Investigating Internalization and Intracellular Trafficking of GPCRs: New Techniques and Real-Time Experimental Approaches

  • Simon R. Foster
  • Hans Bräuner-OsborneEmail author
Part of the Handbook of Experimental Pharmacology book series (HEP, volume 245)


The ability to regulate the interaction between cells and their extracellular environment is essential for the maintenance of appropriate physiological function. For G protein-coupled receptors (GPCRs), this regulation occurs through multiple mechanisms that provide spatial and temporal control for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies that have been used to investigate internalization and intracellular signaling of GPCRs, with a particular focus on emerging real-time techniques. These recent developments have improved our understanding of the complexities of GPCR internalization and intracellular signaling and suggest that the broader biological relevance and potential therapeutic implications of these processes remain to be explored.


Arrestin Endosome G protein-coupled receptor Internalization Signaling SNAP-tag 



The authors would like to acknowledge members of the Bräuner-Osborne lab and Dr. Maria Waldhoer for the helpful discussions. S.R.F. acknowledges funding from the Independent Research Fund Denmark | Medical Sciences, the Lundbeck Foundation, and the Augustinus Foundation.


  1. Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG (2003) Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process. J Cell Biol 160:321–328PubMedPubMedCentralCrossRefGoogle Scholar
  2. Arancibia-Carcamo IL, Fairfax BP, Moss SJ, Kittler JT (2006) Studying the localization, surface stability and endocytosis of neurotransmitter receptors by antibody labeling and biotinylation approaches. In: Kittler JT, Moss SJ (eds) The dynamic synapse: molecular methods in ionotropic receptor biology. CRC Press/Taylor & Francis, Taylor & Francis Group, Boca RatonGoogle Scholar
  3. Barak LS, Ferguson SSG, Zhang J, Caron MG (1997) A β-Arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J Biol Chem 272:27497–27500PubMedCrossRefGoogle Scholar
  4. Beautrait A, Paradis JS, Zimmerman B, Giubilaro J, Nikolajev L, Armando S, Kobayashi H, Yamani L, Namkung Y, Heydenreich FM, Khoury E, Audet M, Roux PP, Veprintsev DB, Laporte SA, Bouvier M (2017) A new inhibitor of the beta-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling. Nat Commun 8:15054PubMedPubMedCentralCrossRefGoogle Scholar
  5. Boivin B, Vaniotis G, Allen BG, Hebert TE (2008) G protein-coupled receptors in and on the cell nucleus: a new signaling paradigm? J Recept Signal Transduct Res 28:15–28PubMedCrossRefGoogle Scholar
  6. Boucrot E, Ferreira AP, Almeida-Souza L, Debard S, Vallis Y, Howard G, Bertot L, Sauvonnet N, McMahon HT (2015) Endophilin marks and controls a clathrin-independent endocytic pathway. Nature 517:460–465PubMedCrossRefGoogle Scholar
  7. Cahill CM, Holdridge SV, Morinville A (2007) Trafficking of delta-opioid receptors and other G-protein-coupled receptors: implications for pain and analgesia. Trends Pharmacol Sci 28:23–31PubMedCrossRefGoogle Scholar
  8. Cahill TJ 3rd, Thomsen AR, Tarrasch JT, Plouffe B, Nguyen AH, Yang F, Huang LY, Kahsai AW, Bassoni DL, Gavino BJ, Lamerdin JE, Triest S, Shukla AK, Berger B, JT L, Antar A, Blanc A, Qu CX, Chen X, Kawakami K, Inoue A, Aoki J, Steyaert J, Sun JP, Bouvier M, Skiniotis G, Lefkowitz RJ (2017) Distinct conformations of GPCR-beta-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc Natl Acad Sci U S A 114:2562–2567PubMedPubMedCentralCrossRefGoogle Scholar
  9. Calebiro D, Nikolaev VO, Gagliani MC, de Filippis T, Dees C, Tacchetti C, Persani L, Lohse MJ (2009) Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 7:e1000172PubMedPubMedCentralCrossRefGoogle Scholar
  10. Calebiro D, Godbole A, Lyga S, Lohse MJ (2015) Trafficking and function of GPCRs in the endosomal compartment. Methods Mol Biol 1234:197–211PubMedCrossRefGoogle Scholar
  11. Cao TT, Mays RW, von Zastrow M (1998) Regulated endocytosis of G-protein-coupled receptors by a biochemically and functionally distinct subpopulation of clathrin-coated pits. J Biol Chem 273:24592–24602PubMedCrossRefGoogle Scholar
  12. Conn PM, Ulloa-Aguirre A (2010) Trafficking of G-protein-coupled receptors to the plasma membrane: insights for pharmacoperone drugs. Trends Endocrinol Metab 21:190–197PubMedCrossRefGoogle Scholar
  13. Conn PM, Ulloa-Aguirre A, Ito J, Janovick JA (2007) G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacol Rev 59:225–250PubMedCrossRefGoogle Scholar
  14. Conn PM, Spicer TP, Scampavia L, Janovick JA (2015) Assay strategies for identification of therapeutic leads that target protein trafficking. Trends Pharmacol Sci 36:498–505PubMedPubMedCentralCrossRefGoogle Scholar
  15. Dale LB, Bhattacharya M, Seachrist JL, Anborgh PH, Ferguson SS (2001) Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is beta-arrestin1 isoform-specific. Mol Pharmacol 60:1243–1253PubMedCrossRefGoogle Scholar
  16. De Wire SM, Yamashita DS, Rominger DH, Liu G, Cowan CL, Graczyk TM, Chen XT, Pitis PM, Gotchev D, Yuan C, Koblish M, Lark MW, Violin JD (2013) A G protein-biased ligand at the mu-opioid receptor is potently analgesic with reduced gastrointestinal and respiratory dysfunction compared with morphine. J Pharmacol Exp Ther 344:708–717CrossRefGoogle Scholar
  17. Diviani D, Lattion AL, Abuin L, Staub O, Cotecchia S (2003) The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis. J Biol Chem 278:19331–19340PubMedCrossRefGoogle Scholar
  18. Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902PubMedCrossRefGoogle Scholar
  19. Drake MT, Shenoy SK, Lefkowitz RJ (2006) Trafficking of G protein-coupled receptors. Circ Res 99:570–582PubMedCrossRefGoogle Scholar
  20. Eichel K, Jullie D, von Zastrow M (2016) Beta-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18:303–310PubMedPubMedCentralCrossRefGoogle Scholar
  21. Eriksen J, Bjorn-Yoshimoto WE, Jorgensen TN, Newman AH, Gether U (2010) Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons. J Biol Chem 285:27289–27301PubMedPubMedCentralCrossRefGoogle Scholar
  22. Fan GH, Yang W, Wang XJ, Qian Q, Richmond A (2001) Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization. Biochemistry 40:791–800PubMedPubMedCentralCrossRefGoogle Scholar
  23. Feinstein TN, Wehbi VL, Ardura JA, Wheeler DS, Ferrandon S, Gardella TJ, Vilardaga JP (2011) Retromer terminates the generation of cAMP by internalized PTH receptors. Nat Chem Biol 7:278–284PubMedPubMedCentralCrossRefGoogle Scholar
  24. Feinstein TN, Yui N, Webber MJ, Wehbi VL, Stevenson HP, King JD Jr, Hallows KR, Brown D, Bouley R, Vilardaga JP (2013) Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin. J Biol Chem 288:27849–27860PubMedPubMedCentralCrossRefGoogle Scholar
  25. Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24PubMedGoogle Scholar
  26. Ferguson SS, Downey WE 3rd, Colapietro AM, Barak LS, Menard L, Caron MG (1996) Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271:363–366PubMedCrossRefGoogle Scholar
  27. Feron O, Smith TW, Michel T, Kelly RA (1997) Dynamic targeting of the agonist-stimulated m2 muscarinic acetylcholine receptor to caveolae in cardiac myocytes. J Biol Chem 272:17744–17748PubMedCrossRefGoogle Scholar
  28. Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ, Vilardaga JP (2009) Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 5:734–742PubMedPubMedCentralCrossRefGoogle Scholar
  29. Fisher GW, Adler SA, Fuhrman MH, Waggoner AS, Bruchez MP, Jarvik JW (2010) Detection and quantification of beta2AR internalization in living cells using FAP-based biosensor technology. J Biomol Screen 15:703–709PubMedCrossRefGoogle Scholar
  30. Fraile-Ramos A, Kohout TA, Waldhoer M, Marsh M (2003) Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway. Traffic 4:243–253PubMedCrossRefGoogle Scholar
  31. Gales C, Rebois RV, Hogue M, Trieu P, Breit A, Hebert TE, Bouvier M (2005) Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods 2:177–184PubMedCrossRefGoogle Scholar
  32. Geppetti P, Veldhuis NA, Lieu T, Bunnett NW (2015) G protein-coupled receptors: dynamic machines for signaling pain and itch. Neuron 88:635–649PubMedCrossRefGoogle Scholar
  33. Goodman OB Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL (1996) Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature 383:447–450PubMedCrossRefGoogle Scholar
  34. Haasemann M, Cartaud J, Muller-Esterl W, Dunia I (1998) Agonist-induced redistribution of bradykinin B2 receptor in caveolae. J Cell Sci 111(Pt 7):917–928PubMedGoogle Scholar
  35. Halls ML, Poole DP, Ellisdon AM, Nowell CJ, Canals M (2015) Detection and quantification of intracellular signaling using FRET-based biosensors and high content imaging. Methods Mol Biol 1335:131–161PubMedCrossRefGoogle Scholar
  36. Halls ML, Yeatman HR, Nowell CJ, Thompson GL, Gondin AB, Civciristov S, Bunnett NW, Lambert NA, Poole DP, Canals M (2016) Plasma membrane localization of the μ-opioid receptor controls spatiotemporal signaling. Sci Signal 9:ra16-ra16CrossRefGoogle Scholar
  37. Hamdan FF, Audet M, Garneau P, Pelletier J, Bouvier M (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J Biomol Screen 10:463–475PubMedCrossRefGoogle Scholar
  38. Harper CB, Martin S, Nguyen TH, Daniels SJ, Lavidis NA, Popoff MR, Hadzic G, Mariana A, Chau N, McCluskey A, Robinson PJ, Meunier FA (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 286:35966–35976PubMedPubMedCentralCrossRefGoogle Scholar
  39. Harper CB, Popoff MR, McCluskey A, Robinson PJ, Meunier FA (2013) Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors. Trends Cell Biol 23:90–101PubMedCrossRefGoogle Scholar
  40. Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM (2013) Response to lack of specificity of commercial antibodies leads to misidentification of angiotensin type-1 receptor protein. Hypertension 61:e32PubMedPubMedCentralCrossRefGoogle Scholar
  41. Hislop JN, von Zastrow M (2011) Analysis of GPCR localization and trafficking. Methods Mol Biol 746:425–440PubMedPubMedCentralCrossRefGoogle Scholar
  42. Holloway AC, Qian H, Pipolo L, Ziogas J, Miura S, Karnik S, Southwell BR, Lew MJ, Thomas WG (2002) Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors. Mol Pharmacol 61:768–777PubMedCrossRefGoogle Scholar
  43. Huang Y, Willars GB (2011) Generation of epitope-tagged GPCRs. Methods Mol Biol 746:53–84PubMedCrossRefGoogle Scholar
  44. Irannejad R, von Zastrow M (2014) GPCR signaling along the endocytic pathway. Curr Opin Cell Biol 27:109–116PubMedCrossRefGoogle Scholar
  45. Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–538PubMedCrossRefGoogle Scholar
  46. Ishizaka N, Griendling KK, Lassegue B, Alexander RW (1998) Angiotensin II type 1 receptor: relationship with caveolae and caveolin after initial agonist stimulation. Hypertension 32:459–466PubMedCrossRefGoogle Scholar
  47. Jacobsen SE, Ammendrup-Johnsen I, Jansen AM, Gether U, Madsen KL, Bräuner-Osborne H (2017) The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway. J Biol Chem 292:6910–6926PubMedCrossRefPubMedCentralGoogle Scholar
  48. Jensen BC, Swigart PM, Simpson PC (2009) Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. Naunyn Schmiedeberg’s Arch Pharmacol 379:409–412CrossRefGoogle Scholar
  49. Jensen DD, Lieu T, Halls ML, Veldhuis NA, Imlach WL, Mai QN, Poole DP, Quach T, Aurelio L, Conner J, Herenbrink CK, Barlow N, Simpson JS, Scanlon MJ, Graham B, McCluskey A, Robinson PJ, Escriou V, Nassini R, Materazzi S, Geppetti P, Hicks GA, Christie MJ, Porter CJH, Canals M, Bunnett NW (2017) Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief. Sci Transl Med 9.
  50. Jorgensen R, Martini L, Schwartz TW, Elling CE (2005) Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype. Mol Endocrinol 19:812–823PubMedCrossRefGoogle Scholar
  51. Kenakin T (2017) Signaling bias in drug discovery. Expert Opin Drug Discov 12:321–333PubMedCrossRefGoogle Scholar
  52. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89PubMedCrossRefGoogle Scholar
  53. Kifor O, Diaz R, Butters R, Kifor I, Brown EM (1998) The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains of bovine parathyroid cells. J Biol Chem 273:21708–21713PubMedCrossRefGoogle Scholar
  54. Klein Herenbrink C, Sykes DA, Donthamsetti P, Canals M, Coudrat T, Shonberg J, Scammells PJ, Capuano B, Sexton PM, Charlton SJ, Javitch JA, Christopoulos A, Lane JR (2016) The role of kinetic context in apparent biased agonism at GPCRs. Nat Commun 7:10842PubMedPubMedCentralCrossRefGoogle Scholar
  55. Kotowski SJ, Hopf FW, Seif T, Bonci A, von Zastrow M (2011) Endocytosis promotes rapid dopaminergic signaling. Neuron 71:278–290PubMedPubMedCentralCrossRefGoogle Scholar
  56. Kuna RS, Girada SB, Asalla S, Vallentyne J, Maddika S, Patterson JT, Smiley DL, Di Marchi RD, Mitra P (2013) Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic beta-cells. Am J Physiol Endocrinol Metab 305:E161–E170PubMedCrossRefGoogle Scholar
  57. Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG, Barak LS (1999) The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A 96:3712–3717PubMedPubMedCentralCrossRefGoogle Scholar
  58. Lee MH, Appleton KM, Strungs EG, Kwon JY, Morinelli TA, Peterson YK, Laporte SA, Luttrell LM (2016) The conformational signature of beta-arrestin2 predicts its trafficking and signalling functions. Nature 531:665–668PubMedPubMedCentralCrossRefGoogle Scholar
  59. Levoye A, Zwier JM, Jaracz-Ros A, Klipfel L, Cottet M, Maurel D, Bdioui S, Balabanian K, Prezeau L, Trinquet E, Durroux T, Bachelerie F (2015) A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate. Front Endocrinol 6:167CrossRefGoogle Scholar
  60. Lobingier BT, Huttenhain R, Eichel K, Miller KB, Ting AY, von Zastrow M, Krogan NJ (2017) An approach to spatiotemporally resolve protein interaction networks in living cells. Cell 169:350–360.e12PubMedPubMedCentralCrossRefGoogle Scholar
  61. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ (1990) Beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248:1547–1550PubMedCrossRefGoogle Scholar
  62. Lohse MJ, Nuber S, Hoffmann C (2012) Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol Rev 64:299–336PubMedCrossRefGoogle Scholar
  63. Luttrell LM, Maudsley S, Bohn LM (2015) Fulfilling the promise of “biased” G protein-coupled receptor agonism. Mol Pharmacol 88:579–588PubMedPubMedCentralCrossRefGoogle Scholar
  64. Lyga S, Volpe S, Werthmann RC, Gotz K, Sungkaworn T, Lohse MJ, Calebiro D (2016) Persistent cAMP signaling by internalized LH receptors in ovarian follicles. Endocrinology 157:1613–1621PubMedCrossRefGoogle Scholar
  65. Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839–850PubMedCrossRefGoogle Scholar
  66. Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prezeau L, Trinquet E, Pin JP (2008) Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods 5:561–567PubMedPubMedCentralCrossRefGoogle Scholar
  67. McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A, Whiting A, Gorgani NN, Lloyd J, Quan A, Moshkanbaryans L, Krishnan S, Perera S, Chircop M, von Kleist L, McGeachie AB, Howes MT, Parton RG, Campbell M, Sakoff JA, Wang X, Sun JY, Robertson MJ, Deane FM, Nguyen TH, Meunier FA, Cousin MA, Robinson PJ (2013) Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic 14:1272–1289PubMedPubMedCentralCrossRefGoogle Scholar
  68. Moore CA, Milano SK, Benovic JL (2007) Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 69:451–482PubMedCrossRefGoogle Scholar
  69. Mulherkar N, Raaben M, de la Torre JC, Whelan SP, Chandran K (2011) The Ebola virus glycoprotein mediates entry via a non-classical dynamin-dependent macropinocytic pathway. Virology 419:72–83PubMedPubMedCentralCrossRefGoogle Scholar
  70. Mullershausen F, Zecri F, Cetin C, Billich A, Guerini D, Seuwen K (2009) Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nat Chem Biol 5:428–434PubMedCrossRefGoogle Scholar
  71. Namkung Y, Le Gouill C, Lukashova V, Kobayashi H, Hogue M, Khoury E, Song M, Bouvier M, Laporte SA (2016) Monitoring G protein-coupled receptor and beta-arrestin trafficking in live cells using enhanced bystander BRET. Nat Commun 7:12178PubMedPubMedCentralCrossRefGoogle Scholar
  72. Nuber S, Zabel U, Lorenz K, Nuber A, Milligan G, Tobin AB, Lohse MJ, Hoffmann C (2016) Beta-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531:661–664PubMedPubMedCentralCrossRefGoogle Scholar
  73. Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem 275:17201–17210PubMedCrossRefGoogle Scholar
  74. Oh P, Horner T, Witkiewicz H, Schnitzer JE (2012) Endothelin induces rapid, dynamin-mediated budding of endothelial caveolae rich in ET-B. J Biol Chem 287:17353–17362PubMedPubMedCentralCrossRefGoogle Scholar
  75. Okamoto Y, Ninomiya H, Miwa S, Masaki T (2000) Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells. J Biol Chem 275:6439–6446PubMedCrossRefGoogle Scholar
  76. Paek J, Kalocsay M, Staus DP, Wingler L, Pascolutti R, Paulo JA, Gygi SP, Kruse AC (2017) Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling. Cell 169:338–349.e11PubMedPubMedCentralCrossRefGoogle Scholar
  77. Paing MM, Temple BR, Trejo J (2004) A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization. J Biol Chem 279:21938–21947PubMedCrossRefGoogle Scholar
  78. Pampillo M, Babwah AV (2015) Quantifying GPCR internalization: a focus on the Kisspeptin receptor. Methods Mol Biol 1272:119–132PubMedCrossRefGoogle Scholar
  79. Pierce KL, Premont RT, Lefkowitz RJ (2002) Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3:639–650PubMedCrossRefGoogle Scholar
  80. Porrello ER, Pfleger KD, Seeber RM, Qian H, Oro C, Abogadie F, Delbridge LM, Thomas WG (2011) Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles. Cell Signal 23:1767–1776PubMedCrossRefGoogle Scholar
  81. Pradhan AA, Tawfik VL, Tipton AF, Scherrer G (2015) In vivo techniques to investigate the internalization profile of opioid receptors. Methods Mol Biol 1230:87–104PubMedPubMedCentralCrossRefGoogle Scholar
  82. Re M, Pampillo M, Savard M, Dubuc C, McArdle CA, Millar RP, Conn PM, Gobeil F Jr, Bhattacharya M, Babwah AV (2010) The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane. PLoS One 5:e11489PubMedPubMedCentralCrossRefGoogle Scholar
  83. Renard HF, Simunovic M, Lemiere J, Boucrot E, Garcia-Castillo MD, Arumugam S, Chambon V, Lamaze C, Wunder C, Kenworthy AK, Schmidt AA, McMahon HT, Sykes C, Bassereau P, Johannes L (2015) Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature 517:493–496PubMedCrossRefGoogle Scholar
  84. Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307:1625–1630PubMedCrossRefGoogle Scholar
  85. Rizzolio S, Tamagnone L (2017) Antibody-feeding assay: a method to track the internalization of neuropilin-1 and other cell surface receptors. Methods Mol Biol 1493:311–319PubMedCrossRefGoogle Scholar
  86. Roed SN, Wismann P, Underwood CR, Kulahin N, Iversen H, Cappelen KA, Schaffer L, Lehtonen J, Hecksher-Soerensen J, Secher A, Mathiesen JM, Bräuner-Osborne H, Whistler JL, Knudsen SM, Waldhoer M (2014) Real-time trafficking and signaling of the glucagon-like peptide-1 receptor. Mol Cell Endocrinol 382:938–949PubMedCrossRefGoogle Scholar
  87. Roed SN, Nøhr AC, Wismann P, Iversen H, Bräuner-Osborne H, Knudsen SM, Waldhoer M (2015) Functional consequences of glucagon-like peptide-1 receptor cross-talk and trafficking. J Biol Chem 290:1233–1243PubMedCrossRefGoogle Scholar
  88. Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP (2017) A comprehensive map of molecular drug targets. Nat Rev Drug Discov 16:19–34PubMedCrossRefGoogle Scholar
  89. Shenoy SK, Lefkowitz RJ (2011) β-arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol Sci 32:521–533PubMedPubMedCentralCrossRefGoogle Scholar
  90. Slessareva JE, Routt SM, Temple B, Bankaitis VA, Dohlman HG (2006) Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome. Cell 126:191–203PubMedCrossRefGoogle Scholar
  91. Smith TH, Coronel LJ, Li JG, Dores MR, Nieman MT, Trejo J (2016) Protease-activated receptor-4 signaling and trafficking is regulated by the clathrin adaptor protein complex-2 independent of ß-arrestins. J Biol Chem 291:18453–18464PubMedPubMedCentralCrossRefGoogle Scholar
  92. Sorkin A, von Zastrow M (2009) Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol 10:609–622PubMedPubMedCentralCrossRefGoogle Scholar
  93. Thomsen AR, Plouffe B, Cahill TJ 3rd, Shukla AK, Tarrasch JT, Dosey AM, Kahsai AW, Strachan RT, Pani B, Mahoney JP, Huang L, Breton B, Heydenreich FM, Sunahara RK, Skiniotis G, Bouvier M, Lefkowitz RJ (2016) GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling. Cell 166:907–919PubMedPubMedCentralCrossRefGoogle Scholar
  94. Tsvetanova NG, von Zastrow M (2014) Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis. Nat Chem Biol 10:1061–1065PubMedPubMedCentralCrossRefGoogle Scholar
  95. Uchida Y, Rutaganira FU, Jullie D, Shokat KM, von Zastrow M (2017) Endosomal phosphatidylinositol 3-kinase is essential for canonical GPCR signaling. Mol Pharmacol 91:65–73PubMedPubMedCentralCrossRefGoogle Scholar
  96. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P (2009) Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 106:18825–18830PubMedPubMedCentralCrossRefGoogle Scholar
  97. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M, McCartney J, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu PA (2011) Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A 108:18843–18848PubMedPubMedCentralCrossRefGoogle Scholar
  98. Vilardaga JP, Bunemann M, Krasel C, Castro M, Lohse MJ (2003) Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat Biotechnol 21:807–812PubMedCrossRefGoogle Scholar
  99. Violin JD, De Wire SM, Yamashita D, Rominger DH, Nguyen L, Schiller K, Whalen EJ, Gowen M, Lark MW (2010) Selectively engaging beta-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance. J Pharmacol Exp Ther 335:572–579PubMedCrossRefGoogle Scholar
  100. von Zastrow M, Kobilka BK (1992) Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem 267:3530–3538Google Scholar
  101. Wager-Miller J, Mackie K (2016) Quantitation of plasma membrane (G protein-coupled) receptor trafficking in cultured cells. Methods Mol Biol 1412:255–266PubMedCrossRefGoogle Scholar
  102. Wainwright CE, Elborn JS, Ramsey BW, Marigowda G, Huang X, Cipolli M, Colombo C, Davies JC, De Boeck K, Flume PA, Konstan MW, McColley SA, McCoy K, McKone EF, Munck A, Ratjen F, Rowe SM, Waltz D, Boyle MP (2015) Lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N Engl J Med 373:220–231PubMedPubMedCentralCrossRefGoogle Scholar
  103. Walther C, Ferguson SS (2013) Arrestins: role in the desensitization, sequestration, and vesicular trafficking of G protein-coupled receptors. Prog Mol Biol Transl Sci 118:93–113PubMedCrossRefGoogle Scholar
  104. Wehbi VL, Stevenson HP, Feinstein TN, Calero G, Romero G, Vilardaga JP (2013) Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gbetagamma complex. Proc Natl Acad Sci U S A 110:1530–1535PubMedPubMedCentralCrossRefGoogle Scholar
  105. Wilden U, Hall SW, Kuhn H (1986) Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc Natl Acad Sci U S A 83:1174–1178PubMedPubMedCentralCrossRefGoogle Scholar
  106. Wu Y, Tapia PH, Jarvik J, Waggoner AS, Sklar LA (2014) Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor. Curr Protoc Cytom 67:9.43Google Scholar
  107. Wyse BD, Prior IA, Qian H, Morrow IC, Nixon S, Muncke C, Kurzchalia TV, Thomas WG, Parton RG, Hancock JF (2003) Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane. J Biol Chem 278:23738–23746PubMedCrossRefGoogle Scholar
  108. Xu J, Corneillie TM, Moore EG, Law GL, Butlin NG, Raymond KN (2011) Octadentate cages of Tb(III) 2-hydroxyisophthalamides: a new standard for luminescent lanthanide labels. J Am Chem Soc 133:19900–19910PubMedPubMedCentralCrossRefGoogle Scholar
  109. Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73PubMedPubMedCentralCrossRefGoogle Scholar
  110. Zhu Y, Watson J, Chen M, Shen DR, Yarde M, Agler M, Burford N, Alt A, Jayachandra S, Cvijic ME, Zhang L, Dyckman A, Xie J, O’Connell J, Banks M, Weston A (2014) Integrating high-content analysis into a multiplexed screening approach to identify and characterize GPCR agonists. J Biomol Screen 19:1079–1089PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Department of Drug Design and Pharmacology, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagenDenmark

Personalised recommendations