Real-Time Monitoring of GPCR/cAMP Signalling by FRET and Single-Molecule Microscopy

Abstract

G-protein-coupled receptors (GPCRs), located on the surface of virtually every cell in our organism, mediate the effects of many hormones and neurotransmitters. Although GPCRs have been extensively studied for more than 4 decades using pharmacological and biochemical methods, the recent introduction of optical methods such as fluorescence resonance energy transfer (FRET) and single-molecule microscopy is fostering novel and important discoveries in the field. Here, we review the use of such optical methods, focusing on some recent examples of their application to important and still unresolved questions concerning the spatial organisation and dynamics of GPCR signalling.

Key words

G-protein-coupled receptor - thyroid-stimulating hormone - fluorescence resonance energy transfer - single-molecule microscopy

• References

• 1 Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 2002; 3: 639-650
• 2 Lefkowitz RJ. A brief history of G-protein coupled receptors (Nobel Lecture). Angew Chem Int Ed 2013; 52: 6366-6378
• 3 Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, Kobilka BK. Structure of a nanobody-stabilized active state of the β₂ adrenoceptor. Nature 2011; 469: 175-180
• 4 Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK. Crystal structure of the human β₂ adrenergic G-protein-coupled receptor. Nature 2007; 450: 383-387
• 5 Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK. GPCR engineering yields high-resolution structural insights into β₂-adrenergic receptor function. Science 2007; 318: 1266-1273
• 6 Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK. Crystal structure of the β₂ adrenergic receptor-Gs protein complex. Nature 2011; 477: 549-555
• 7 Kobilka B. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew Chem Int Ed 2013; 52: 6380-6388
• 8 Gether U, Lin S, Kobilka BK. Fluorescent labeling of purified β₂ adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem 1995; 270: 28268-28275
• 9 Swaminath G, Xiang Y, Lee TW, Steenhuis J, Parnot C, Kobilka BK. Sequential binding of agonists to the β₂ adrenoceptor. Kinetic evidence for intermediate conformational states. J Biol Chem 2004; 279: 686-691
• 10 Kobilka BK, Deupi X. Conformational complexity of G-protein-coupled receptors. Trends Pharmacol Sci 2007; 28: 397-406
• 11 Deupi X, Kobilka BK. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. Physiology (Bethesda) 2010; 25: 293-303
• 12 Swaminath G, Deupi X, Lee TW, Zhu W, Thian FS, Kobilka TS, Kobilka B. Probing the β₂ adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists. J Biol Chem 2005; 280: 22165-22171
• 13 Deupi X, Kobilka B. Activation of G protein-coupled receptors. Adv Protein Chem 2007; 74: 137-166
• 14 Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO, Mildorf TJ, Shaw DE, Kobilka BK, Prosser RS. The role of ligands on the equilibria between functional states of a G protein-coupled receptor. J Am Chem Soc 2013; 135: 9465-9474
• 15 Nygaard R, Zou Y, Dror RO, Mildorf TJ, Arlow DH, Manglik A, Pan AC, Liu CW, Fung JJ, Bokoch MP, Thian FS, Kobilka TS, Shaw DE, Mueller L, Prosser RS, Kobilka BK. The dynamic process of β₂-adrenergic receptor activation. Cell 2013; 152: 532-542
• 16 Kohlhoff KJ, Shukla D, Lawrenz M, Bowman GR, Konerding DE, Belov D, Altman RB, Pande VS. Cloud-based simulations on Google Exacycle reveal ligand modulation of GPCR activation pathways. Nat Chem 2014; 6: 15-21
• 17 Lohse MJ, Nikolaev VO, Hein P, Hoffmann C, Vilardaga JP, Bünemann M. Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol Sci 2008; 29: 159-165
• 18 Lohse MJ, Nuber S, Hoffmann C. Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol Rev 2012; 64: 299-336
• 19 Lohse MJ, Maiellaro I, Calebiro D. Kinetics and mechanism of G protein-coupled receptor activation. Curr Opin Cell Biol 2013; 27C: 87-93
• 20 Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, Persani L, Lohse MJ. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 2009; 7: e1000172
• 21 Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ, Vilardaga JP. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 2009; 5: 734-742
• 22 Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M. Conformational biosensors reveal GPCR signalling from endosomes. Nature 2013; 495: 534-538
• 23 Calebiro D, Nikolaev VO, Persani L, Lohse MJ. Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 2010; 31: 221-228
• 24 Hern JA, Baig AH, Mashanov GI, Birdsall B, Corrie JE, Lazareno S, Molloy JE, Birdsall NJ. Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules. Proc Natl Acad Sci USA 2010; 107: 2693-2698
• 25 Kasai RS, Suzuki KG, Prossnitz ER, Koyama-Honda I, Nakada C, Fujiwara TK, Kusumi A. Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J Cell Biol 2011; 192: 463-480
• 26 Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A, Cocucci E, Zurn A, Lohse MJ. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci USA 2013; 110: 743-748
• 27 Castro M, Nikolaev VO, Palm D, Lohse MJ, Vilardaga JP. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism. Proc Natl Acad Sci USA 2005; 102: 16084-16089
• 28 Werthmann RC, Volpe S, Lohse MJ, Calebiro D. Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells. FASEB J 2012; 26: 2043-2048
• 29 Hein P, Frank M, Hoffmann C, Lohse MJ, Bünemann M. Dynamics of receptor/G protein coupling in living cells. EMBO J 2005; 24: 4106-4114
• 30 Hein P, Rochais F, Hoffmann C, Dorsch S, Nikolaev VO, Engelhardt S, Berlot CH, Lohse MJ, Bünemann M. Gs activation is time-limiting in initiating receptor-mediated signaling. J Biol Chem 2006; 281: 33345-33351
• 31 Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 1991; 349: 694-697
• 32 Bacskai BJ, Hochner B, Mahaut-Smith M, Adams SR, Kaang BK, Kandel ER, Tsien RY. Spatially resolved dynamics of cAMP and protein kinase A subunits in Aplysia sensory neurons. Science 1993; 260: 222-226
• 33 Giepmans BN, Adams SR, Ellisman MH, Tsien RY. The fluorescent toolbox for assessing protein location and function. Science 2006; 312: 217-224
• 34 Vilardaga JP, Bünemann M, Krasel C, Castro M, Lohse MJ. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat Biotechnol 2003; 21: 807-812
• 35 Hoffmann C, Gaietta G, Bünemann M, Adams SR, Oberdorff-Maass S, Behr B, Vilardaga JP, Tsien RY, Ellisman MH, Lohse MJ. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2005; 2: 171-176
• 36 Bünemann M, Frank M, Lohse MJ. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc Natl Acad Sci USA 2003; 100: 16077-16082
• 37 Nikolaev VO, Bünemann M, Hein L, Hannawacker A, Lohse MJ. Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 2004; 279: 37215-37218
• 38 Nikolaev VO, Bünemann M, Schmitteckert E, Lohse MJ, Engelhardt S. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching β₁-adrenergic but locally confined β₂-adrenergic receptor-mediated signaling. Circ Res 2006; 99: 1084-1091
• 39 Nikolaev VO, Gambaryan S, Lohse MJ. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods 2006; 3: 23-25
• 40 DiPilato LM, Cheng X, Zhang J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments. Proc Natl Acad Sci USA 2004; 101: 16513-16518
• 41 Violin JD, DiPilato LM, Yildirim N, Elston TC, Zhang J, Lefkowitz RJ. β₂-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics. J Biol Chem 2008; 283: 2949-2961
• 42 Ponsioen B, Zhao J, Riedl J, Zwartkruis F, van der Krogt G, Zaccolo M, Moolenaar WH, Bos JL, Jalink K. Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep 2004; 5: 1176-1180
• 43 Norris RP, Ratzan WJ, Freudzon M, Mehlmann LM, Krall J, Movsesian MA, Wang H, Ke H, Nikolaev VO, Jaffe LA. Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 2009; 136: 1869-1878
• 44 Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T. A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000; 2: 25-29
• 45 Zaccolo M, Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 2002; 295: 1711-1715
• 46 Mongillo M, McSorley T, Evellin S, Sood A, Lissandron V, Terrin A, Huston E, Hannawacker A, Lohse MJ, Pozzan T, Houslay MD, Zaccolo M. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 2004; 95: 67-75
• 47 Allen MD, Zhang J. Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun 2006; 348: 716-721
• 48 Zhang J, Ma Y, Taylor SS, Tsien RY. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci USA 2001; 98: 14997-15002
• 49 Depry C, Allen MD, Zhang J. Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 2011; 7: 52-58
• 50 Zhang J, Hupfeld CJ, Taylor SS, Olefsky JM, Tsien RY. Insulin disrupts β₂-adrenergic signalling to protein kinase A in adipocytes. Nature 2005; 437: 569-573
• 51 Hempel CM, Vincent P, Adams SR, Tsien RY, Selverston AI. Spatio-temporal dynamics of cyclic AMP signals in an intact neural circuitum. Nature 1996; 384: 166-169
• 52 Gorbunova YV, Spitzer NC. Dynamic interactions of cyclic AMP transients and spontaneous Ca(2 +) spikes. Nature 2002; 418: 93-96
• 53 Webb RJ, Marshall F, Swann K, Carroll J. Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes. Dev Biol 2002; 246: 441-454
• 54 Takeda N, Kyozuka K, Deguchi R. Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae. Dev Biol 2006; 298: 248-258
• 55 Goaillard JM, Vincent PV, Fischmeister R. Simultaneous measurements of intracellular cAMP and L-type Ca2+ current in single frog ventricular myocytes. J Physiol 2001; 530: 79-91
• 56 Nikolaev VO, Gambaryan S, Engelhardt S, Walter U, Lohse MJ. Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis. J Biol Chem 2005; 280: 1716-1719
• 57 Castro LR, Gervasi N, Guiot E, Cavellini L, Nikolaev VO, Paupardin-Tritsch D, Vincent P. Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons. J Neurosci 2010; 30: 6143-6151
• 58 Castro LR, Brito M, Guiot E, Polito M, Korn CW, Hervé D, Girault JA, Paupardin-Tritsch D, Vincent P. Striatal neurones have a specific ability to respond to phasic dopamine release. J Physiol 2013; 591: 3197-3214
• 59 Dunn TA, Wang CT, Colicos MA, Zaccolo M, DiPilato LM, Zhang J, Tsien RY, Feller MB. Imaging of cAMP levels and protein kinase A activity reveals that retinal waves drive oscillations in second-messenger cascades. J Neurosci 2006; 26: 12807-12815
• 60 Klarenbeek JB, Goedhart J, Hink MA, Gadella TW, Jalink K. A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS One 2011; 6: e19170
• 61 Polito M, Klarenbeek J, Jalink K, Paupardin-Tritsch D, Vincent P, Castro LR. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons. Front Cell Neurosci 2013; 7: 211
• 62 Jacobs S, Calebiro D, Nikolaev VO, Lohse MJ, Schulz S. Real-time monitoring of somatostatin receptor-cAMP signaling in live pituitary. Endocrinology 2010; 151: 4560-4565
• 63 Müllershausen F, Zecri F, Cetin C, Billich A, Guerini D, Seuwen K. Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nat Chem Biol 2009; 5: 428-434
• 64 Lohse MJ, Calebiro D. Cell biology: Receptor signals come in waves. Nature 2013; 495: 457-458
• 65 Wachten S, Masada N, Ayling LJ, Ciruela A, Nikolaev VO, Lohse MJ, Cooper DM. Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells. J Cell Sci 2010; 123: 95-106
• 66 Dyachok O, Isakov Y, Sagetorp J, Tengholm A. Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells. Nature 2006; 439: 349-352
• 67 Sample V, DiPilato LM, Yang JH, Ni Q, Saucerman JJ, Zhang J. Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP. Nat Chem Biol 2012; 8: 375-382
• 68 Liu S, Zhang J, Xiang YK. FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes. Biochem Biophys Res Commun 2011; 404: 581-586
• 69 Di Benedetto G, Scalzotto E, Mongillo M, Pozzan T. Mitochondrial Ca(2)(+) uptake induces cyclic AMP generation in the matrix and modulates organelle ATP levels. Cell Metab 2013; 17: 965-975
• 70 Lefkimmiatis K, Leronni D, Hofer AM. The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics. J Cell Biol 2013; 202: 453-462
• 71 Di Benedetto G, Zoccarato A, Lissandron V, Terrin A, Li X, Houslay MD, Baillie GS, Zaccolo M. Protein kinase A type I and type II define distinct intracellular signaling compartments. Circ Res 2008; 103: 836-844
• 72 Herget S, Lohse MJ, Nikolaev VO. Real-time monitoring of phosphodiesterase inhibition in intact cells. Cell Signal 2008; 20: 1423-1431
• 73 Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 2003; 21: 86-89