations of the AT1R and have important implications for understanding pathophysiological AT1R signaling.
    Preview · Article · Aug 2014 · Journal of Biological Chemistry
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    ABSTRACT: G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, 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 cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.
    Full-text · Article · Jun 2014 · Nature
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    Preview · Article · Apr 2014 · Journal of the American College of Cardiology
  • James W Wisler · Kunhong Xiao · Alex Rb Thomsen · Robert J Lefkowitz
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    ABSTRACT: 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.
    No preview · Article · Apr 2014 · Current opinion in cell biology
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    ABSTRACT: 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 β-arrestin (βarr)) 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-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies which 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.
    Full-text · Article · Mar 2014 · Journal of Biological Chemistry
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    ABSTRACT: 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 β2 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 β2 adrenergic receptor (β2AR). Here we set out to study the functional interaction of 18 related nanobodies with the β2 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 β2AR-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, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-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.
    Full-text · Article · Dec 2013 · Molecular pharmacology
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    ABSTRACT: 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 β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-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 β-arrestins and found that β-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 β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-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 β-arrestin biased ligand [Sar(1),Ile(4),Ile(8)]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility.
    Full-text · Article · Nov 2013 · PLoS ONE
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    ABSTRACT: G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.
    Full-text · Article · Oct 2013 · ACS Medicinal Chemistry Letters
  • Robert J. Lefkowitz
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    ABSTRACT: Das Konzept der Rezeptoren fasziniert Wissenschaftler seit mehr als einem Jahrhundert. Heute weiß man, dass die G-Protein-gekoppelten Rezeptoren (GPCRs) die bei weitem größte, vielfältigste und ubiquitärste Gruppe unter den verschiedenen Familien von Plasmamembranrezeptoren sind. Die Erforschung der GPCRs wurde mit dem Chemie-Nobelpreis 2012 gewürdigt.
    No preview · Article · Jun 2013 · Angewandte Chemie
  • Robert J Lefkowitz
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    ABSTRACT: The idea of receptors has fascinated scientists for more than a century. Today it is known that the G-protein coupled receptors (GPCRs) represent by far the largest, most versatile and most ubiquitous of the several families of plasma membrane receptors. The Nobel Prize for Chemistry 2012 was awarded for studies on GPCRs.
    No preview · Article · Jun 2013 · Angewandte Chemie International Edition
  • Robert J Lefkowitz
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    ABSTRACT: Visual arrestin and the two β-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 β2-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 β-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.
    No preview · Article · Jun 2013 · Progress in molecular biology and translational science
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    ABSTRACT: 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 β-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of β-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 β-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 β-arrestin-1. The structure of the β-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in β-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 β-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on β-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.
    Full-text · Article · Apr 2013 · Nature
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    ABSTRACT: A prospective, large library virtual screen against an activated β2AR structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound 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 DRD2 activated model templated on the activated β2AR structure returned few hits of only marginal potency.
    Full-text · Article · Mar 2013 · ACS Chemical Biology
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    ABSTRACT: β-Arrestins were initially discovered as negative regulators of G protein-coupled receptor signaling. Although β-arrestins have more recently been implicated as scaffold proteins that interact with various mitogenic and developmental signals, the genetic role of β-arrestins in driving oncogenesis is not known. Here we have investigated the role of β-arrestin in hematologic malignancies and have found that although both β-arrestin1 and -2 are expressed in the hematopoietic system, loss of β-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 β-arrestin2 leads to a significant inhibition of β-catenin stabilization, and ectopic activation of Wnt signaling reverses the defects observed in the β-arrestin2 mutant cells. These data cumulatively show that β-arrestin2 is essential for CML disease propagation and indicate that β-arrestins and the Wnt/β-catenin pathway lie in a signaling hierarchy in the context of CML cancer stem cell maintenance.
    Full-text · Article · Jul 2012 · Proceedings of the National Academy of Sciences
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    Preview · Dataset · Jun 2012
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    Dataset: SBML model

    Preview · Dataset · Jun 2012
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    ABSTRACT: Supplementary Figures S1–21, Supplementary Tables S1–2
    Preview · Dataset · Jun 2012
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    ABSTRACT: 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 β-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 β-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.
    Full-text · Article · Jun 2012 · Molecular Systems Biology

Publication Stats

98k Citations
7,630.41 Total Impact Points


  • 1976-2015
    • Duke University
      • • Department of Surgery
      • • Department of Medicine
      Durham, North Carolina, United States
  • 1979-2014
    • Howard Hughes Medical Institute
      Ашбърн, Virginia, United States
  • 1974-2014
    • Duke University Medical Center
      • • Department of Biochemistry
      • • Department of Medicine
      • • Department of Cell Biology
      • • Division of Medical Oncology
      • • Department of Surgery
      • • Division of Cardiology
      Durham, North Carolina, United States
  • 2003
    • University of Texas at Austin
      • Institute for Cellular and Molecular Biology
      Texas City, TX, United States
  • 1996-2002
    • Emory University
      • • Department of Pharmacology
      • • School of Medicine
      Atlanta, GA, United States
  • 1997
    • Hohenheim University
      • Institute of Physiology
      Stuttgart, Baden-Württemberg, Germany
    • University of Houston
      • Department of Pharmacological and Pharmaceutical Sciences
      Houston, Texas, United States
  • 1995
    • Johns Hopkins University
      • Department of Neuroscience
      Baltimore, MD, United States
  • 1984-1995
    • Baylor College of Medicine
      Houston, Texas, United States
  • 1994
    • University of Texas Southwestern Medical Center
      Dallas, Texas, United States
  • 1993
    • University of Cincinnati
      Cincinnati, Ohio, United States
  • 1991
    • National Tsing Hua University
      • Institute of Biomedical Sciences
      Hsin-chu-hsien, Taiwan, Taiwan
    • University of California, Berkeley
      • Department of Molecular and Cell Biology
      Berkeley, California, United States
  • 1987-1988
    • Yale University
      • School of Medicine
      New Haven, Connecticut, United States
  • 1980
    • Baltimore City Public Schools
      Baltimore, Maryland, United States
  • 1973
    • Massachusetts General Hospital
      • Department of Medicine
      Boston, Massachusetts, United States
    • Harvard University
      Cambridge, Massachusetts, United States
  • 1972
    • University of Miami Miller School of Medicine
      • Division of Hospital Medicine
      Miami, Florida, United States