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cAMP Signaling: Methods and Protocols PDF

227 Pages·2015·6.899 MB·English
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Methods in Molecular Biology 1294 Manuela Zaccolo Editor cAMP Signaling Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 cAMP Signaling Methods and Protocols Edited by Manuela Zaccolo Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, UK Editor Manuela Z accolo Department of Physiology, Anatomy, and Genetics University of Oxford Oxford, UK ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2536-0 ISBN 978-1-4939-2537-7 (eBook) DOI 10.1007/978-1-4939-2537-7 Library of Congress Control Number: 2015934164 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2 015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Cover Illustration: Epifl uorescence image of a neonatal cardiac myocyte expressing a FRET-based reporter for detection of cAMP levels in real-time. The panels on the left and centre show the pseudocolor image of the cell in the cyan (480nm) and yellow (540nm) channels, respectively, acquired on excitation of the sample at 430nm. The panel on the right shows the image of the 480nm/535 nm FRET signal from the same cell, showing in red subcellular compartments with higher cAMP levels. The myocytes was treated with 10nM isoproterenol to trigger generation of cAMP. Image: M Zaccolo (University of Oxford, UK). Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Prefa ce Adenosine 3′,5′-monophosphate (cAMP), the prototypical intracellular second messenger, regulates a large variety of cellular functions and biological processes, including gene tran- scription, cell metabolism, proliferation, development, as well as more specialized functions depending on the cell type. In its simpler formulation, the cAMP signaling pathway involves a hormone (the “fi rst” messenger) that binds and activates a specifi c G protein-coupled receptor that in turn activates adenylyl cyclases to synthesize cAMP. The intracellular (or “second”) messenger cAMP then binds to a limited number of intracellular effectors, most notably to protein kinase A (PKA), which phosphorylates downstream targets leading to a specifi c functional outcome. Signal termination is mediated by phosphodiesterases (that hydrolyze cAMP) and phosphatases (that dephosphorylate PKA targets), enzymes that are modulated by complex regulatory mechanisms. In the last 15 years, the fi eld of cAMP signaling has witnessed an exciting development with accumulating evidence demonstrating that cAMP is compartmentalized and that spa- tial regulation of cAMP signals is critical for faithful signal propagation and for specifi city of response. This realization has changed our understanding of cAMP signaling from a model where a linear pathway connects the receptor located at the plasma membrane with an effector and its function to a model where signal propagation occurs within a complex net- work of cAMP-dependent signaling pathways simultaneously operating within the same cell. The pathway or pathways the cAMP signal travels along are dictated by the overall state of the cell at the time the cAMP signal is generated, depending on the activity of on/off signals that operate on individual routes at that particular time. Based on this new model, the functional outcome of a signal mediated by cAMP depends strictly on local and tempo- ral regulation. The hormonal specifi city of cAMP action results from the generation of distinct pools of the second messenger which in turn mediate different functional outcomes via activation of different subsets of the cAMP effector PKA. PKA is largely localized to different subcellular compartments via binding to a family of scaffolding proteins known as A Kinase Anchoring Proteins (AKAPs). Apart from their common ability to anchor PKA, AKAPs show a high degree of structural variability which allows for different subcellular localization and binding to a variety of other signaling components. As a result, AKAPs serve as signaling centers, where elements of the cAMP signaling pathway and other regula- tory molecules are organized for a particular task. The realization of this extremely complex spatial organization and local regulation is providing novel mechanistic insight into cell physiology and is producing a novel frame- work for the identifi cation of disease mechanisms. This new model also offers the potential to establish original avenues for the treatment of disease. New approaches have been devel- oped that allow researchers to gain information that goes beyond a measure of cAMP activ- ity at the whole cell or cell population level. In preparing this volume, I have tried to encompass new technological developments that specifi cally address questions related to cAMP compartmentalization, that probe relevant protein–protein interactions, that increase the spatial and temporal resolution of cAMP signals detection, and that can facilitate integration of the mounting complexity of the information that is becoming available on this signaling system. v vi Preface I am extremely grateful to all authors for living with my deadlines and providing excel- lent and comprehensive methods and extensive notes with essential “tricks of the trade” that are so precious when troubleshooting a new technique. Finally, I thank the Senior Editor, John Walker, for giving me the opportunity to compile this volume in the excellent series, Methods in Molecular Biology . I hope the selection of methods will prove appealing and will be a real resource to researchers in the fi eld. Oxford, UK M anuela Zaccolo Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x 1 Simultaneous Assessment of cAMP Signaling Events in Different Cellular Compartments Using FRET-Based Reporters. . . . . . . . . . . . . . . . . . . . . . . . . 1 Alex B urdyga and Konstantinos L efkimmiatis 2 R ecording Intracellular cAMP Levels with EPAC-Based FRET Sensors by Fluorescence Lifetime Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Marcel R aspe , Jeffrey K larenbeek , and Kees Jalink 3 A Novel Approach Combining Real-Time Imaging and the Patch-Clamp Technique to Calibrate FRET-Based Reporters for cAMP in Their Cellular Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5 Andreas K oschinski and Manuela Zaccolo 4 Structure-Based, In Silico Approaches for the Development of Novel cAMP FRET Reporters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Matías M achado and Sergio P antano 5 A utomated Image Analysis of FRET Signals for Subcellular cAMP Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 9 Silas J. Leavesley , Arie N akhmani , Yi G ao , and T homas C. R ich 6 Channel-Based Reporters for cAMP Detection . . . . . . . . . . . . . . . . . . . . . . . . 7 1 Thomas C. Rich , Wenkuan X in , Silas J . L eavesley , and Mark S. Taylor 7 I maging Sub-plasma Membrane cAMP Dynamics with Fluorescent Translocation Reporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5 Anders T engholm and Olof I devall-Hagren 8 A denoviral Transduction of FRET-Based Biosensors for cAMP in Primary Adult Mouse Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Oliver Lomas , Marcella B rescia , R icardo Carnicer , Stefania M onterisi , Nicoletta C . Surdo , and Manuela Z accolo 9 G eneration of Transgenic Mice Expressing FRET Biosensors. . . . . . . . . . . . . . 1 17 Daniela Hübscher and Viacheslav O. N ikolaev 10 P hotoactivatable Adenylyl Cyclases (PACs) as a Tool to Study cAMP Signaling In Vivo: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Marina Efetova and Martin Schwärzel 11 Selective Disruption of the AKAP Signaling Complexes. . . . . . . . . . . . . . . . . . 1 37 Eileen J . Kennedy and John D . S cott 12 Screening for Small Molecule Disruptors of AKAP–PKA Interactions . . . . . . . 1 51 Carolin S chächterle , Frank Christian , J oão Miguel Parente Fernandes , and Enno Klussmann vii viii Contents 13 Structure-Based Bacteriophage Screening for AKAP-S elective PKA Regulatory Subunit Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67 Ryan Walker-Gray and Matthew G . G old 14 A Yeast-Based High-Throughput Screen for Modulators of Phosphodiesterase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 81 Ana Santos de Medeiros and Charles S. H offman 15 Separation of PKA and PKG Signaling Nodes by Chemical Proteomics . . . . . . 191 Eleonora Corradini , Albert J. R . H eck , and Arjen S cholten 16 Development of Computational Models of cAMP Signaling . . . . . . . . . . . . . . 2 03 Susana R . N eves-Zaph and R oy S . Song Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 19 Contributors MARCELLA BRESCIA • Department of Physiology, Anatomy, and Genetics, U niversity of Oxford , O xford , U K ALEX BURDYGA • Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, BHF Centre of Research Excellence, University of Oxford, Sherrington Building, Parks Road, Oxford, UK RICARDO CARNICER • Department of Cardiovascular Medicine , U niversity of Oxford , Oxford , U K FRANK CHRISTIAN • Institute of Cardiovascular and Medical Sciences, University of Glasgow , Glasgow, U K ELEONORA CORRADINI • Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Netherlands Proteomics Centre and Utrecht Institute for Pharmaceutical Sciences, Utrecht University , Domplein, Utrecht, T he Netherlands MARINA EFETOVA • Institute for Biology Neurobiology, F reie Universität Berlin , B erlin , Germany YI G AO • Department of Biomedical Informatics, S tony Brook University , Stony Brook, NY, U SA MATTHEW G. GOLD , P H.D. • Department of Neuroscience, Physiology, & Pharmacology, University College London , L ondon, UK OLOF IDEVALL-HAGREN • Department of Medical Cell Biology, Biomedical Centre, Uppsala University , U ppsala , S weden ALBERT J.R. HECK • Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Netherlands Proteomics Centre and Utrecht Institute for Pharmaceutical Sciences, U trecht University , U trecht, T he Netherlands CHARLES S. HOFFMAN • Biology Department , B oston College , C hestnut Hill, M A , U SA DANIELA HÜBSCHER • Heart Research Center Göttingen, Georg August University Medical Center , U niversity of Göttingen , G öttingen, G ermany KEES JALINK • Department of Cell Biology , T he Netherlands Cancer Institute , Amsterdam, The Netherlands EILEEN J . KENNEDY • Department of Pharmaceutical and Biomedical Sciences, U niversity of Georgia College of Pharmacy , A thens , GA , U SA JEFFREY KLARENBEEK • Department of Cell Biology , T he Netherlands Cancer Institute , Amsterdam, T he Netherlands ENNO KLUSSMANN • Max Delbruck Center for Molecular Medicine Berlin (MDC) , Berlin, Germany ANDREAS KOSCHINSKI • Department of Physiology, Anatomy, and Genetics, U niversity of Oxford , O xford , U K SILAS J . LEAVESLEY • Department of Chemical and Biomolecular Engineering, Center for Lung Biology, U niversity of South Alabama , M obile , A L, U SA ; D epartment of Pharmacology, Center for Lung Biology, University of South Alabama , Mobile, AL, U SA KONSTANTINOS L EFKIMMIATIS • Department of Physiology, Anatomy and Genetics, Burdon Sanderson Cardiac Science Centre, BHF Centre of Research Excellence, University of Oxford, Sherrington Building, Parks Road, Oxford, UK ix

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