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Nuclear Localisation and Function of the G Protein-Coupled Receptor Kinase 4 Subfamily

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Nuclear Localisation and Function of the G Protein-Coupled Receptor Kinase 4 Subfamily Nuclear localisation and function of the G protein-coupled Receptor Kinase 4 subfamily Laura R Johnson MRC Laboratory for Molecular Cell Biology and Cell Biology Unit & Department of Pharmacology, University College London Submitted for the degree of Doctor of Philosophy December 2006 1 UMI Number: U591752 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591752 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 I, Laura Ruth Johnson, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis 2 Publications Papers G protein-coupled receptor kinase 5 contains a DNA-binding nuclear localisation sequence. Johnson, L.R., Scott, M.G.H. & Pitcher, J.A. Mol Cell Biol. 2004 Dec; 24(23): 10169-79. GRK5 DNA-binding ability and kinase activity are required for inhibition of hypertrophy in myocytes. Johnson, L.R. & Pitcher, J.A. PNAS paper in revision. The G protein-coupled receptor kinase 4 subfamily members contain nuclear localization sequences. Johnson, L.R. & Pitcher, J.A. JBC paper in preparation. Posters/Presentations Nuclear Localisation of the GRK4 Subfamily of GRKs. Johnson, L.R. & Pitcher, J.A. Keystone Symposium, G Protein-Coupled Receptors: Evolving Concepts and New Techniques. February 12 - 17, 2006. Keystone, Colorado. Selected for oral presentation. Differential Regulation of the Nuclear Localisation of GRK4 Subfamily Members. Johnson, L.R. & Pitcher, J.A. BioScience 2005: From genes to systems. July 17-21, 2005. Glasgow, UK. Differential Regulation of the Nuclear Localisation of G Protein-Coupled Receptor Kinase 4 Subfamily Members. Johnson, L.R. & Pitcher, J.A. 4th BPS Focused Meeting on Cell Signalling. April 11 - 12, 2005. Leicester, UK. Selected for oral presentation. 3 Abstract G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate and mediate densensitisation of agonist-occupied G protein- coupled receptors (GPCRs). Recent evidence suggests, however, additional cellular functions for these enzymes. The seven mammalian GRKs are divided into the GRK1-, GRK2- and GRK4-like subfamilies. I have discovered that GRK4 subfamily members contain functional nuclear localisation sequences (NLSs). The location of the NLSs of GRK5 and 6 is similar whereas the NLS of GRK4 is more N-terminal. Although all members of the GRK4 subfamily contain NLSs the nuclear localisation of these kinases is differentially regulated by Ca 2+/CaM. Furthermore, the NLS of GRKs 5 and 6 , but not GRK4, mediate DNA-binding in vitro, suggesting differential nuclear signalling roles for these enzymes. I investigated structural determinants of GRK5 required for DNA-binding and nuclear localisation. The ability of GRK5 to bind DNA requires an intact NLS and an N-terminally located calmodulin (CaM) binding domain. Additionally, CaM- dependent autophosphorylation of GRK5 reduces the affinity of GRK5 for DNA. Overexpression of GRK5 in cultured rat neonatal cardiac myocytes rescues hypertrophy induced by phenylephrine, an a-adrenergic receptor agonist, or by activation of PLC. Hypertrophy is not rescued by over expression of GRK5 with a mutant NLS (GRK5ANLS), mutant N-terminal CaM binding site (GRK5NTPB) or a catalytically in active version of the kinase (GRK5K215R). That GRK5 and GRK5ANLS show similar kinase activity in vitro suggests that both the kinase activity and the DNA-binding ability of GRK5 contributes to its role in rescuing hypertrophy. Since GRK5 rescues hypertrophy induced downstream of adrenergic receptors, by PLC activation, GRK5 DNA-binding ability and kinase activity in the nucleus would appear to be required for this effect. Clearly, the GRK4 subfamily of GRKs is more diverse than previously appreciated and play a role in cellular signalling in the nucleus distinct from GPCR desensitisation. 5 To Julie... .. .for being an inspirational scientist. The amount you give to your field is outstanding. Without your unwavering support and guidance I would never have completed this journey in one piece! There are no words to describe how thankful I am that you agreed to be my PhD supervisor - 1 could not have envisaged such a fun filled journey without you. Thank you from the bottom of my heart for all you have given to me. Matt... .. .for unending love and support. I could not have completed this without you. The best decision I ever made was not going to do this in Manchester! Love as always. Mum & Dad... .. .for giving me the vision to reach as far as I could. Thank you for all your support, love and encouragement for the last twenty-six years; it is because of you that I am here now. My PhD Year Group... .. .Rosy, Uli, Karina and Emma - thanks for always being there! 6 Abbreviations used in this thesis ocAR: a-adrenergic receptor ocibAR: aie-adrenergic receptor PAR: p-adrenergic receptor parr: Parrestin p-MHC: P-myosin heavy chain a-MHC: a-myosin heavy chain 7TMRs: seven transmembrane receptors AC: adenylyl cyclase ACE: angiotensin converting enzyme ACh: acetylcholine ANF: atrial natriuretic factor Angll: angiotensin II AnglliAR: angiotensin II type IA receptor Ao: angiotensinogen AP: autophosphorylation aPKC: atypical protein kinase C AR: adrenergic receptor 7 ATP: adenosine triphosphate ATR: angiotensin receptor bHLH: basic helix-loop-helix BSA: bovine serum albumin Ca2+/CaM: calcium-calmodulin Ca2+: calcium CALM: clathrin assembly lymphoid myeloid leukemia CaM: calmodulin CaMBP/m: cytosolic CaM binding sequestrant CaMBP/n: nuclear CaM binding sequestrant cAMP: cyclic adenosine monophosphate CAMTA2: calmodulin binding transcription activator 2 CASTing: cyclic amplification of targets CBP: cAMP response element-binding-protein CCPs: clathrin coated pits CCR: chemokine receptor CDKs: cyclin dependent kinases cGMP: cyclic guanosine monophosphate CHF: chronic heart failure ChIP: chromatin immunoprecipitation CHO: Chinese hamster ovary CHO-M3: Chinese hamster ovary cells expressing muscarinic M3 receptor cPKC: classical PKC CRM: chromosomal region maintenance CsA: cyclosporine A CSP: calcium sensor protein CTPB: C-terminal polybasic CXCR: chemokine receptor DAG: diacylglycerol DMEM: Dulbecco’s Modified Eagle Medium DNA: deoxyribonucleic acid E. coli: Escherichia coli EGF: epidermal growth factor EGFR: epidermal growth factor receptor ENaCs: epithelial sodium channels ER: endoplasmic reticulum ERK: extracellular signal-regulated kinase ERM: ezrin-radixin-moesin 9 ETi: endothelin -1 ETiR: endothelin-1 receptor EYFP: enhanced yellow fluorescent protein FCS: foetal calf serum G protein: GTP-binding protein GAP: GTPase activating protein GEF: guanine nucleotide exchange factor GIRK: G protein-coupled inwardly rectifying potassium channel GIT: G protein-coupled receptor kinase-interacting protein GPCR: G protein-coupled receptor GRK: G protein-coupled receptor kinase HATs: histone acetyltransferases HCC: hepatocellular carcinoma HD AC: histone deacetylase HEK-293: human embryonic kidney 293 HEp2: human epithelial 2 HF: heart failure IP3: 1,4,5-trisphosphate Ik B: inhibitor of kappa B JAK: janus-family tyrosine kinase JNK: c-Jun amino-terminal kinase LB: luria broth M2MR: M2 muscarinic receptor M3MR: M3 muscarinic receptor MAPK: mitogen-activated protein kinase MEF2: myocyte enhancer factor 2 MEK1: MAPK/ERK kinase 1 MK2: MAPK-activated protein kinase 2 MKK: mitogen activated protein kinase kinase MR: muscarinic receptor mRNA: messenger ribonucleic acid NES: nuclear export sequence NFAT: nuclear factor of activated T cells NF k B: nuclear factor kappa B NHERF: Na+/H+ exchanger regulatory factor NLS: nuclear localisation sequence NPC: nuclear pore complex nPKC: novel PKC NTPB: N-terminal polybasic PBS: phosphate-buffered saline PCR: polymerase chain reaction PDZ: PSD-95, Dig, ZO-1 homology PE: phenylephrine PH: pleckstrin homology PI3K: phosphoinositide 3-kinases PIP2 : phosphatidylinositol 4,5-bisphosphate PIP3 : phosphatidylinositol 3,4,5-trisphosphate PKA: protein kinase A, cAMP-dependent protein kinase PKC: protein kinase C or Ca2+-dependent protein kinase PKI: protein kinase C inhibitor PLC: phospholipase C PM A: phorbol ester 12-tetradecanoylphorbol-13 acetate PMSF: phenylmethanesulfonyl fluoride RGS: regulator of G protein signalling RH: regulator of G protein signalling homology domain RNA: ribonucleic acid RNAi: ribonucleic acid interference ROS: rod outer segments RPMI: Roswell Park Memorial Institute R-Smads: receptor activated smads RT: room temperature RTK: receptor tyrosine kinase SDS: sodium dodecyl sulphate SDS-PAGE: SDS polyacrylamide gel electrophoresis SHHF: spontaneously hypertensive heart failure SR: sarcoplasmic reticulum SRF: serum response factor ssDNA: single stranded deoxyribonucleic acid STAT: signal transducers and activators of transcription TEMED: N, N, N', N'-tetramethyl-ethylenediamine TF: transcription factor TPA: 12-o-tetradecanoylphorbol-13 -acetate TSA: trichostatin A WT: wildtype 13 Table of Contents 1. Introduction
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