DOCSLIB.ORG

The G-Protein-Coupled Receptor Kinase 5 Inhibits NF B Transcriptional Activity by Inducing Nuclear Accumulation Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The G-Protein-Coupled Receptor Kinase 5 Inhibits NF B Transcriptional Activity by Inducing Nuclear Accumulation Of The G-protein-coupled receptor kinase 5 inhibits NF␬B transcriptional activity by inducing nuclear accumulation of I␬B␣ Daniela Sorrientoa, Michele Ciccarellia, Gaetano Santullia, Alfonso Campanilea, Giovanna Giuseppina Altobellia, Vincenzo Ciminib, Gennaro Galassoa, Dalila Astonec, Federico Piscionea, Lucio Pastorec, Bruno Trimarcoa, and Guido Iaccarinoa,1 Dipartimento di aMedicina Clinica, Scienze Cardiovascolari, ed Immunologiche, and bScienze Biomorfologiche e Funzionali, Universita`Federico II, 80134 Naples, Italy; and cCentro di Ingegneria Genetica –Biotecnologie Avanzate, 80145 Naples, Italy Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved September 12, 2008 (received for review May 12, 2008) G-protein-coupled receptor (GPCR) kinases, GRKs, are known as the nucleus and to bind to specific sequences in the promoter or serine/threonine kinases that regulate GPCR signaling, but recent enhancer regions of target genes (11). Recently this view has been findings propose functions for these kinases besides receptor desen- partially revisited by the description of NES and NLS sequences on sitization. Indeed, GRK5 can translocate to the nucleus by means of a the NF␬B/I␬B␣ complex that allow a continuous shuttling between nuclear localization sequence, suggesting that this kinase regulates the nucleus and the cytosol by means of nuclear transporters, such transcription events in the nucleus. To evaluate the effect of GRK5– as CRM1 (1, 3). New partners of this complex are described along I␬B␣ interaction on NF␬B signaling, we induced the overexpression the way: ␤-arrestin 2 interacts with the NF␬B/I␬B␣ complex within and the knockdown of GRK5 in cell cultures. GRK5 overexpression the cytosol, causing the blockade of NF␬B transcriptional activity causes nuclear accumulation of I␬B␣, leading to the inhibition of NF␬B (16, 17). Studying a NF␬B cognate precursor—NF␬B p105, which transcriptional activity. Opposite results are achieved by GRK5 knock- actively participates in cytosolic signal transduction but lacks tran- down through siRNA. A physical interaction between GRK5 and I␬B␣, scriptional activity—Parameswaran et al. (18) recently described the rather than phosphorylative events, appears as the underlying mech- ability of GRK5 to physically interact with NF␬B p105. These anism. We identify the regulator of gene protein signaling homology authors did not investigate the eventual effects of GRK5 on NF␬B domain of GRK5 (RH) and the N-terminal domain of I␬B␣ as the transcriptional activity. regions involved in such interaction. To confirm the biological rele- NF␬B drives VEGF, bFGF, IL-8, and other cytokines’ expres- vance of this mechanism of regulation for NF␬B, we evaluated the sions (19). In the endothelium, cytokine production sustains tissue effects of GRK5-RH on NF␬B-dependent phenotypes. In particular, regeneration by providing the start to neoangiogenesis and there- GRK5-RH overexpression impairs apoptosis protection and cytokine fore blood and nutrient support. Failure of NF␬B activation is production in vitro and inflammation and tissue regeneration in vivo. associated with impaired cytokine production and angiogenesis Our results reveal an unexpected role for GRK5 in the regulation of (20, 21). NF␬B transcription activity. Placing these findings in perspective, this On the basis of these preexisting observations, we hypothesized mechanism may represent a therapeutic target for all those condi- that GRK5 could regulate NF␬B transcriptional activity. In par- tions involving excessive NF␬B activity. ticular, we investigated whether GRK5 affects I␬B␣ cellular levels and NF␬B activity and the molecular basis for GRK5 and I␬B␣ angiogenesis ͉ gene transcription ͉ inflammation ͉ signal transduction physical interaction. Also, we assessed the effects on NF␬B- dependent phenotypes such as apoptosis, cytokine production, and -protein-coupled receptor (GPCR) kinases, GRKs, constitute in vivo and in vitro angiogenesis. a large family of serine/threonine protein kinases that regulate G Results GPCR signaling (1–3), consisting of 7 isoforms that share structural and functional similarities (4). A central catalytic domain is flanked GRK5 Overexpression Causes I␬B␣ Nuclear Accumulation. We evalu- ␬ ␣ by an N-terminal domain that includes a region of homology to ated GRK5 and I B cellular localization by Western blot in bovine regulators of G-protein signaling (RH) and a C-terminal domain of aorta endothelial cells (BAEC) overexpressing the WT bovine ␬ ␣ variable length (5, 6). The catalytic domain of GRKs is relatively GRK5 gene. The overexpression of GRK5-WT increases I B ␬ ␣ well-conserved among the members of different subfamilies levels in whole-cell extracts by inducing I B nuclear accumulation (Ϸ45% sequence identity), whereas N-terminal RH domains dis- and does not change cytosol levels (Fig. 1A). Fig. 1B shows that the play weak homology (Ϸ27%) and C termini have little or no total and nuclear levels of GRK5 after transfection increase in a sequence homology. GRKs have different tissue distribution, sub- time-dependent manner and are associated with a progressive ␬ ␣ cellular localization, and kinase activity regulation (7, 8). GRKs accumulation of I B levels both in whole-cell lysates and nuclear ␬ mostly localize at the plasma membrane (2), but recently Johnson extracts. Accordingly, NF B activity decreases over time, synchro- ␬ ␣ et al. (7) demonstrated that GRK4–6 (but not other GRKs) can nized with the increase of I B levels. These results suggest that shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence Author Contributions: G.I. designed research; D.S., M.C., G.S., A.C., G.G.A., V.C., G.G., D.A., (NES), thus suggesting a nuclear effect for the GRK4–6 subfamily. F.P., and L.P. performed research; D.S., B.T., and G.I. analyzed data; and D.S., B.T., and G.I. NF␬B is an ubiquitously expressed and highly regulated dimeric wrote the paper. transcription factor (3) regulating the expression of genes respon- The authors declare no conflict of interest. sible for innate and adaptive immunity, tissue regeneration, stress This article is a PNAS Direct Submission. responses, apoptosis, cell proliferation, and differentiation (9–14). 1 ␬ To whom correspondence should be addressed at: Medicina Clinica, Scienze Cardiovascolari Within the canonical view of the regulation of the NF B activity, ed Immunologiche, Federico II University, Via Pansini 5; Edificio 2, 80131 Naples, Italy. E-mail: the inactive NF␬B/I␬B␣ complex localizes in the cytosol until an [email protected]. extracellular stimulus, such as TNF␣ or LPS, induces I␬B kinase This article contains supporting information online at www.pnas.org/cgi/content/full/ (IKK)-mediated I␬B␣ phosphorylation and subsequent degrada- 0804446105/DCSupplemental. tion by the proteasome (15). NF␬B is therefore free to shuttle into © 2008 by The National Academy of Sciences of the USA 17818–17823 ͉ PNAS ͉ November 18, 2008 ͉ vol. 105 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804446105 Downloaded by guest on October 1, 2021 B BAEC BAEC C BAEC whole cell lysates nuclear extracts whole cell lysates GRK5 0 0.5 1 2 ug GRK5 0 12 24 36 48 0 12 24 36 48 hrs GRK5 GRK5 GRK5 I B A BAEC I B I B Actin H3 Actin GRK5 WT - + - + - + 450 GRK5 450 400 400 I B 350 350 300 300 B activity Actin B activity 250 250 H3 200 200 Whole Cytosol Nucleus 150 150 100 100 50 50 Relative NF Relative NF Relative D HEK293 0 0 whole cell lysates 0 12 24 36 48 hrs 0 0.5 1 2 GRK5 ( g) CTRL GRK5 GRK5 HEK293 BAEC WT siRNA E F whole cell lysates nuclear extracts GRK5 CTRL GRK5 GRK5 GRK5 0 5 10 20 ug WT K215R I B siRNA GRK5 GRK5 Actin I B I B 800 700 Actin NF B 600 H3 500 B activity 800 400 1200 700 300 1000 600 200 800 500 B activity B Relative NF 100 B activity 400 600 0 300 CTRL GRK5 GRK5 400 200 WT siRNA 200 Relative NF 100 Relative NF 0 0 CTRL GRK5 GRK2 0 5 10 20 siRNA ( g) GRK5 WT K215R Fig. 1. GRK5 binds and stabilizes I␬B␣, causing inhibition of NF␬B activity. (A)I␬B␣ and GRK5 levels were analyzed in whole, cytosolic, and nuclear extracts by Western blot in BAEC overexpressing GRK5-WT. The overexpression of GRK5-WT increases I␬B␣ levels in whole-cell extracts and induces I␬B␣ nuclear accumulation. (B)To evaluate the effects of GRK5 on I␬B␣ turnover, we analyzed GRK5 and I␬B␣ expression in a time-course of GRK5 transfection. The increase of GRK5 levels induces a progressive increase of I␬B␣ levels and a reduction of NF␬B activity (*P Ͻ 0.05 vs. control). (C) A time course of transfection of GRK5 was performed in BAEC and GRK5 and I␬B␣ levels analyzed both in whole- and nuclear-cell extracts. The time-dependent increase of GRK5 levels associates to I␬B␣ stabilization and nuclear localization and subsequently NF␬B activity inhibition (*P Ͻ 0.05 vs. control). (D) HEK293 transfected with GRK5-WT or GRK5siRNA were analyzed by WB for GRK5 and I␬B␣ expression. As in BAEC, GRK5 overexpression induces I␬B␣ accumulation; conversely, GRK5 knockdown by GRK5siRNA associates to I␬B␣ degradation. Actin is shown for protein-loading control. HEK293 were transfected with ␬B-Luc plasmid and GRK5-WT or GRK5-siRNA and luciferase activity was measured. GRK5-WT overexpression causes inhibition of NF␬B activity, whereas GRK5 knockdown increases NF␬B transcription levels. The data in the bar graph are expressed as meanϮSEM and are representative of 3 experiments (*P Ͻ 0.05 vs. control). (E) GRK5 and I␬B␣ expression and luciferase activity were assessed in HEK293 transfected with increasing doses of GRK5siRNA.
Load more

Recommended publications
  • Defective Lymphocyte Chemotaxis in Я-Arrestin2- and GRK6-Deficient Mice
    Defective lymphocyte chemotaxis in ␤-arrestin2- and GRK6-deficient mice Alan M. Fong*, Richard T. Premont*, Ricardo M. Richardson*, Yen-Rei A. Yu†, Robert J. Lefkowitz*‡§, and Dhavalkumar D. Patel*†¶ Departments of *Medicine, ‡Biochemistry, and †Immunology, and §Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710 Contributed by Robert J. Lefkowitz, April 4, 2002 Lymphocyte chemotaxis is a complex process by which cells move kinase, extracellular receptor kinase, and c-jun terminal kinase within tissues and across barriers such as vascular endothelium and activation (9–12), they might also act as positive regulators of is usually stimulated by chemokines such as stromal cell-derived chemotaxis. To evaluate the role of the GRK-arrestin pathway factor-1 (CXCL12) acting via G protein-coupled receptors. Because in chemotaxis, we studied the chemotactic responses of lym- members of this receptor family are regulated (‘‘desensitized’’) by phocytes from ␤-arrestin- and GRK-deficient mice toward G protein-coupled receptor kinase (GRK)-mediated receptor phos- gradients of stromal cell-derived factor 1 (CXCL12), a well ␤ phorylation and -arrestin binding, we examined signaling and characterized chemokine whose receptor is CXCR4, a core- chemotactic responses in splenocytes derived from knockout mice ceptor for HIV. deficient in various ␤-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of ␤-arrestin2, Materials and Methods GRK5, and GRK6 were examined because all three proteins are :expressed at high levels in purified mouse CD3؉ T and B220؉ B Mice. The following mouse strains were used in this study splenocytes. CXCL12 stimulation of membrane GTPase activity was ␤-arrestin2-deficient (back-crossed for six generations onto the unaffected in splenocytes derived from GRK5-deficient mice but C57͞BL6 background; ref.
    [Show full text]
  • Supplementary Figure S1. Intracellular Ca2+ Levels Following Decursin Treatment in F11 Cells in the Presence of Menthol
    Supplementary Figure S1. Intracellular Ca2+ levels following decursin treatment in F11 cells in the presence of menthol (A) Intracellular Ca2+ levels after treatment with decursin every 3 s. The red arrow indicates the duration of treatment with 200 μM of menthol and decursin. NC: The negative control treated with DMSO only; PC: The positive control treated with 200 μM menthol without decursin. (B) Average intracellular Ca2+ levels after treatment with decursin. The average was quantified from the normalized Δ340/380 ratio for 10 cycles after treatment with the decursin solution at the 10th cycle, as shown in Fig. 1A. The normalized Δ340/380 ratio was calculated using the following for- mula: [ratio of fluorescence intensity at 510 nm (emission) to that at 340 nm (excitation)]/[ratio of fluorescence intensity at 510 nm (emission) to that at a wavelength of 380 nm (excitation)]. Cells 2021, 10, 547. https://doi.org/10.3390/cells10030547 www.mdpi.com/journal/cells Cells 2021, 10, 547 2 of 5 Table S1. List of protein targets of decursin detected by the SwissTargetPrediction web tool Common Target Uniprot ID ChEMBL ID Target Class Probability name Poly [ADP-ribose] polymerase-1 PARP1 P09874 CHEMBL3105 Enzyme 0.104671941 N-acylsphingosine-amidohydro- NAAA Q02083 CHEMBL4349 Enzyme 0.104671941 lase Acid ceramidase ASAH1 Q13510 CHEMBL5463 Enzyme 0.104671941 Family A G protein- Neuropeptide Y receptor type 5 NPY5R Q15761 CHEMBL4561 0.104671941 coupled receptor Family A G protein- Melatonin receptor 1A MTNR1A P48039 CHEMBL1945 0.104671941 coupled
    [Show full text]
  • A Novel Kinase Inhibitor Establishes a Predominant Role for Protein Kinase D As a Cardiac Class Iia Histone Deacetylase Kinase
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 631–637 journal homepage: www.FEBSLetters.org A novel kinase inhibitor establishes a predominant role for protein kinase D as a cardiac class IIa histone deacetylase kinase Lauren Monovich a,*, Richard B. Vega a, Erik Meredith a, Karl Miranda a, Chang Rao a, Michael Capparelli a, Douglas D. Lemon b, Dillon Phan b, Keith A. Koch b, Joseph A. Chapo b, David B. Hood b, Timothy A. McKinsey b,* a Novartis Institutes for Biomedical Research, 3333 Walnut Street, Boulder, CO 80301, United States b Gilead Colorado, Inc., 3333 Walnut Street, Boulder, CO 80301, United States article info abstract Article history: Class IIa histone deacetylases (HDACs) repress genes involved in pathological cardiac hypertrophy. Received 13 November 2009 The anti-hypertrophic action of class IIa HDACs is overcome by signals that promote their phosphor- Revised 8 December 2009 ylation-dependent nuclear export. Several kinases have been shown to phosphorylate class IIa Accepted 11 December 2009 HDACs, including calcium/calmodulin-dependent protein kinase (CaMK), protein kinase D (PKD) Available online 14 December 2009 and G protein-coupled receptor kinase (GRK). However, the identity of the kinase(s) responsible Edited by Ivan Sadowski for phosphorylating class IIa HDACs during cardiac hypertrophy has remained controversial. We describe a novel and selective small molecule inhibitor of PKD, bipyridyl PKD inhibitor (BPKDi). BPKDi blocks signal-dependent phosphorylation and nuclear export of class IIa HDACs in cardio- Keywords: Kinase myocytes and concomitantly suppresses hypertrophy of these cells.
    [Show full text]
  • Comprehensive Assessment of the Association of WNK4
    OPEN Comprehensive Assessment of the SUBJECT AREAS: Association of WNK4 Polymorphisms GENETICS RESEARCH RENOVASCULAR HYPERTENSION with Hypertension: Evidence from a EPIDEMIOLOGY Meta-Analysis Received Xiao-gang Guo1, Jie Ding1, Hui Xu1,2, Tian-ming Xuan1, Wei-quan Jin1, Xiang Yin1, Yun-peng Shang1, 22 April 2014 Fu-rong Zhang1, Jian-hua Zhu1 & Liang-rong Zheng1 Accepted 15 September 2014 1Department of Cardiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China, Published 2Xiuzhou District, Gaozhao Street Community Health Service Center, Jiaxing 314031, China. 30 September 2014 The relationship between with-no-lysine [K] kinase 4 (WNK4) gene polymorphisms and hypertension has been widely investigated, However, the studies yielded contradictory results. To evaluate these inconclusive Correspondence and findings comprehensively, we therefore performed a meta-analysis. Ten articles encompassing 16 requests for materials independent case-control studies with 6089 hypertensive cases and 4881 normotensive controls were should be addressed to selected for this meta-analysis. Four WNK4 gene polymorphisms were identified (G1155942T, G1156666A, X.-G.G. (gxg22222@ T1155547C, and C6749T). The results showed statistically significant associations of G1155942T polymorphism (allelic genetic model: odds ration or OR51.62, 95% confidence interval or CI: 1.11–2.38, zju.edu.cn) P50.01; dominant model: OR51.85, 95% CI: 1.07–3.19, P50.03) and C6749T polymorphism (allele contrast: OR52.04, 95% CI: 1.60–2.59, P,0.01; dominant model: OR52.04, 95%CI: 1.59–2.62, P,0.01; and homozygous model: OR55.01, 95% CI: 1.29–19.54, P50.02) with hypertension risk. However, neither C1155547T nor G1156666A was associated significantly with hypertension susceptibility.
    [Show full text]
  • Comprehensive Assessment of Indian Variations in the Druggable Kinome Landscape Highlights Distinct Insights at the Sequence, Structure and Pharmacogenomic Stratum
    SUPPLEMENTARY MATERIAL Comprehensive assessment of Indian variations in the druggable kinome landscape highlights distinct insights at the sequence, structure and pharmacogenomic stratum Gayatri Panda1‡, Neha Mishra1‡, Disha Sharma2,3, Rahul C. Bhoyar3, Abhinav Jain2,3, Mohamed Imran2,3, Vigneshwar Senthilvel2,3, Mohit Kumar Divakar2,3, Anushree Mishra3, Priyanka Banerjee4, Sridhar Sivasubbu2,3, Vinod Scaria2,3, Arjun Ray1* 1 Department of Computational Biology, Indraprastha Institute of Information Technology, Okhla, India. 2 Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India. 3 CSIR-Institute of Genomics and Integrative Biology, Mathura Road, Delhi-110020, India. 4 Institute for Physiology, Charite-University of Medicine, Berlin, 10115 Berlin, Germany. ‡These authors contributed equally to this work. * [email protected] TABLE OF CONTENTS Name Title Supplemental_Figure_S1 Fauchere and Pliska hydrophobicity scale for variations in structure data Supplemental_Figure_S2 Phenotypic drug-drug correlogram Supplemental_Table_S1 545 kinase coding genes used in the study Supplemental_Table_S2 Classes and count of kinase coding genes Supplemental_Table_S3 Allele frequency Indian v/s other populations from 1000 genome data(1000g2015). Supplemental_Table_S4 IndiGen Structure Data- consisting of 12 genes and their 22 variants Supplemental_Table_S5 Genes, PDB ids, mutations in IndiGen data and associated drugs (FDA approved) Supplemental_Table_S6 Data used for docking and binding pocket similarity analysis Supplemental_Table_S7
    [Show full text]
  • G Protein-Coupled Receptor Kinase 4 Gene Variants in Human Essential Hypertension
    G protein-coupled receptor kinase 4 gene variants in human essential hypertension Robin A. Felder*, Hironobu Sanada*†, Jing Xu‡, Pei-Ying Yu‡, Zheng Wang‡, Hidetsuna Watanabe*, Laureano D. Asico*, Wei Wang‡, Shaopeng Zheng‡, Ikuyo Yamaguchi‡, Scott M. Williams§, James Gainer¶, Nancy J. Brown¶, Debra Hazen-Martinʈ, Lee-Jun C. Wong**, Jean E. Robillard††, Robert M. Carey‡‡, Gilbert M. Eisner‡, and Pedro A. Jose‡ *Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA 22908; ‡Department of Pediatrics and Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007; §Department of Microbiology, Meharry Medical College, Nashville, TN 37208; ¶Department of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232; ʈDepartment of Pathology, Medical University of South Carolina, Charleston, SC 29403; **Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007; ††Department of Pediatrics, University of Michigan College of Medicine, Ann Arbor, MI 48109; and ‡‡Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908 Communicated by Maria Iandolo New, Weill Medical College of Cornell University, New York, NY, December 21, 2001 (received for review August 10, 2001) Essential hypertension has a heritability as high as 30–50%, but its abnormal renal sodium transporters (3, 8, 13, 17). Also, the genetic cause(s) has not been determined despite intensive inves- coding region of the D1 receptor is unchanged in hypertensive tigation. The renal dopaminergic system exerts a pivotal role in subjects (16), as well as in rodents with genetic hypertension maintaining fluid and electrolyte balance and participates in the (unpublished studies). pathogenesis of genetic hypertension.
    [Show full text]
  • GRK4 Protein Recombinant Human Protein Expressed in Sf9 Cells
    Catalog # Aliquot Size G02-31G-20 20 µg G02-31G-50 50 µg GRK4 Protein Recombinant human protein expressed in Sf9 cells Catalog # G02-31G Lot # P1749-5 Product Description Purity Recombinant human GRK4 (140-end) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. The gene accession number for GRK4 is BC117320. The purity of GRK4 was determined to be >70% by densitometry, GRK4 Gene Aliases approx. MW 75kDa. GPRK2L, GPRK4, GRK4a, IT11 Formulation Recombinant protein stored in 50mM Tris-HCl, pH 7.5, 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. Storage and Stability o Store product at –70 C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Scientific Background GRK4 or G protein-coupled receptor kinase 4 is a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase subfamily of the Ser/Thr protein kinase family which phosphorylates the activated forms of G protein-coupled receptors thus initiating its deactivation and it also play an important role in receptor desensitization. GRK4 protein has kinase activity GRK4 Protein and that it interacts with and is inhibitable by calmodulin Recombinant human protein expressed in Sf9 cells (1).GRK4 has been linked to both genetic and acquired Catalog # hypertension. GRK4 is highly expressed in testis (2). G02-31G Lot # P1749-5 References Purity >70% Concentration 0.05 µg/µl Stability 1yr at –70oC from date of shipment 1.
    [Show full text]
  • Adrenergic Receptor Transactivation of the EGFR Confers Cardioprotection
    β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection Takahisa Noma, … , Robert J. Lefkowitz, Howard A. Rockman J Clin Invest. 2007;117(9):2445-2458. https://doi.org/10.1172/JCI31901. Research Article Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β1AR signaling to the EGFR. This β-arrestin–dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein–coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin–mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family. Find the latest version: https://jci.me/31901/pdf Related Commentary, page 2396 Research article β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection Takahisa Noma,1 Anthony Lemaire,1 Sathyamangla V. Naga Prasad,1 Liza Barki-Harrington,1 Douglas G. Tilley,1 Juhsien Chen,1 Philippe Le Corvoisier,1 Jonathan D. Violin,1 Huijun Wei,1 Robert J. Lefkowitz,1,2 and Howard A. Rockman1,3,4 1Department of Medicine, 2Howard Hughes Medical Institute, 3Department of Cell Biology, and 4Department of Molecular Genetics, Duke University Medical Center, Durham, North Carolina, USA.
    [Show full text]
  • Receptor Kinase 6 Mice Deficient for G Protein-Coupled
    Increased Acute Inflammation, Leukotriene B 4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 This information is current as of September 29, 2021. Annemieke Kavelaars, Anne Vroon, Roel P. Raatgever, Alan M. Fong, Richard T. Premont, Dhavalkumar D. Patel, Robert J. Lefkowitz and Cobi J. Heijnen J Immunol 2003; 171:6128-6134; ; doi: 10.4049/jimmunol.171.11.6128 Downloaded from http://www.jimmunol.org/content/171/11/6128 References This article cites 32 articles, 22 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/171/11/6128.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 29, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 Annemieke Kavelaars,1* Anne Vroon,1* Roel P.
    [Show full text]
  • Supplementary Table S1. Kinase Selectivity of MPI-0479605. (A)
    A B In-house kinase profiling Invitrogen SelectScreen Profiling % % % Kinase IC50 ( M) Kinase IC50 ( M) inhibition inhibition inhibition AKT3 >5 IKK-epsilon >5 Kinase at 500 nM Kinase at 500 nM Kinase at 500 nM ALK 0.26 INSR 0.38 ABL1 7 GRK6 -7 PAK1 4 AUR-A >5 JAK1 >5 GRK2 4 HCK 7 PASK 6 AUR-B >5 JNK1 0.11 AKT1 2 HIPK2 9 PHKG1 8 AUR-C >5 MEK1 >5 AXL 6 HIPK4 -6 PRKCB1 -2 B-RAF 3.2 MST4 >5 BMX 14 IGF1R 37 PRKCE 0 CDK1 >5 NEK2 >5 BRSK1 -9 IRAK4 0 PRKCG 3 CDK2 >5 PDK1 >5 CAMK2D -1 ITK 9 PRKCI -2 CDK6 >5 PKA >5 CDC42 BPA 0 LCK 29 PRKCN 6 CHK1 >5 PKC-delta >5 CDK5/p25 -1 LIMK1 17 PRKCZ -5 CHK2 >5 PLK1 >5 CSF1R 82 MAP2K1 11 PRKG2 2 C-MET >5 PLK4 3.3 CSK 1 MAP2K6 2 PTK6 15 C-SRC >5 ROCK2 >5 CSNK1D 10 MAP3K3 17 RET 11 DYRK2 >5 RSK2 >5 CSNK1G1 -1 MAP3K5 -1 RPS6KA2 23 ERK2 3.9 STK33 1.1 CSNK2A2 33 MAP3K8 12 RPS6KA5 2 FAK1 2.7 TAK1 >5 DCAMKL2 2 MAPK1 0 SGK2 4 FER 0.59 TAO1 >5 DYRK1B 1 MAPK11 2 SNF1LK2 3 FLT3 0.080 TBK1 >5 EEF2K 2 MAPK12 5 SRPK2 -3 HGK >5 TRKB >5 EGFR 14 MAPK3 -2 STK22B 11 IKK-alpha >5 YES1 >5 EPHA1 6 MAPK9 61 STK23 -1 IKK-beta >5 EPHA4 -10 MAPKAPK2 9 STK24 -2 EPHB1 -33 MARK4 3 STK25 0 ERBB2 5 MELK 6 TEK 6 FGFR1 15 MERTK 7 TYK2 10 FGR 17 MKNK1 2 TYRO3 -2 FLT4 15 MST1R 3 ZAP70 5 FRAP1 7 NEK1 1 GRK4 3 NEK4 -6 Supplementary Table S1.
    [Show full text]
  • MONOCLONAL ANTI-GRK 4-6 CLONE A16/17 Purified Mouse Immunoglobulin
    MONOCLONAL ANTI-GRK 4-6 CLONE A16/17 Purified Mouse Immunoglobulin Product Number G4540 Product Description association of GRK2 with membranes.8 Conversely, Monoclonal Anti-GRK 4-6 (mouse IgG1k isotype) was GRK5 is significantly inhibited after phosphorylation by produced using a GST fusion protein corresponding to PKC because of a decreased activity and decreased 9 residues 463-590 of bovine GRK5 as immunogen. This affinity for receptor. In the visual system, GRK1 has sequence shares 95% homology with human and rat been shown to be inhibited by the Ca2+-binding protein 10 GRK5 and approximately 80% homology with human recoverin. Although regulation by recoverin may be and rat GRK4 and GRK6. The antibody is purified using specific for GRK1, a universal mediator of calcium ammonium sulfate precipitation. signaling, calmodulin, appears to inhibit all other GRK subtypes, with GRK5 being the most sensitive. Monoclonal Anti-GRK 4-6 specifically reacts with human, mouse and rat GRK 4-6. It may be used to Reagents identify GRK4, GRK5 and GRK6 by immunoblotting1 The product is supplied as purified mouse and immunoprecipitation. immunoglobulin in 0.1M Tris-glycine, pH 7.0, containing 0.035% sodium azide and 30% glycerol G-protein coupled receptor kinases (GRKs) are a family Antibody concentration is approximately 0.7 mg/ml. of serine/threonine kinases that play a role in desensitization by phosphorylation of G protein-coupled Precautions and Disclaimer receptors. Receptors are phosphorylated by GRKs in Due to the sodium azide content a material safety data vitro on intracellular loops and/or the carboxyl-terminal sheet (MSDS) has been sent to the attention of the tail.
    [Show full text]
  • GRK5 Is an Essential Co-Repressor for Cardiac Mineralocorticoid
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 17 September 2020 doi:10.20944/preprints202009.0374.v1 1 Article 2 GRK5 is an Essential Co-repressor for Cardiac 3 Mineralocorticoid Receptor Antagonism induced by 4 Finerenone but not Eplerenone 5 Victoria L. Desimine 1,+, Jennifer Ghandour 1, Natalie Cora 1, Celina M. Pollard 1, Rachel Valiente 6 1, Krysten E. Ferraino 1, Janelle Pereyra 1, Daniela Pi Noa 1, Yanelys Duarte 1, Yaimiry Martinez 1, 7 Jennifer Maning 1,‡, Barbara M. Parker 1, Ava R. Brill 1, Valentina Guidi 2, Beatrix Aukszi 2, and 8 Anastasios Lymperopoulos 1,* 9 1 Laboratory for the Study of Neurohormonal Control of the Circulation, Department of Pharmaceutical 10 Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL 33328, USA; 11 [email protected] (V.L.D.); [email protected] (J.G.); [email protected] (N.C.); 12 [email protected] (C.M.P.); [email protected] (R.V.); [email protected] (K.E.F.); 13 [email protected] (J.P.); [email protected] (D.P.N.); [email protected] (Y.D.); 14 [email protected] (Y.M.); [email protected] (J.M.); [email protected] (B.M.P.); 15 [email protected] (A.R.B.) 16 2 Department of Chemistry and Physics, Halmos College of Natural Sciences and Oceanography, Nova 17 Southeastern University, Fort Lauderdale, FL 33328, USA; [email protected] (V.G.); [email protected] 18 (B.A.) 19 * Correspondence: [email protected]; Tel.: +1-954-262-1338; Fax: +1-954-262-2278 20 † Present address: James A.
    [Show full text]