Α-Dystrobrevin-1 Recruits Α-Catulin to the Α1d- Adrenergic Receptor/Dystrophin-Associated Protein Complex Signalosome

Total Page:16

File Type:pdf, Size:1020Kb

Α-Dystrobrevin-1 Recruits Α-Catulin to the Α1d- Adrenergic Receptor/Dystrophin-Associated Protein Complex Signalosome α-Dystrobrevin-1 recruits α-catulin to the α1D- adrenergic receptor/dystrophin-associated protein complex signalosome John S. Lyssanda, Jennifer L. Whitingb, Kyung-Soon Leea, Ryan Kastla, Jennifer L. Wackera, Michael R. Bruchasa, Mayumi Miyatakea, Lorene K. Langebergb, Charles Chavkina, John D. Scottb, Richard G. Gardnera, Marvin E. Adamsc, and Chris Haguea,1 Departments of aPharmacology and cPhysiology and Biophysics, University of Washington, Seattle, WA 98195; and bDepartment of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 Edited by Robert J. Lefkowitz, Duke University Medical Center/Howard Hughes Medical Institute, Durham, NC, and approved October 29, 2010 (received for review July 22, 2010) α1D-Adrenergic receptors (ARs) are key regulators of cardiovascu- pression increases in patients with benign prostatic hypertrophy lar system function that increase blood pressure and promote vas- (12). Through proteomic screening, we discovered that α1D-ARs cular remodeling. Unfortunately, little information exists about are scaffolded to the dystrophin-associated protein complex the signaling pathways used by this important G protein-coupled (DAPC) via the anchoring protein syntrophin (10). Coexpression α “ α receptor (GPCR). We recently discovered that 1D-ARs form a sig- with syntrophins increases 1D-AR plasma membrane expression, nalosome” with multiple members of the dystrophin-associated drug binding, and activation of Gαq/11 signaling after agonist protein complex (DAPC) to become functionally expressed at the activation. Moreover, syntrophin knockout mice lose α1D-AR– plasma membrane and bind ligands. However, the molecular stimulated increases in blood pressure, demonstrating the im- α α mechanism by which the DAPC imparts functionality to the 1D- portance of these essential GIPs for 1D-AR function in vivo (10). AR signalosome remains a mystery. To test the hypothesis that Proper organization of signaling molecules within cells by the previously unidentified molecules are recruited to the α1D-AR sig- DAPC is essential for the maintenance of cellular homeostasis at nalosome, we performed an extensive proteomic analysis on each synaptic junctions (13). Mutations in DAPC result in severe PHARMACOLOGY member of the DAPC. Bioinformatic analysis of our proteomic data muscle wasting diseases, such as Duchenne muscular dystrophy/ sets detected a common interacting protein of relatively unknown Becker muscular dystrophy, and as a result, the role of this function, α-catulin. Coimmunoprecipitation and blot overlay complex for proper skeletal muscle function has been thoroughly assays indicate that α-catulin is directly recruited to the α1D-AR studied (14). However, the DAPC performs many other func- signalosome by the C-terminal domain of α-dystrobrevin-1 and tions: it facilitates proper water transport across the blood–brain not the closely related splice variant α-dystrobrevin-2. Proteomic barrier by anchoring aquaporin (15, 16), clusters nicotinic ace- and biochemical analysis revealed that α-catulin supersensitizes tylcholine receptors to ensure signal transmission at para- α1D-AR functional responses by recruiting effector molecules to sympathetic synapses (13), and anchors neuronal NOS at the cell the signalosome. Taken together, our study implicates α-catulin membrane in cardiac myocytes to permit cardiodilation (17). We as a unique regulator of GPCR signaling and represents a unique previously demonstrated that α1D-ARs form a complex with the expansion of the intricate and continually evolving array of GPCR DAPC (10), but why this interaction is necessary for α1D-AR signaling networks. functional coupling is unknown. In this study, we postulated that molecules necessary for α protein-coupled receptors (GPCRs) are seven-transmembrane 1D-AR signaling are recruited by the DAPC. Using a sequential fi α spanning proteins that are responsible for communicating in- proteomic screening approach, we identi ed -catulin as a G α formation in the form of extracellular stimuli across lipid mem- unique member of the 1D-AR signalosome. The goal of these α branes into distinct intracellular signals with precise accuracy. After experiments was to understand how -catulin integrates into this ligand binding, GPCRs signal through the canonical heterotrimeric growing GPCR protein complex and to decipher the purpose of G protein signaling pathway to activate a diverse array of down- this relatively unstudied protein in GPCR signaling networks. stream effectors (1). Recently, it has become evident that most Results GPCRs collaborate with one or more additional proteins at specific α α points in their lifecycle. These GPCR interacting proteins (or GIPs) -Catulin: Unique Member of the 1D-AR/DAPC Signalosome. We α are largely receptor subtype and cell context specific, include both previously demonstrated that syntrophins are required for 1D- α membrane and cytosolic proteins, and typically play a highly specific AR function in vitro and in vivo by anchoring 1D-ARs to the fi DAPC (10). Our working hypothesis is that the DAPC facilitates supporting role for GPCR function (i.e., traf cking, ligand binding, α enhancing signaling, signal termination, and/or degradation) (2–4). 1D-AR function by acting as a multiprotein scaffold to arrange Recently, we used yeast two-hybrid and proteomic screens to signaling molecules in close proximity to the receptor. To test this hypothesis, we fused tandem-affinity purification (TAP) epitopes identify GIPs for a clinically important GPCR, the α1D-adrenergic receptor (AR) (5). A member of the adrenergic family (α1, α2, β), α1D-ARs are ubiquitously expressed on blood vessels and are responsible for increasing blood pressure during exercise, injury, Author contributions: J.S.L., J.L. Whiting, J.L. Wacker, M.R.B., M.M., L.K.L., C.C., J.D.S., R.G.G., α M.E.A., and C.H. designed research; J.S.L., J.L. Whiting, K.-S.L., R.K., M.R.B., M.M., L.K.L., stress, or cardiovascular disease (6). 1D-AR knockout mice are M.E.A., and C.H. performed research; J.S.L., J.L. Whiting, J.L. Wacker, M.R.B., C.C., J.D.S., hypotensive and resistant to high salt diet-induced hypertension R.G.G., M.E.A., and C.H. analyzed data; and J.S.L., J.D.S., R.G.G., M.E.A., and C.H. wrote the (7, 8), yet this GPCR has been largely ignored over the past 20 y paper. fl because after transfection into cell culture α1D-ARs are seques- The authors declare no con ict of interest. tered in the endoplasmic reticulum (9, 10). Clinical interest in This article is a PNAS Direct Submission. the α1D-AR as a drug target has recently increased with the dis- 1To whom correspondence should be addressed. E-mail: [email protected]. α coveries that 1D-ARs are the predominant subtype expressed in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. epicardial coronary arteries (11) and that α1D-AR prostate ex- 1073/pnas.1010819107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1010819107 PNAS Early Edition | 1of6 α α α containing streptavidin/calmodulin-binding proteins to the α1D- -Catulin Is Recruited to the 1D-AR Signalosome by -DB1. We α α α β β AR and all members of the DAPC, including, α-syntrophin, β - detected -catulin as a positive interactor in the 1D-AR, / 1/ 2- 1 α fi syntrophin, β -syntrophin, α-dystrobrevin-1 (α-DB1), and α-DB2. syntrophin, and -DB1 proteomic screens, making it dif cult to 2 α HEK293 cell lines stably expressing each individual clone were predict which protein directly binds and recruits -catulin to the α created. TAP-tagged and associated proteins were purified from 1D-AR/DAPC signalosome. An important clue was that α-catulin was not detectable in the α-DB2 screen (Fig. 1A and lysates of each stable cell line, digested with trypsin, and the Table S1). α-DB1 and α-DB2 are C-terminal splice variants of tryptic peptides subjected to liquid chromatography (LC) tandem the α-DB gene (20). Following their common 504-aa core, mass spectrometry (MS/MS). α-DB1 has a unique 184-aa C terminus, whereas α-DB2 has a 9- fi Analysis of the peptides identi ed by LC-MS/MS revealed aa unique C terminus. Because α-catulin interacts with α-DB1 both predicted and unique components of the scaffold (Fig. 1A but not with α-DB2, we suspected that α-catulin is most likely and Table S1). The core components of the scaffold (dystrophin, recruited by association with the α-DB1 C-terminal domain. utrophin, α/β-DB, and α/β1/β2-syntrophin) were cross-identified To test this possibility, we used HEK293 cells stably expressing in each of the individual pulldowns. Our experiments identified TAP-α-DB1 or TAP-α-DB2. Coimmunoprecipitation experi- α known interactors [i.e., CASK and LIN7C with β2-syntrophin, ments revealed that TAP- -DB1 interacts with both endogenous Gα subunits with α-syntrophin (18, 19)] for each bait protein, and transiently transfected α-catulin-flag, whereas TAP-α-DB2 confirming the validity of our experimental data sets. One pro- failed to coimmunoprecipitate either protein (Fig. 2A). These tein, α-catulin (CTNNAL1) was consistently identified in the results were corroborated by experiments demonstrating that α-catulin-flag coimmunoprecipitates specifically with endoge- majority of our proteomic screens (Table S1). α α Next, we confirmed these findings using immunoprecipitation nous -DB1 but not -DB2 (Fig. 2B). Next, we used nitrocellulose blot overlay assays to test whether experiments of full-length proteins in living cells. Shown in Fig. α α α the interaction between -DB1 and -catulin is direct. TAP- 1B, lysates from HEK293 cells stably expressing TAP- 1D-ARs α-DB1, TAP-α-DB2, or untransfected HEK293 cell lysates were α α were precipitated and probed for -catulin, -DB1, and syntro- separated by SDS/PAGE, transferred to nitrocellulose, and in- phin.
Recommended publications
  • Supplemental Information to Mammadova-Bach Et Al., “Laminin Α1 Orchestrates VEGFA Functions in the Ecosystem of Colorectal Carcinogenesis”
    Supplemental information to Mammadova-Bach et al., “Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinogenesis” Supplemental material and methods Cloning of the villin-LMα1 vector The plasmid pBS-villin-promoter containing the 3.5 Kb of the murine villin promoter, the first non coding exon, 5.5 kb of the first intron and 15 nucleotides of the second villin exon, was generated by S. Robine (Institut Curie, Paris, France). The EcoRI site in the multi cloning site was destroyed by fill in ligation with T4 polymerase according to the manufacturer`s instructions (New England Biolabs, Ozyme, Saint Quentin en Yvelines, France). Site directed mutagenesis (GeneEditor in vitro Site-Directed Mutagenesis system, Promega, Charbonnières-les-Bains, France) was then used to introduce a BsiWI site before the start codon of the villin coding sequence using the 5’ phosphorylated primer: 5’CCTTCTCCTCTAGGCTCGCGTACGATGACGTCGGACTTGCGG3’. A double strand annealed oligonucleotide, 5’GGCCGGACGCGTGAATTCGTCGACGC3’ and 5’GGCCGCGTCGACGAATTCACGC GTCC3’ containing restriction site for MluI, EcoRI and SalI were inserted in the NotI site (present in the multi cloning site), generating the plasmid pBS-villin-promoter-MES. The SV40 polyA region of the pEGFP plasmid (Clontech, Ozyme, Saint Quentin Yvelines, France) was amplified by PCR using primers 5’GGCGCCTCTAGATCATAATCAGCCATA3’ and 5’GGCGCCCTTAAGATACATTGATGAGTT3’ before subcloning into the pGEMTeasy vector (Promega, Charbonnières-les-Bains, France). After EcoRI digestion, the SV40 polyA fragment was purified with the NucleoSpin Extract II kit (Machery-Nagel, Hoerdt, France) and then subcloned into the EcoRI site of the plasmid pBS-villin-promoter-MES. Site directed mutagenesis was used to introduce a BsiWI site (5’ phosphorylated AGCGCAGGGAGCGGCGGCCGTACGATGCGCGGCAGCGGCACG3’) before the initiation codon and a MluI site (5’ phosphorylated 1 CCCGGGCCTGAGCCCTAAACGCGTGCCAGCCTCTGCCCTTGG3’) after the stop codon in the full length cDNA coding for the mouse LMα1 in the pCIS vector (kindly provided by P.
    [Show full text]
  • Genetic Mutations and Mechanisms in Dilated Cardiomyopathy
    Genetic mutations and mechanisms in dilated cardiomyopathy Elizabeth M. McNally, … , Jessica R. Golbus, Megan J. Puckelwartz J Clin Invest. 2013;123(1):19-26. https://doi.org/10.1172/JCI62862. Review Series Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of congestive heart failure. In hypertrophic cardiomyopathy (HCM), cardiac output is limited by the thickened myocardium through impaired filling and outflow. Mutations in the genes encoding the thick filament components myosin heavy chain and myosin binding protein C (MYH7 and MYBPC3) together explain 75% of inherited HCMs, leading to the observation that HCM is a disease of the sarcomere. Many mutations are “private” or rare variants, often unique to families. In contrast, dilated cardiomyopathy (DCM) is far more genetically heterogeneous, with mutations in genes encoding cytoskeletal, nucleoskeletal, mitochondrial, and calcium-handling proteins. DCM is characterized by enlarged ventricular dimensions and impaired systolic and diastolic function. Private mutations account for most DCMs, with few hotspots or recurring mutations. More than 50 single genes are linked to inherited DCM, including many genes that also link to HCM. Relatively few clinical clues guide the diagnosis of inherited DCM, but emerging evidence supports the use of genetic testing to identify those patients at risk for faster disease progression, congestive heart failure, and arrhythmia. Find the latest version: https://jci.me/62862/pdf Review series Genetic mutations and mechanisms in dilated cardiomyopathy Elizabeth M. McNally, Jessica R. Golbus, and Megan J. Puckelwartz Department of Human Genetics, University of Chicago, Chicago, Illinois, USA. Genetic mutations account for a significant percentage of cardiomyopathies, which are a leading cause of conges- tive heart failure.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • The Biomarkers of Key Mirnas and Target Genes Associated with Acute Myocardial Infarction
    The biomarkers of key miRNAs and target genes associated with acute myocardial infarction Qi Wang1, Bingyan Liu2,3, Yuanyong Wang4, Baochen Bai1, Tao Yu3 and Xian–ming Chu1,5 1 Department of Cardiology, The Affiliated hospital of Qingdao University, Qingdao, China 2 School of Basic Medicine, Qingdao University, Qingdao, China 3 Institute for Translational Medicine, Qingdao University, Qingdao, China 4 Department of Thoracic Surgery, Affiliated Hospital of Qingdao University, Qingdao, China 5 Department of Cardiology, The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, China ABSTRACT Background. Acute myocardial infarction (AMI) is considered one of the most prominent causes of death from cardiovascular disease worldwide. Knowledge of the molecular mechanisms underlying AMI remains limited. Accurate biomarkers are needed to predict the risk of AMI and would be beneficial for managing the incidence rate. The gold standard for the diagnosis of AMI, the cardiac troponin T (cTnT) assay, requires serial testing, and the timing of measurement with respect to symptoms affects the results. As attractive candidate diagnostic biomarkers in AMI, circulating microRNAs (miRNAs) are easily detectable, generally stable and tissue specific. Methods. The Gene Expression Omnibus (GEO) database was used to compare miRNA expression between AMI and control samples, and the interactions between miRNAs and mRNAs were analysed for expression and function. Furthermore, a protein-protein interaction (PPI) network was constructed. The miRNAs identified in the bioinformatic analysis were verified by RT-qPCR in an H9C2 cell line. The miRNAs in plasma samples from patients with AMI (n D 11) and healthy controls (n D 11) were used to construct Submitted 23 December 2019 receiver operating characteristic (ROC) curves to evaluate the clinical prognostic value Accepted 14 April 2020 of the identified miRNAs.
    [Show full text]
  • Multiomic Approaches to Uncover the Complexities of Dystrophin-Associated Cardiomyopathy
    International Journal of Molecular Sciences Review Multiomic Approaches to Uncover the Complexities of Dystrophin-Associated Cardiomyopathy Aoife Gowran 1,*, Maura Brioschi 2, Davide Rovina 1 , Mattia Chiesa 3,4 , Luca Piacentini 3,* , Sara Mallia 1, Cristina Banfi 2,* , Giulio Pompilio 1,5,6,* and Rosaria Santoro 1,4 1 Unit of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy; [email protected] (D.R.); [email protected] (S.M.); [email protected] (R.S.) 2 Unit of Cardiovascular Proteomics, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy; [email protected] 3 Bioinformatics and Artificial Intelligence Facility, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy; [email protected] 4 Department of Electronics, Information and Biomedical Engineering, Politecnico di Milano, 20133 Milan, Italy 5 Department of Cardiac Surgery, Centro Cardiologico Monzino-IRCCS, 20138 Milan, Italy 6 Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20122 Milan, Italy * Correspondence: [email protected] (A.G.); [email protected] (L.P.); cristina.banfi@cardiologicomonzino.it (C.B.); [email protected] (G.P.) Abstract: Despite major progress in treating skeletal muscle disease associated with dystrophinopathies, cardiomyopathy is emerging as a major cause of death in people carrying dystrophin gene mutations that remain without a targeted cure even with new treatment directions and advances in modelling Citation: Gowran, A.; Brioschi, M.; abilities. The reasons for the stunted progress in ameliorating dystrophin-associated cardiomyopathy Rovina, D.; Chiesa, M.; Piacentini, L.; (DAC) can be explained by the difficulties in detecting pathophysiological mechanisms which can also Mallia, S.; Banfi, C.; Pompilio, G.; Santoro, R.
    [Show full text]
  • Ryanodine Receptors Are Part of the Myospryn Complex in Cardiac Muscle Received: 10 March 2017 Matthew A
    www.nature.com/scientificreports OPEN Ryanodine receptors are part of the myospryn complex in cardiac muscle Received: 10 March 2017 Matthew A. Benson1, Caroline L. Tinsley2, Adrian J. Waite 2, Francesca A. Carlisle2, Steve M. Accepted: 12 June 2017 M. Sweet3, Elisabeth Ehler4, Christopher H. George5, F. Anthony Lai5,6, Enca Martin-Rendon7 Published online: 24 July 2017 & Derek J. Blake 2 The Cardiomyopathy–associated gene 5 (Cmya5) encodes myospryn, a large tripartite motif (TRIM)- related protein found predominantly in cardiac and skeletal muscle. Cmya5 is an expression biomarker for a number of diseases afecting striated muscle and may also be a schizophrenia risk gene. To further understand the function of myospryn in striated muscle, we searched for additional myospryn paralogs. Here we identify a novel muscle-expressed TRIM-related protein minispryn, encoded by Fsd2, that has extensive sequence similarity with the C-terminus of myospryn. Cmya5 and Fsd2 appear to have originated by a chromosomal duplication and are found within evolutionarily-conserved gene clusters on diferent chromosomes. Using immunoafnity purifcation and mass spectrometry we show that minispryn co-purifes with myospryn and the major cardiac ryanodine receptor (RyR2) from heart. Accordingly, myospryn, minispryn and RyR2 co-localise at the junctional sarcoplasmic reticulum of isolated cardiomyocytes. Myospryn redistributes RyR2 into clusters when co-expressed in heterologous cells whereas minispryn lacks this activity. Together these data suggest a novel role for the myospryn complex in the assembly of ryanodine receptor clusters in striated muscle. Te unique cytoskeletal organisation of striated muscle is dependent upon the formation of specialised interac- tions between proteins that have both structural and signalling functions1.
    [Show full text]
  • Updates on Myopia
    Updates on Myopia A Clinical Perspective Marcus Ang Tien Y. Wong Editors Updates on Myopia Marcus Ang • Tien Y. Wong Editors Updates on Myopia A Clinical Perspective Editors Marcus Ang Tien Y. Wong Singapore National Eye Center Singapore National Eye Center Duke-NUS Medical School Duke-NUS Medical School National University of Singapore National University of Singapore Singapore Singapore This book is an open access publication. ISBN 978-981-13-8490-5 ISBN 978-981-13-8491-2 (eBook) https://doi.org/10.1007/978-981-13-8491-2 © The Editor(s) (if applicable) and The Author(s) 2020, corrected publication 2020 Open Access This book is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this book are included in the book's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the book's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifc statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.
    [Show full text]
  • 'Clustering of Nicotinic Acetylcholine Receptors: from the Neuromuscular
    Huh, K H; Fuhrer, C. Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses. Mol. Neurobiol. 2002, 25(1):79-112. Postprint available at: http://www.zora.unizh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.unizh.ch Originally published at: Mol. Neurobiol. 2002, 25(1):79-112 Winterthurerstr. 190 CH-8057 Zurich http://www.zora.unizh.ch Year: 2002 Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses Huh, K H; Fuhrer, C Huh, K H; Fuhrer, C. Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses. Mol. Neurobiol. 2002, 25(1):79-112. Postprint available at: http://www.zora.unizh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.unizh.ch Originally published at: Mol. Neurobiol. 2002, 25(1):79-112 Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses Abstract Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds.
    [Show full text]
  • Individual Protomers of a G Protein-Coupled Receptor Dimer Integrate Distinct Functional Modules
    OPEN Citation: Cell Discovery (2015) 1, 15011; doi:10.1038/celldisc.2015.11 © 2015 SIBS, CAS All rights reserved 2056-5968/15 ARTICLE www.nature.com/celldisc Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules Nathan D Camp1, Kyung-Soon Lee2, Jennifer L Wacker-Mhyre2, Timothy S Kountz2, Ji-Min Park2, Dorathy-Ann Harris2, Marianne Estrada2, Aaron Stewart2, Alejandro Wolf-Yadlin1, Chris Hague2 1Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USA; 2Department of Pharmacology, University of Washington School of Medicine, Seattle, WA, USA Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)—a key regulator of cardiovascular, urinary and CNS function—binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand inter- action, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.
    [Show full text]
  • Human Stem Cell Models Identify Targets of Healthy
    HUMAN STEM CELL MODELS IDENTIFY TARGETS OF HEALTHY AND MALIGNANT HEMATOPOIETIC REGULATION BY JENNIFER C REID, BSc A Thesis Submitted to the School of Graduate Studies In Partial Fulfillment of the Requirements For the Degree Doctor of Philosophy McMaster University ÓCopyright by Jennifer C Reid, April 2020 JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry Descriptive Note McMaster University BACHELOR OF SCIENCE (2015) Hamilton, Ontario (Biomedical Research Specialization, Co-op) Title: Use of human stem cell models to identify targets of healthy and malignant hematopoietic regulation Author: Jennifer C. Reid Supervisor: Dr. Mickie Bhatia Number of pages: xii, 163 ii JC Reid <- PhD Thesis ❖ McMaster University $ Biochemistry AbstrAct Hematopoiesis is the highly regenerative process of producing billions of blood cells each day, including white blood cells, red blood cells, and platelets. Given the relatively short life span of these mature cells, hematopoiesis is dependent on stem and progenitor cells to generate renewed progeny, which represents a tightly regulated process. This includes cell intrinsic and external factors, and where dysregulation can lead to anemia and cancer. As such, the hematopoietic hierarchy has been intensely studied for nearly a century and represents a gold standard model of cell fate and developmental biology, in research and clinical applications. Cellular models, such as in vitro culture and human-mouse xenografts in vivo, have been developed to explain complex phenomena pertaining to hematopoiesis and also interrogate processes which are too invasive to study in humans. Hematopoietic generation is required beyond sustaining homeostasis, and progenitors can be damaged through cytotoxic injuries such as radiation and standard chemotherapy, and also undergo leukemic transformation.
    [Show full text]
  • Dystrophin Complex Functions As a Scaffold for Signalling Proteins☆
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1838 (2014) 635–642 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Review Dystrophin complex functions as a scaffold for signalling proteins☆ Bruno Constantin IPBC, CNRS/Université de Poitiers, FRE 3511, 1 rue Georges Bonnet, PBS, 86022 Poitiers, France article info abstract Article history: Dystrophin is a 427 kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and Received 27 May 2013 incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex Received in revised form 22 August 2013 (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins Accepted 28 August 2013 and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force trans- Available online 7 September 2013 duction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaf- fold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling Keywords: Dystrophin-associated protein complex (DAPC) processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. syntrophin The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle Sodium channel cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More TRPC channel generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins.
    [Show full text]
  • Supplementary Table 1
    Supplementary Table 1. Large-scale quantitative phosphoproteomic profiling was performed on paired vehicle- and hormone-treated mTAL-enriched suspensions (n=3). A total of 654 unique phosphopeptides corresponding to 374 unique phosphoproteins were identified. The peptide sequence, phosphorylation site(s), and the corresponding protein name, gene symbol, and RefSeq Accession number are reported for each phosphopeptide identified in any one of three experimental pairs. For those 414 phosphopeptides that could be quantified in all three experimental pairs, the mean Hormone:Vehicle abundance ratio and corresponding standard error are also reported. Peptide Sequence column: * = phosphorylated residue Site(s) column: ^ = ambiguously assigned phosphorylation site Log2(H/V) Mean and SE columns: H = hormone-treated, V = vehicle-treated, n/a = peptide not observable in all 3 experimental pairs Sig. column: * = significantly changed Log 2(H/V), p<0.05 Log (H/V) Log (H/V) # Gene Symbol Protein Name Refseq Accession Peptide Sequence Site(s) 2 2 Sig. Mean SE 1 Aak1 AP2-associated protein kinase 1 NP_001166921 VGSLT*PPSS*PK T622^, S626^ 0.24 0.95 PREDICTED: ATP-binding cassette, sub-family A 2 Abca12 (ABC1), member 12 XP_237242 GLVQVLS*FFSQVQQQR S251^ 1.24 2.13 3 Abcc10 multidrug resistance-associated protein 7 NP_001101671 LMT*ELLS*GIRVLK T464, S468 -2.68 2.48 4 Abcf1 ATP-binding cassette sub-family F member 1 NP_001103353 QLSVPAS*DEEDEVPVPVPR S109 n/a n/a 5 Ablim1 actin-binding LIM protein 1 NP_001037859 PGSSIPGS*PGHTIYAK S51 -3.55 1.81 6 Ablim1 actin-binding
    [Show full text]