Ligand-induced activation of G protein–coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen–binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.


Article metrics loading...

Loading full text...

Full text loading...


Literature Cited

  1. Lagerström MC, Schioth HB. 1.  2008. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7:339–57 [Google Scholar]
  2. Lefkowitz RJ. 2.  2004. Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol. Sci. 25:413–22 [Google Scholar]
  3. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H. 3.  et al. 2000. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–45 [Google Scholar]
  4. Katritch V, Cherezov V, Stevens RC. 4.  2013. Structure-function of the G protein–coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53:531–56 [Google Scholar]
  5. Pierce KL, Premont RT, Lefkowitz RJ. 5.  2002. Seven-transmembrane receptors. Nat. Rev. Mol. Cell. Biol. 3:639–50 [Google Scholar]
  6. Wisler JW, Xiao K, Thomsen ARB, Lefkowitz RJ. 6.  2014. Recent developments in biased agonism. Curr. Opin. Cell Biol. 27:18–24 [Google Scholar]
  7. Mombaerts P. 7.  2004. Genes and ligands for odorant, vomeronasal and taste receptors. Nat. Rev. Neurosci. 5:263–78 [Google Scholar]
  8. Overington JP, Al-Lazikani B, Hopkins AL. 8.  2006. How many drug targets are there?. Nat. Rev. Drug Discov. 5:993–96 [Google Scholar]
  9. Rask-Andersen M, Masuram S, Schioth HB. 9.  2014. The druggable genome: Evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu. Rev. Pharmacol. Toxicol. 54:9–26 [Google Scholar]
  10. 10. Int. Union Basic Clin. Pharmacol./Br. Pharmacol. Soc. (IUPHAR/BPS). 2016. The IUPHAR/BPS Guide to PHARMACOLOGY Edinburgh, UK: IUPHAR/BPS http://www.guidetopharmacology.org/
  11. De Lean A, Stadel JM, Lefkowitz RJ. 11.  1980. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol. Chem. 255:7108–17 [Google Scholar]
  12. Flock T, Ravarani CN, Sun D, Venkatakrishnan AJ, Kayikci M. 12.  et al. 2015. Universal allosteric mechanism for Gα activation by GPCRs. Nature 524:173–79 [Google Scholar]
  13. Tesmer JJ, Sunahara RK, Gilman AG, Sprang SR. 13.  1997. Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsα·GTPγS. Science 278:1907–16 [Google Scholar]
  14. Waldo GL, Ricks TK, Hicks SN, Cheever ML, Kawano T. 14.  et al. 2010. Kinetic scaffolding mediated by a phospholipase C–β and Gq signaling complex. Science 330:974–80 [Google Scholar]
  15. Berridge MJ, Irvine RF. 15.  1989. Inositol phosphates and cell signalling. Nature 341:197–205 [Google Scholar]
  16. Kang DS, Tian X, Benovic JL. 16.  2014. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking. Curr. Opin. Cell Biol. 27:63–71 [Google Scholar]
  17. Premont RT, Gainetdinov RR. 17.  2007. Physiological roles of G protein–coupled receptor kinases and arrestins. Annu. Rev. Physiol. 69:511–34 [Google Scholar]
  18. Lohse MJ. 18.  2010. Dimerization in GPCR mobility and signaling. Curr. Opin. Pharmacol. 10:53–58 [Google Scholar]
  19. Ferré S, Casadó V, Devi LA, Filizola M, Jockers R. 19.  et al. 2014. G protein–coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol. Rev. 66:413–34 [Google Scholar]
  20. Kniazeff J, Bessis AS, Maurel D, Ansanay H, Prezeau L, Pin JP. 20.  2004. Closed state of both binding domains of homodimeric mGlu receptors is required for full activity. Nat. Struct. Mol. Biol. 11:706–13 [Google Scholar]
  21. Isberg V, Mordalski S, Munk C, Rataj K, Harpsøe K. 21.  et al. 2016. GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res 44:D356–64 [Google Scholar]
  22. Maeda S, Schertler GF. 22.  2013. Production of GPCR and GPCR complexes for structure determination. Curr. Opin. Struct. Biol. 23:381–92 [Google Scholar]
  23. Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS. 23.  et al. 2007. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318:1266–73 [Google Scholar]
  24. Chun E, Thompson AA, Liu W, Roth CB, Griffith MT. 24.  et al. 2012. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20:967–76 [Google Scholar]
  25. Serrano-Vega MJ, Magnani F, Shibata Y, Tate CG. 25.  2008. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. PNAS 105:877–82 [Google Scholar]
  26. Tate CG. 26.  2012. A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem. Sci. 37:343–52 [Google Scholar]
  27. Vaidehi N, Grisshammer R, Tate CG. 27.  2016. How can mutations thermostabilize G-protein-coupled receptors?. Trends Pharmacol. Sci. 37:37–46 [Google Scholar]
  28. Caffrey M, Cherezov V. 28.  2009. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4:706–31 [Google Scholar]
  29. Bowler MW, Guijarro M, Petitdemange S, Baker I, Svensson O. 29.  et al. 2010. Diffraction cartography: Applying microbeams to macromolecular crystallography sample evaluation and data collection. Acta Crystallogr. D Biol. Crystallogr. 66:855–64 [Google Scholar]
  30. Liu W, Wacker D, Gati C, Han GW, James D. 30.  et al. 2013. Serial femtosecond crystallography of G protein-coupled receptors. Science 342:1521–24 [Google Scholar]
  31. Weichert D, Gmeiner P. 31.  2015. Covalent molecular probes for class A G protein-coupled receptors: advances and applications. ACS Chem. Biol. 10:1376–86 [Google Scholar]
  32. Chae PS, Rasmussen SGF, Rana RR, Gotfryd K, Chandra R. 32.  et al. 2010. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7:1003–8 [Google Scholar]
  33. Cooke RM, Brown AJ, Marshall FH, Mason JS. 33.  2015. Structures of G protein-coupled receptors reveal new opportunities for drug discovery. Drug Discov. Today 20:1355–64 [Google Scholar]
  34. Kobilka BK. 34.  2011. Structural insights into adrenergic receptor function and pharmacology. Trends Pharmacol. Sci. 32:213–18 [Google Scholar]
  35. Leff P. 35.  1995. The two-state model of receptor activation. Trends Pharmacol. Sci. 16:89–97 [Google Scholar]
  36. Deupi X, Kobilka B. 36.  2007. Activation of G protein-coupled receptors. Adv. Protein Chem. 74:137–66 [Google Scholar]
  37. Galandrin S, Oligny-Longpre G, Bouvier M. 37.  2007. The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol. Sci. 28:423–30 [Google Scholar]
  38. Kenakin T, Christopoulos A. 38.  2012. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 12:205–16 [Google Scholar]
  39. Chachisvilis M, Zhang YL, Frangos JA. 39.  2006. G protein-coupled receptors sense fluid shear stress in endothelial cells. PNAS 103:15463–68 [Google Scholar]
  40. Dawaliby R, Trubbia C, Delporte C, Masureel M, Van Antwerpen P. 40.  et al. 2016. Allosteric regulation of G protein-coupled receptor activity by phospholipids. Nat. Chem. Biol. 12:35–39 [Google Scholar]
  41. Oates J, Watts A. 41.  2011. Uncovering the intimate relationship between lipids, cholesterol and GPCR activation. Curr. Opin. Struct. Biol. 21:802–7 [Google Scholar]
  42. Mahaut-Smith MP, Martinez-Pinna J, Gurung IS. 42.  2008. A role for membrane potential in regulating GPCRs?. Trends Pharmacol. Sci. 29:421–29 [Google Scholar]
  43. Ghanouni P, Schambye H, Seifert R, Lee TW, Rasmussen SGF. 43.  et al. 2000. The effect of pH on β2 adrenoceptor function: evidence for protonation-dependent activation. J. Biol. Chem. 275:3121–27 [Google Scholar]
  44. Liu W, Chun E, Thompson AA, Chubukov P, Xu F. 44.  et al. 2012. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337:232–36 [Google Scholar]
  45. Isogai S, Deupi X, Opitz C, Heydenreich FM, Tsai CJ. 45.  et al. 2016. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 530:237–41 [Google Scholar]
  46. Sounier R, Mas C, Steyaert J, Laeremans T, Manglik A. 46.  et al. 2015. Propagation of conformational changes during μ-opioid receptor activation. Nature 524:375–78 [Google Scholar]
  47. Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K. 47.  2012. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–10 [Google Scholar]
  48. Nygaard R, Zou Y, Dror RO, Mildorf TJ, Arlow DH. 48.  et al. 2013. The dynamic process of β2-adrenergic receptor activation. Cell 152:532–42 [Google Scholar]
  49. Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z. 49.  et al. 2015. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161:1101–11 [Google Scholar]
  50. Lamichhane R, Liu JJ, Pljevaljcic G, White KL, van der Schans E. 50.  et al. 2015. Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR. PNAS 112:14254–59 [Google Scholar]
  51. Bockenhauer S, Furstenberg A, Yao XJ, Kobilka BK, Moerner WE. 51.  2011. Conformational dynamics of single G protein-coupled receptors in solution. J. Phys. Chem. B 115:13328–38 [Google Scholar]
  52. Shukla AK, Westfield GH, Xiao K, Reis RI, Huang LY. 52.  et al. 2014. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512:218–22 [Google Scholar]
  53. Westfield GH, Rasmussen SGF, Su M, Dutta S, DeVree BT. 53.  et al. 2011. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. PNAS 108:16086–91 [Google Scholar]
  54. Kang Y, Zhou XE, Gao X, He Y, Liu W. 54.  et al. 2015. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523:561–67 [Google Scholar]
  55. Yang L, Yang D, de Graaf C, Moeller A, West GM. 55.  et al. 2015. Conformational states of the full-length glucagon receptor. Nat. Commun. 6:7859 [Google Scholar]
  56. Deupi X, Kobilka BK. 56.  2010. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. Physiology 25:293–303 [Google Scholar]
  57. Dror RO, Arlow DH, Maragakis P, Mildorf TJ, Pan AC. 57.  et al. 2011. Activation mechanism of the β2-adrenergic receptor. PNAS 108:18684–89 [Google Scholar]
  58. Manglik A, Kobilka BK. 58.  2014. The role of protein dynamics in GPCR function: insights from the β2AR and rhodopsin. Curr. Opin. Cell Biol. 27C:136–43 [Google Scholar]
  59. Kenakin T. 59.  2002. Efficacy at G-protein-coupled receptors. Nat. Rev. Drug Discov. 1:103–10 [Google Scholar]
  60. Kahsai AW, Xiao K, Rajagopal S, Ahn S, Shukla AK. 60.  et al. 2011. Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat. Chem. Biol. 7:692–700 [Google Scholar]
  61. Kobilka B. 61.  2013. The structural basis of G-protein-coupled receptor signaling (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 52:6380–88 [Google Scholar]
  62. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C. 62.  et al. 1993. Naturally occurring antibodies devoid of light chains. Nature 363:446–48First description of functional heavy-chain-only antibodies in camelids. [Google Scholar]
  63. Muyldermans S. 63.  2013. Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 82:775–97 [Google Scholar]
  64. Arbabi-Ghahroudi M, Tanha J, MacKenzie R. 64.  2005. Prokaryotic expression of antibodies. Cancer Metastasis Rev 24:501–19 [Google Scholar]
  65. Frenken LGJ, van der Linden RHJ, Hermans PWJJ, Bos JW, Ruuls RC. 65.  et al. 2000. Isolation of antigen specific Llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J. Biotechnol. 78:11–21 [Google Scholar]
  66. Frenken LGJ, Hessing JGM, Van den Hondel CAMJJ, Verrips CT. 66.  1998. Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. Res. Immunol. 149:589–99 [Google Scholar]
  67. Joosten V, Gouka RJ, Van den Hondel CAMJJ, Verrips CT, Lokman BC. 67.  2005. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Appl. Microbiol. Biotechnol. 66:384–92 [Google Scholar]
  68. Agrawal V, Slivac I, Perret S, Bisson L, St-Laurent G. 68.  et al. 2012. Stable expression of chimeric heavy chain antibodies in CHO cells. Single Domain Antibodies: Methods and Protocols D Saerens, S Muyldermans 287–303 Totowa, NJ: Humana Press [Google Scholar]
  69. Ismaili A, Jalali-Javaran M, Rasaee MJ, Rahbarizadeh F, Forouzandeh-Moghadam M, Memari HR. 69.  2007. Production and characterization of anti-(mucin MUC1) single-domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi). Biotechnol. Appl Biochem. 47:11–19 [Google Scholar]
  70. De Buck S, Nolf J, De Meyer T, Virdi V, De Wilde K. 70.  et al. 2013. Fusion of an Fc chain to a VHH boosts the accumulation levels in Arabidopsis seeds. Plant Biotechnol. J. 11:1006–16 [Google Scholar]
  71. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K. 71.  et al. 2002. Single-domain antibody fragments with high conformational stability. Protein Sci. 11:500–15 [Google Scholar]
  72. Pardon E, Laeremans T, Triest S, Rasmussen SGF, Wohlkonig A. 72.  et al. 2014. A general protocol for the generation of nanobodies for structural biology. Nat. Protoc. 9:674–93Laboratory protocols for the generation of in vivo matured nanobodies. [Google Scholar]
  73. Ryckaert S, Pardon E, Steyaert J, Callewaert N. 73.  2010. Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris. J. Biotechnol. 145:93–98 [Google Scholar]
  74. Fleetwood F, Devoogdt N, Pellis M, Wernery U, Muyldermans S. 74.  et al. 2013. Surface display of a single-domain antibody library on Gram-positive bacteria. Cell Mol. Life Sci. 70:1081–93 [Google Scholar]
  75. Pellis M, Pardon E, Zolghadr K, Rothbauer U, Vincke C. 75.  et al. 2012. A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional nanobodies. Arch. Biochem. Biophys. 526:114–23 [Google Scholar]
  76. Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W. 76.  et al. 1998. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J 17:3512–20 [Google Scholar]
  77. De Genst E, Silence K, Decanniere K, Conrath K, Loris R. 77.  et al. 2006. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. PNAS 103:4586–91 [Google Scholar]
  78. Domanska K, Vanderhaegen S, Srinivasan V, Pardon E, Dupeux F. 78.  et al. 2011. Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant. PNAS 108:1314–19 [Google Scholar]
  79. Abskharon RN, Giachin G, Wohlkonig A, Soror SH, Pardon E. 79.  et al. 2014. Probing the N-terminal β-sheet conversion in the crystal structure of the human prion protein bound to a nanobody. J. Am. Chem. Soc. 136:937–44 [Google Scholar]
  80. Korotkov KV, Pardon E, Steyaert J, Hol WG. 80.  2009. Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure 17:255–65 [Google Scholar]
  81. Loris R, Marianovsky I, Lah J, Laeremans T, Engelberg-Kulka H. 81.  et al. 2003. Crystal structure of the intrinsically flexible addiction antidote MazE. J. Biol. Chem. 278:28252–57 [Google Scholar]
  82. Rivera-Calzada A, Fronzes R, Savva CG, Chandran V, Lian PW. 82.  et al. 2013. Structure of a bacterial type IV secretion core complex at subnanometre resolution. EMBO J. 32:1195–204 [Google Scholar]
  83. Ehrnstorfer IA, Geertsma ER, Pardon E, Steyaert J, Dutzler R. 83.  2014. Crystal structure of a SLC11 (NRAMP) transporter reveals the basis for transition-metal ion transport. Nat. Struct. Mol. Biol. 21:990–96 [Google Scholar]
  84. Smirnova I, Kasho V, Jiang X, Pardon E, Steyaert J, Kaback HR. 84.  2014. Outward-facing conformers of LacY stabilized by nanobodies. PNAS 111:18548–53 [Google Scholar]
  85. Geertsma ER, Chang YN, Shaik FR, Neldner Y, Pardon E. 85.  et al. 2015. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family. Nat. Struct. Mol. Biol. 22:803–8 [Google Scholar]
  86. Hassaine G, Deluz C, Grasso L, Wyss R, Tol MB. 86.  et al. 2014. X-ray structure of the mouse serotonin 5-HT3 receptor. Nature 512:276–81 [Google Scholar]
  87. Li L, Park E, Ling J, Ingram J, Ploegh H, Rapoport TA. 87.  2016. Crystal structure of a substrate-engaged SecY protein-translocation channel. Nature 531:395–99 [Google Scholar]
  88. Rostislavleva K, Soler N, Ohashi Y, Zhang L, Pardon E. 88.  et al. 2015. Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes. Science 350:aac7365 [Google Scholar]
  89. Pathare GR, Nagy I, Sledz P, Anderson DJ, Zhou HJ. 89.  et al. 2014. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. PNAS 111:2984–89 [Google Scholar]
  90. Rasmussen SGF, DeVree BT, Zou Y, Kruse AC, Chung KY. 90.  et al. 2011. Crystal structure of the β2 adrenergic receptor–Gs protein complex. Nature 477:549–55First structure of a GPCR·G protein complex solved by nanobody-assisted crystallography. [Google Scholar]
  91. Rasmussen SGF, Choi HJ, Fung JJ, Pardon E, Casarosa P. 91.  et al. 2011. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469:175–80First description of a G protein–mimicking nanobody that stabilizes the agonist-bound active state of β2AR. [Google Scholar]
  92. Kruse AC, Ring AM, Manglik A, Hu J, Hu K. 92.  et al. 2013. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504:101–6 [Google Scholar]
  93. Ring AM, Manglik A, Kruse AC, Enos MD, Weis WI. 93.  et al. 2013. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502:575–79 [Google Scholar]
  94. Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN. 94.  et al. 2015. Structural insights into μ-opioid receptor activation. Nature 524:315–21 [Google Scholar]
  95. Burg JS, Ingram JR, Venkatakrishnan AJ, Jude KM, Dukkipati A. 95.  et al. 2015. Structural basis for chemokine recognition and activation of a viral G protein–coupled receptor. Science 347:1113–17 [Google Scholar]
  96. Rajewsky K. 96.  1996. Clonal selection and learning in the antibody system. Nature 381:751–58 [Google Scholar]
  97. Smirnova I, Kasho V, Jiang X, Pardon E, Steyaert J, Kaback HR. 97.  2015. Transient conformers of LacY are trapped by nanobodies. PNAS 112:13839–44 [Google Scholar]
  98. Palczewski K. 98.  2006. G protein–coupled receptor rhodopsin. Annu. Rev. Biochem. 75:743–67 [Google Scholar]
  99. Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP. 99.  2008. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454:183–87 [Google Scholar]
  100. Vogel R, Siebert F. 100.  2001. Conformations of the active and inactive states of opsin. J. Biol. Chem. 276:38487–93 [Google Scholar]
  101. Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauß N. 101.  et al. 2008. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455:497–502 [Google Scholar]
  102. Choe HW, Kim YJ, Park JH, Morizumi T, Pai EF. 102.  et al. 2011. Crystal structure of metarhodopsin II. Nature 471:651–55 [Google Scholar]
  103. Standfuss J, Edwards PC, D'Antona A, Fransen M, Xie G. 103.  et al. 2011. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656–60 [Google Scholar]
  104. Yao XJ, Vélez Ruiz G, Whorton MR, Rasmussen SGF, DeVree BT. 104.  et al. 2009. The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. PNAS 106:9501–6 [Google Scholar]
  105. Rosenbaum DM, Zhang C, Lyons JA, Holl R, Aragao D. 105.  et al. 2011. Structure and function of an irreversible agonist-β2 adrenoceptor complex. Nature 469:236–40 [Google Scholar]
  106. Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ. 106.  et al. 2011. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474:521–25 [Google Scholar]
  107. White JF, Noinaj N, Shibata Y, Love J, Kloss B. 107.  et al. 2012. Structure of the agonist-bound neurotensin receptor. Nature 490:508–13 [Google Scholar]
  108. Lebon G, Warne T, Tate CG. 108.  2012. Agonist-bound structures of G protein-coupled receptors. Curr. Opin. Struct. Biol. 22:482–90 [Google Scholar]
  109. Steyaert J, Kobilka BK. 109.  2011. Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21:567–72 [Google Scholar]
  110. Murphree LJ, Marshall MA, Rieger JM, MacDonald TL, Linden J. 110.  2002. Human A2A adenosine receptors: high-affinity agonist binding to receptor-G protein complexes containing Gβ4. Mol. Pharmacol. 61:455–62 [Google Scholar]
  111. Isberg V, de Graaf C, Bortolato A, Cherezov V, Katritch V. 111.  et al. 2015. Generic GPCR residue numbers – aligning topology maps while minding the gaps. Trends Pharmacol. Sci. 36:22–31 [Google Scholar]
  112. Deupi X, Standfuss J. 112.  2011. Structural insights into agonist-induced activation of G-protein-coupled receptors. Curr. Opin. Struct. Biol. 21:541–51 [Google Scholar]
  113. Ghanouni P, Gryczynski Z, Steenhuis JJ, Lee TW, Farrens DL. 113.  et al. 2001. Functionally different agonists induce distinct conformations in the G protein coupling domain of the β2 adrenergic receptor. J. Biol. Chem. 276:24433–36 [Google Scholar]
  114. Yao X, Parnot C, Deupi X, Ratnala VR, Swaminath G. 114.  et al. 2006. Coupling ligand structure to specific conformational switches in the β2-adrenoceptor. Nat. Chem. Biol. 2:417–22 [Google Scholar]
  115. Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R. 115.  et al. 2010. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–12 [Google Scholar]
  116. Horst R, Stanczak P, Stevens RC, Wüthrich K. 116.  2013. β2-Adrenergic receptor solutions for structural biology analyzed with microscale NMR diffusion measurements. Angew. Chem. Int. Ed. Engl. 52:331–35 [Google Scholar]
  117. Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K. 117.  et al. 2012. Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region. Nat. Commun. 3:1045 [Google Scholar]
  118. Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO. 118.  et al. 2013. The role of ligands on the equilibria between functional states of a G protein-coupled receptor. J. Am. Chem. Soc. 135:9465–74 [Google Scholar]
  119. Chung KY, Day PW, Vélez-Ruiz G, Sunahara RK, Kobilka BK. 119.  2013. Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach. PLOS ONE 8:e54942 [Google Scholar]
  120. Misquitta LV, Misquitta Y, Cherezov V, Slattery O, Mohan JM. 120.  et al. 2004. Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12:2113–24 [Google Scholar]
  121. Zou Y, Weis WI, Kobilka BK. 121.  2012. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor. PLOS ONE 7:e46039 [Google Scholar]
  122. Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L. 122.  et al. 2006. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3:887–89 [Google Scholar]
  123. Vercruysse T, Pawar S, De Borggraeve W, Pardon E, Pavlakis GN. 123.  et al. 2011. Measuring cooperative Rev protein-protein interactions on Rev responsive RNA by fluorescence resonance energy transfer. RNA Biol. 8:316–24 [Google Scholar]
  124. Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP. 124.  et al. 2013. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–38First application of nanobodies as biosensors to monitor conformational changes of GPCRs in living cells. [Google Scholar]
  125. Staus DP, Wingler LM, Strachan RT, Rasmussen SGF, Pardon E. 125.  et al. 2014. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol. Pharmacol. 85:472–81 [Google Scholar]
  126. Jähnichen S, Blanchetot C, Maussang D, Gonzalez-Pajuelo M, Chow KY. 126.  et al. 2010. CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. PNAS 107:20565–70 [Google Scholar]
  127. Maussang D, Mujic-Delic A, Descamps FJ, Stortelers C, Vanlandschoot P. 127.  et al. 2013. Llama-derived single variable domains (nanobodies) directed against chemokine receptor CXCR7 reduce head and neck cancer cell growth in vivo. J. Biol. Chem. 288:29562–72 [Google Scholar]
  128. Bradley ME, Dombrecht B, Manini J, Willis J, Vlerick D. 128.  et al. 2015. Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action. Mol. Pharmacol. 87:251–62 [Google Scholar]
  129. Chapman AP, Antoniw P, Spitali M, West S, Stephens S, King DJ. 129.  1999. Therapeutic antibody fragments with prolonged in vivo half-lives. Nat. Biotechnol. 17:780–83 [Google Scholar]
  130. Holt LJ, Herring C, Jespers LS, Woolven BP, Tomlinson IM. 130.  2003. Domain antibodies: proteins for therapy. Trends Biotechnol 21:484–90 [Google Scholar]
  131. Saerens D, Ghassabeh GH, Muyldermans S. 131.  2008. Single-domain antibodies as building blocks for novel therapeutics. Curr. Opin. Pharmacol. 8:600–8 [Google Scholar]
  132. Lawson AD. 132.  2012. Antibody-enabled small-molecule drug discovery. Nat. Rev. Drug Discov. 11:519–25 [Google Scholar]
  133. Henzler-Wildman K, Kern D. 133.  2007. Dynamic personalities of proteins. Nature 450:964–72 [Google Scholar]
  134. Taly A, Corringer PJ, Guedin D, Lestage P, Changeux JP. 134.  2009. Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat. Rev. Drug Discov. 8:733–50 [Google Scholar]
  135. Zhou HX, McCammon JA. 135.  2010. The gates of ion channels and enzymes. Trends Biochem. Sci. 35:179–85 [Google Scholar]
  136. Chene P. 136.  2008. Challenges in design of biochemical assays for the identification of small molecules to target multiple conformations of protein kinases. Drug Discov. Today 13:522–29 [Google Scholar]
  137. Gronemeyer H, Bourguet W. 137.  2009. Allosteric effects govern nuclear receptor action: DNA appears as a player. Sci. Signal. 2:pe34 [Google Scholar]
  138. Lefranc MP, Pommié C, Ruiz M, Giudicelli V, Foulquier E. 138.  et al. 2003. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev. Comp. Immunol. 27:55–77 [Google Scholar]

Data & Media loading...

  • Article Type: Review Article
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error