Showing papers by "Robert J. Lefkowitz published in 2001"
TL;DR: The data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
Abstract: Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of β-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered β-arrestin-2 binding to the receptor and internalization of AT1aR-β-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-β-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, β-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged β-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with β-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with β-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to β-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in β-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to β-arrestin-2, and the association of β-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
838 citations
TL;DR: It is indicated that ubiquitination of the receptor and of β-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.
Abstract: Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.
829 citations
TL;DR: The signalling roles of arrestins have been expanded even further with the discovery that the formation of stable receptor–arrestin complexes initiates photoreceptor apoptosis in Drosophila, leading to retinal degeneration.
Abstract: In the classical model of G-protein-coupled receptor (GPCR) regulation, arrestins terminate receptor signalling. After receptor activation, arrestins desensitize phosphorylated GPCRs, blocking further activation and initiating receptor internalization. This function of arrestins is exemplified by studies on the role of arrestins in the development of tolerance to, but not dependence on, morphine. Arrestins also link GPCRs to several signalling pathways, including activation of the non-receptor tyrosine kinase SRC and mitogen-activated protein kinase. In these cascades, arrestins function as adaptors and scaffolds, bringing sequentially acting kinases into proximity with each other and the receptor. The signalling roles of arrestins have been expanded even further with the discovery that the formation of stable receptor-arrestin complexes initiates photoreceptor apoptosis in Drosophila, leading to retinal degeneration. Here we review our current understanding of arrestin function, discussing both its classical and newly discovered roles.
472 citations
TL;DR: In this article, the authors investigate the role of β-adrenergic receptor (AR) and β-AR subtypes in regulating cardiomyocyte survival and apoptosis and explore underlying mechanisms.
Abstract: The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.
458 citations
TL;DR: The discovery that β-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs.
Abstract: Activation of classical second messenger cascades cannot fully explain the recently appreciated roles of heptahelical, or G-protein coupled receptors (GPCRs), in stimulation of mitogen activated protein kinase (MAPK) cascades. Rather, several distinct signaling mechanisms appear to contribute to GPCR-mediated MAPK activation. These include transactivation of the Epidermal Growth Factor Receptor (EGFR) via the autocrine/paracrine release of EGF-like ligands at the cell surface and scaffolding of MAPK cascades. A significant advance in the understanding of how GPCRs activate MAPK cascades is the discovery that β-arrestin, a protein well known for its roles in both receptor desensitization and internalization, serves as a scaffolding protein for at least two GPCR stimulated MAPK cascades, the extracellular signal regulated kinase (ERK) cascade and the c-jun N-terminal kinase 3 (JNK3) cascade. Together, these novel mechanisms of GPCR-mediated MAPK regulation may permit GPCRs in specific situations to control the temporal and spatial activity of MAPKs and thereby determine the consequences of GPCR stimulation with respect to transcriptional activation, cell proliferation and apoptosis.
407 citations
TL;DR: Results suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.
Abstract: The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.
353 citations
TL;DR: These recent discoveries indicate that the beta-arrestins play widespread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from heptahelical receptors.
335 citations
TL;DR: A significant synergy between an established heart-failure treatment and the strategy of βARK1 inhibition is demonstrated, resulting in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with β-blocker therapy.
Abstract: Chronic human heart failure is characterized by abnormalities in β-adrenergic receptor (βAR) signaling, including increased levels of βAR kinase 1 (βARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of βARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of βARK1 (βARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca2+-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 ± 1 weeks). In contrast, CSQ/βARKct mice exhibited a significant increase in mean survival age (15 ± 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/βARKct, left ventricular end diastolic dimension 5.60 ± 0.17 mm vs. 4.19 ± 0.09 mm, P < 0.005; % fractional shortening, 15 ± 2 vs. 36 ± 2, P < 0.005). The enhancement of the survival rate in CSQ/βARKct mice was substantially potentiated by chronic treatment with the βAR antagonist metoprolol (CSQ/βARKct nontreated vs. CSQ/βARKct metoprolol treated, 15 ± 1 weeks vs. 25 ± 2 weeks, P < 0.0001). Thus, overexpression of the βARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with β-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of βARK1 inhibition.
265 citations
TL;DR: The agonists-driven formation of a complex including β-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.
230 citations
TL;DR: This work has developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells, and demonstrates that EGFR-dependent activation of the ERK cascade after α2AAR stimulation requires Gβγ subunits upstream and dynamin-dependent endocytosis downstream of HB- EGF shedding and EGFR activation.
215 citations
TL;DR: In vivo delivery of Adeno-&bgr;ARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of &bgr ;ARKct results in marked reversal of ventricular dysfunction, providing a novel treatment strategy for improving the cardiac performance of the post-MI heart.
Abstract: Background—Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo β-adrenergic receptor kinase (βARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). Methods and Results—Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5×1011 viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of βARK1 (Adeno-βARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-βARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the βARKct. Baseline in vivo LV systolic perfo...
TL;DR: Three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.
Abstract: The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including β-adrenergic receptor (βAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in βAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the β2AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the βAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of βAR signaling or excitation-contraction coupling can provide therapeutic benefit.
TL;DR: It is shown that PKA directly phosphorylates GRK2 on serine 685 and increases Gβγ subunit binding toGRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor.
TL;DR: The direct binding of the β2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.
TL;DR: A mechanism by which β-arrestin2 functions as a scaffold protein in the JNK3 signaling pathway is delineated and the conserved docking site in β-Arrestin1 is implicate as an important factor in binding J NK3 and stimulating the phosphorylation of JNK 3 by MKK4.
TL;DR: 1) the Src kinase Yes can induce tyrosine phosphorylation of Gαq/11in response to ET-1 stimulation, and 2) β-arrestin 1 and SRC kinase form a molecular complex with the ETA receptor to mediate ET- 1 signaling to GαQ/11 with subsequent glucose transport stimulation.
TL;DR: The pace of current progress in clinical and basic research is such that remarkable improvement in the quality and length of life for those at risk for cardiovascular disease is likely.
Abstract: Cardiovascular disease is the greatest threat to human life and health. The past decade has seen remarkable progress in clinical and basic cardiovascular research, and many areas of opportunity are promising. The pace of current progress in clinical and basic research is such that remarkable improvement in the quality and length of life for those at risk for cardiovascular disease is likely.
TL;DR: BetaArrestins have been shown to act as molecular scaffolds that coordinate the assembly of certain MAP kinase complexes that lead to the stimulation of either ERK1/2 or JNK3 and long-term accumulation of arrestin-rhodopsin complexes, in photoreceptor cells has been shows to trigger apoptosis.
TL;DR: Cardiac overexpression of human &bgr;3ARs results in positive inotropy only on stimulation with a &b gr;3 AR agonist.
Abstract: Background Stimulation of β1- and β2-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the β3AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. Methods and Results To better understand the role of β3ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human β3AR (TGβ3 mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous β1- and β2ARs, isoproterenol resulted in an increase in contractility in the TGβ3 mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human β3AR agonist L-755,507 significantly increased contractility in the TGβ3 mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and b...
TL;DR: βarr1 is identified as a regulator of Dvl-dependent LEF transcription and suggested that βarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.
Abstract: One aspect of the function of the β-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as β-arrestin1 (βarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3β (GSK-3β), stabilization of β-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with βarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with βarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1ɛ and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of βarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas βarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of βarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3β kinase activity indicate that the step affected by βarr1 is upstream of GSK-3β and most likely at the level of Dvl. These results identify βarr1 as a regulator of Dvl-dependent LEF transcription and suggest that βarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.
TL;DR: The data suggest that PE-treated Tgα43 mice have chronic activation of the cardiac sympathetic nervous system, which may be responsible for the appearance of apparent maladaptive hypertrophy with an evolution towards HF and sudden death.
TL;DR: Evidence from the Drosophila visual system that suggests that intracellular rhodopsin and arrestin2 complexes induce apoptosis is discussed, pointing to a mechanism by which proliferative or proapoptotic signals can be mediated largely independent from G protein activation.
Abstract: Recent data suggest that internalized receptor and arrestin complexes are actively involved in signal transduction. Miller and Lefkowitz discuss evidence from the Drosophila visual system that suggests that intracellular rhodopsin and arrestin2 complexes induce apoptosis. Experiments with activated mammalian G protein-coupled receptor and arrestin complexes point to a mechanism by which proliferative or proapoptotic signals can be mediated largely independent from G protein activation.
01 Jan 2001
TL;DR: This work has developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells, and demonstrates that EGFR-dependent activation of the ERK cascade after a2AAR stimulation requires Gbg subunits upstream and dynamin-dependent endocytosis downstream of HBEGF Shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-egF and for HB- EGF
Abstract: “Transactivation” of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/ paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, a2A-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, a2AAR expressing “donor” cells are co-cultured with “acceptor” cells lacking the a2AAR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the a2AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the a2AAR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after a2AAR stimulation requires Gbg subunits upstream and dynamin-dependent endocytosis downstream of HBEGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.
Journal Article•