Pharmacological Assays for Investigating the NOP Receptor

  • Davide Malfacini
  • Girolamo CalóEmail author
Part of the Handbook of Experimental Pharmacology book series (HEP, volume 254)


The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a G protein-coupled receptor involved in the regulation of several physiological functions and pathological conditions. Thus, researchers from academia and industry are pursuing NOP to discover and study novel pharmacological entities. In a multidisciplinary effort of pharmacologists, medicinal chemists, and molecular and structural biologists the mechanisms of NOP activation and inhibition have been, at least partially, disentangled. Here, we review the in vitro methodologies employed, which have contributed to our understanding of this target. We hope this chapter guides the reader through the mostly established assay platforms to investigate NOP pharmacology, and gives some hints taking advantage from what has already illuminated the function of other GPCRs. We analyzed the pharmacological results obtained with a large panel of NOP ligands investigated in several assays including receptor binding, stimulation of GTPγS binding, decrease of cAMP levels, calcium flux stimulation via chimeric G proteins, NOP/G protein and NOP/β-arrestin interaction, label-free assays such as dynamic mass redistribution, and bioassays such as the electrically stimulated mouse vas deferens.


N/OFQ Pharmacological assays Recombinant and native NOP receptors Signal transduction 



We would like to thank T. Kenakin (Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina) for helpful discussion.

Details of Authors’ Contributions

DM and GC wrote the chapter and approved its final version.

Declaration of Interests

DM and GC have nothing to declare.


  1. Arduin M, Spagnolo B, Calo G, Guerrini R, Carra G, Fischetti C, Trapella C, Marzola E, McDonald J, Lambert DG, Regoli D, Salvadori S (2007) Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Calpha,alpha-dialkylated amino acids. Bioorg Med Chem 15(13):4434–4443. CrossRefPubMedGoogle Scholar
  2. Barnes TA, McDonald J, Rowbotham DJ, Duarte TL, Lambert DG (2007) Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system. Naunyn Schmiedebergs Arch Pharmacol 376(3):217–225. CrossRefPubMedGoogle Scholar
  3. Berger H, Calo G, Albrecht E, Guerrini R, Bienert M (2000) [Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain. J Pharmacol Exp Ther 294(2):428–433PubMedGoogle Scholar
  4. Berzetei-Gurske IP, Schwartz RW, Toll L (1996) Determination of activity for nociceptin in the mouse vas deferens. Eur J Pharmacol 302(1–3):R1–R2CrossRefGoogle Scholar
  5. Bevan N, Scott S, Shaw PE, Lee MG, Marshall FH, Rees S (1998) Nociception activates Elk-1 and Sap1a following expression of the ORL1 receptor in Chinese hamster ovary cells. Neuroreport 9(12):2703–2708CrossRefGoogle Scholar
  6. Bigoni R, Calo G, Rizzi A, Guerrini R, De Risi C, Hashimoto Y, Hashiba E, Lambert DG, Regoli D (2000) In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist. Naunyn Schmiedebergs Arch Pharmacol 361(5):565–568CrossRefGoogle Scholar
  7. Bigoni R, Calo G, Guerrini R, Strupish JW, Rowbotham DJ, Lambert DG (2001) Effects of nociceptin and endomorphin 1 on the electrically stimulated human vas deferens. Br J Clin Pharmacol 51(4):355–358CrossRefGoogle Scholar
  8. Bird M, Guerrini R, Calò G, Lambert D (2018) Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594; a novel fluorescent probe for the NOP receptor. Br J Pharmacol 175(24):4496–4506CrossRefGoogle Scholar
  9. Bokoch GM, Katada T, Northup JK, Ui M, Gilman AG (1984) Purification and properties of the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. J Biol Chem 259(6):3560–3567PubMedGoogle Scholar
  10. Bonacci TM, Mathews JL, Yuan C, Lehmann DM, Malik S, Wu D, Font JL, Bidlack JM, Smrcka AV (2006) Differential targeting of Gbetagamma-subunit signaling with small molecules. Science 312(5772):443–446. CrossRefPubMedGoogle Scholar
  11. Briddon SJ, Kilpatrick LE, Hill SJ (2018) Studying GPCR pharmacology in membrane microdomains: fluorescence correlation spectroscopy comes of age. Trends Pharmacol Sci 39(2):158–174. CrossRefPubMedGoogle Scholar
  12. Brown BL, Albano JD, Ekins RP, Sgherzi AM (1971) A simple and sensitive saturation assay method for the measurement of adenosine 3′:5′-cyclic monophosphate. Biochem J 121(3):561–562CrossRefGoogle Scholar
  13. Calebiro D, Sungkaworn T (2018) Single-molecule imaging of GPCR interactions. Trends Pharmacol Sci 39(2):109–122. CrossRefPubMedGoogle Scholar
  14. Calo G, Rizzi A, Bogoni G, Neugebauer V, Salvadori S, Guerrini R, Bianchi C, Regoli D (1996) The mouse vas deferens: a pharmacological preparation sensitive to nociceptin. Eur J Pharmacol 311(1):R3–R5CrossRefGoogle Scholar
  15. Calo G, Rizzi A, Bodin M, Neugebauer W, Salvadori S, Guerrini R, Bianchi C, Regoli D (1997) Pharmacological characterization of nociceptin receptor: an in vitro study. Can J Physiol Pharmacol 75(6):713–718CrossRefGoogle Scholar
  16. Calo G, Bigoni R, Rizzi A, Guerrini R, Salvadori S, Regoli D (2000a) Nociceptin/orphanin FQ receptor ligands. Peptides 21(7):935–947CrossRefGoogle Scholar
  17. Calo G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H, Bianchi C, Lambert DG, Salvadori S, Regoli D (2000b) Characterization of [Nphe(1)]nociceptin(1-13)NH(2), a new selective nociceptin receptor antagonist. Br J Pharmacol 129(6):1183–1193. CrossRefPubMedGoogle Scholar
  18. Calo G, Rizzi A, Rizzi D, Bigoni R, Guerrini R, Marzola G, Marti M, McDonald J, Morari M, Lambert DG, Salvadori S, Regoli D (2002) [Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br J Pharmacol 136(2):303–311. CrossRefPubMedGoogle Scholar
  19. Calo’ G, Guerrini R (2013) Medicinal chemistry, pharmacology, and biological actions of peptide ligands selective for the nociceptin/orphanin FQ receptor. In: Research and Development of opioid-related ligands, ACS symposium series, vol 1131. American Chemical Society, Washington, pp 275–325. CrossRefGoogle Scholar
  20. Camarda V, Fischetti C, Anzellotti N, Molinari P, Ambrosio C, Kostenis E, Regoli D, Trapella C, Guerrini R, Severo S, Calo G (2009) Pharmacological profile of NOP receptors coupled with calcium signaling via the chimeric protein G alpha qi5. Naunyn Schmiedebergs Arch Pharmacol 379(6):599–607. CrossRefPubMedGoogle Scholar
  21. Chan JS, Yung LY, Lee JW, Wu YL, Pei G, Wong YH (1998) Pertussis toxin-insensitive signaling of the ORL1 receptor: coupling to Gz and G16 proteins. J Neurochem 71(5):2203–2210CrossRefGoogle Scholar
  22. Chang SD, Brieaddy LE, Harvey JD, Lewin AH, Mascarella SW, Seltzman HH, Reddy PA, Decker AM, McElhinny CJ Jr, Zhong D, Peterson EE, Navarro HA, Bruchas MR, Carroll FI (2015a) Novel synthesis and pharmacological characterization of NOP receptor agonist 8-[(1S,3aS)-2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198). ACS Chem Neurosci 6(12):1956–1964. CrossRefPubMedGoogle Scholar
  23. Chang SD, Mascarella SW, Spangler SM, Gurevich VV, Navarro HA, Carroll FI, Bruchas MR (2015b) Quantitative signaling and structure-activity analyses demonstrate functional selectivity at the nociceptin/orphanin FQ opioid receptor. Mol Pharmacol 88(3):502–511. CrossRefPubMedGoogle Scholar
  24. Charest PG, Terrillon S, Bouvier M (2005) Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET. EMBO Rep 6(4):334–340. CrossRefPubMedGoogle Scholar
  25. Charlton SJ, Vauquelin G (2010) Elusive equilibrium: the challenge of interpreting receptor pharmacology using calcium assays. Br J Pharmacol 161(6):1250–1265. CrossRefPubMedGoogle Scholar
  26. Civelli O, Reinscheid RK, Zhang Y, Wang Z, Fredriksson R, Schioth HB (2013) G protein-coupled receptor deorphanizations. Annu Rev Pharmacol Toxicol 53:127–146. CrossRefPubMedGoogle Scholar
  27. Conklin BR, Farfel Z, Lustig KD, Julius D, Bourne HR (1993) Substitution of three amino acids switches receptor specificity of Gq alpha to that of Gi alpha. Nature 363(6426):274–276. CrossRefPubMedGoogle Scholar
  28. Connor M, Vaughan CW, Chieng B, Christie MJ (1996) Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro. Br J Pharmacol 119(8):1614–1618CrossRefGoogle Scholar
  29. Corbani M, Gonindard C, Meunier JC (2004) Ligand-regulated internalization of the opioid receptor-like 1: a confocal study. Endocrinology 145(6):2876–2885. CrossRefPubMedGoogle Scholar
  30. Corder G, Castro DC, Bruchas MR, Scherrer G (2018) Endogenous and exogenous opioids in pain. Annu Rev Neurosci 41:453–473. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Coward P, Chan SD, Wada HG, Humphries GM, Conklin BR (1999) Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal Biochem 270(2):242–248. CrossRefPubMedGoogle Scholar
  32. Dooley CT, Houghten RA (1996) Orphanin FQ: receptor binding and analog structure activity relationships in rat brain. Life Sci 59(1):PL23–PL29CrossRefGoogle Scholar
  33. Dooley CT, Houghten RA (2000) Orphanin FQ/nociceptin receptor binding studies. Peptides 21(7):949–960CrossRefGoogle Scholar
  34. Fang Y (2011) Label-free receptor assays. Drug Discov Today Technol 7(1):e5–e11. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Ferrari F, Cerlesi MC, Malfacini D, Asth L, Gavioli EC, Journigan BV, Kamakolanu UG, Meyer ME, Yasuda D, Polgar WE, Rizzi A, Guerrini R, Ruzza C, Zaveri NT, Calo G (2016) In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations. Eur J Pharmacol 793:1–13. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Ferrari F, Malfacini D, Journigan BV, Bird MF, Trapella C, Guerrini R, Lambert DG, Calo G, Zaveri NT (2017) In vitro pharmacological characterization of a novel unbiased NOP receptor-selective nonpeptide agonist AT-403. Pharmacol Res Perspect 5(4). CrossRefGoogle Scholar
  37. Filizola M, Devi LA (2013) Grand opening of structure-guided design for novel opioids. Trends Pharmacol Sci 34(1):6–12. CrossRefPubMedGoogle Scholar
  38. Fischetti C, Camarda V, Rizzi A, Pela M, Trapella C, Guerrini R, McDonald J, Lambert DG, Salvadori S, Regoli D, Calo G (2009) Pharmacological characterization of the nociceptin/orphanin FQ receptor non peptide antagonist Compound 24. Eur J Pharmacol 614(1–3):50–57. CrossRefPubMedGoogle Scholar
  39. Fukuda K, Shoda T, Morikawa H, Kato S, Mori K (1997) Activation of mitogen-activated protein kinase by the nociceptin receptor expressed in Chinese hamster ovary cells. FEBS Lett 412(2):290–294CrossRefGoogle Scholar
  40. 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(3):177–184. CrossRefPubMedGoogle Scholar
  41. Garbison KE, Heinz BA, Lajiness ME, Weidner JR, Sittampalam GS (2004) Phospho-ERK assays. In: Sittampalam GS, Coussens NP, Brimacombe K et al (eds) Assay guidance manual. National Institutes of Health, BethesdaGoogle Scholar
  42. Giuliani S, Maggi CA (1996) Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide. Br J Pharmacol 118(7):1567–1569CrossRefGoogle Scholar
  43. Giuliani S, Lecci A, Maggi CA (2000) Nociceptin and neurotransmitter release in the periphery. Peptides 21(7):977–984CrossRefGoogle Scholar
  44. Grundmann M (2017) Label-free dynamic mass redistribution and bio-impedance methods for drug discovery. Curr Protoc Pharmacol 77(9):1–21. CrossRefGoogle Scholar
  45. Grundmann M, Merten N, Malfacini D, Inoue A, Preis P, Simon K, Ruttiger N, Ziegler N, Benkel T, Schmitt NK, Ishida S, Muller I, Reher R, Kawakami K, Inoue A, Rick U, Kuhl T, Imhof D, Aoki J, Konig GM, Hoffmann C, Gomeza J, Wess J, Kostenis E (2018) Lack of beta-arrestin signaling in the absence of active G proteins. Nat Commun 9(1):341. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Guerrini R, Calo G, Rizzi A, Bigoni R, Bianchi C, Salvadori S, Regoli D (1998) A new selective antagonist of the nociceptin receptor. Br J Pharmacol 123(2):163–165. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Gulati S, Jin H, Masuho I, Orban T, Cai Y, Pardon E, Martemyanov KA, Kiser PD, Stewart PL, Ford CP, Steyaert J, Palczewski K (2018) Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor. Nat Commun 9(1):1996. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Hashiba E, Harrison C, Galo G, Guerrini R, Rowbotham DJ, Smith G, Lambert DG (2001) Characterisation and comparison of novel ligands for the nociceptin/orphanin FQ receptor. Naunyn Schmiedebergs Arch Pharmacol 363(1):28–33CrossRefGoogle Scholar
  49. Hashimoto Y, Calo G, Guerrini R, Smith G, Lambert DG (2000) Antagonistic effects of [Nphe1]nociceptin(1-13)NH2 on nociceptin receptor mediated inhibition of cAMP formation in Chinese hamster ovary cells stably expressing the recombinant human nociceptin receptor. Neurosci Lett 278(1–2):109–112CrossRefGoogle Scholar
  50. Hashimoto Y, Calo G, Guerrini R, Smith G, Lambert DG (2002) Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese hamster ovary cells expressing human nociceptin/orphanin FQ receptors. Eur J Pharmacol 449(1–2):17–22CrossRefGoogle Scholar
  51. Hawes BE, Fried S, Yao X, Weig B, Graziano MP (1998) Nociceptin (ORL-1) and mu-opioid receptors mediate mitogen-activated protein kinase activation in CHO cells through a Gi-coupled signaling pathway: evidence for distinct mechanisms of agonist-mediated desensitization. J Neurochem 71(3):1024–1033CrossRefGoogle Scholar
  52. Hillger JM, Lieuw WL, Heitman LH, AP IJ (2017) Label-free technology and patient cells: from early drug development to precision medicine. Drug Discov Today 22(12):1808–1815. CrossRefPubMedGoogle Scholar
  53. Hirao A, Imai A, Sugie Y, Yamada Y, Hayashi S, Toide K (2008) Pharmacological characterization of the newly synthesized nociceptin/orphanin FQ-receptor agonist 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole as an anxiolytic agent. J Pharmacol Sci 106(3):361–368CrossRefGoogle Scholar
  54. Ikeda K, Kobayashi K, Kobayashi T, Ichikawa T, Kumanishi T, Kishida H, Yano R, Manabe T (1997) Functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated K+ (GIRK) channel. Brain Res Mol Brain Res 45(1):117–126CrossRefGoogle Scholar
  55. Johnson GL, Lapadat R (2002) Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298(5600):1911–1912. CrossRefPubMedGoogle Scholar
  56. Kam KW, New DC, Wong YH (2002) Constitutive activation of the opioid receptor-like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region. J Neurochem 83(6):1461–1470CrossRefGoogle Scholar
  57. Katritch V, Fenalti G, Abola EE, Roth BL, Cherezov V, Stevens RC (2014) Allosteric sodium in class A GPCR signaling. Trends Biochem Sci 39(5):233–244. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Kenakin TP (2014a) Chapter 4 - Pharmacological assay formats: binding. In: Kenakin TP (ed) A pharmacology primer, 4th edn. Academic Press, San Diego, pp 63–83. CrossRefGoogle Scholar
  59. Kenakin TP (2014b) Chapter 5 - Agonists: the measurement of affinity and efficacy in functional assays. In: Kenakin TP (ed) A pharmacology primer, 4th edn. Academic Press, San Diego, pp 85–117. CrossRefGoogle Scholar
  60. Lambert DG (2008) The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential. Nat Rev Drug Discov 7(8):694–710. CrossRefPubMedPubMedCentralGoogle Scholar
  61. Lee MY, Mun J, Lee JH, Lee S, Lee BH, Oh KS (2014) A comparison of assay performance between the calcium mobilization and the dynamic mass redistribution technologies for the human urotensin receptor. Assay Drug Dev Technol 12(6):361–368. CrossRefPubMedPubMedCentralGoogle Scholar
  62. Liao YY, Lee CW, Ho IK, Chiou LC (2012) Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells. Life Sci 90(7–8):306–312. CrossRefPubMedGoogle Scholar
  63. Liu JJ, Sharma K, Zangrandi L, Chen C, Humphrey SJ, Chiu YT, Spetea M, Liu-Chen LY, Schwarzer C, Mann M (2018) In vivo brain GPCR signaling elucidated by phosphoproteomics. Science 360(6395). CrossRefGoogle Scholar
  64. Lou LG, Ma L, Pei G (1997) Nociceptin/orphanin FQ activates protein kinase C, and this effect is mediated through phospholipase C/Ca2+ pathway. Biochem Biophys Res Commun 240(2):304–308. CrossRefPubMedGoogle Scholar
  65. Lowry WE, Huang J, Ma YC, Ali S, Wang D, Williams DM, Okada M, Cole PA, Huang XY (2002) Csk, a critical link of g protein signals to actin cytoskeletal reorganization. Dev Cell 2(6):733–744CrossRefGoogle Scholar
  66. Lundstrom K (2017) Cell-impedance-based label-free technology for the identification of new drugs. Expert Opin Drug Discovery 12(4):335–343. CrossRefGoogle Scholar
  67. Mahmoud S, Margas W, Trapella C, Calo G, Ruiz-Velasco V (2010) Modulation of silent and constitutively active nociceptin/orphanin FQ receptors by potent receptor antagonists and Na+ ions in rat sympathetic neurons. Mol Pharmacol 77(5):804–817. CrossRefPubMedPubMedCentralGoogle Scholar
  68. Mahmoud S, Yun JK, Ruiz-Velasco V (2012) Gbeta2 and Gbeta4 participate in the opioid and adrenergic receptor-mediated Ca2+ channel modulation in rat sympathetic neurons. J Physiol 590(19):4673–4689. CrossRefPubMedPubMedCentralGoogle Scholar
  69. Mahmoud S, Farrag M, Ruiz-Velasco V (2016) Ggamma7 proteins contribute to coupling of nociceptin/orphanin FQ peptide (NOP) opioid receptors and voltage-gated Ca(2+) channels in rat stellate ganglion neurons. Neurosci Lett 627:77–83. CrossRefPubMedPubMedCentralGoogle Scholar
  70. Malfacini D, Ambrosio C, Gro MC, Sbraccia M, Trapella C, Guerrini R, Bonora M, Pinton P, Costa T, Calo G (2015) Pharmacological profile of nociceptin/orphanin FQ receptors interacting with G-proteins and beta-arrestins 2. PLoS One 10(8):e0132865. CrossRefPubMedPubMedCentralGoogle Scholar
  71. Malfacini D, Simon K, Trapella C, Guerrini R, Zaveri NT, Kostenis E, Calo G (2018) NOP receptor pharmacological profile – a dynamic mass redistribution study. PLoS One 13(8):e0203021. CrossRefPubMedPubMedCentralGoogle Scholar
  72. McDonald J, Lambert DG (2010) Binding of GTPgamma[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor. Br J Pharmacol 159(6):1286–1293. CrossRefPubMedPubMedCentralGoogle Scholar
  73. McDonald J, Barnes TA, Okawa H, Williams J, Calo G, Rowbotham DJ, Lambert DG (2003a) Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system. Br J Pharmacol 140(1):61–70. CrossRefPubMedPubMedCentralGoogle Scholar
  74. McDonald J, Calo G, Guerrini R, Lambert DG (2003b) UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPgamma(35)S binding studies. Naunyn Schmiedebergs Arch Pharmacol 367(2):183–187. CrossRefPubMedGoogle Scholar
  75. Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B et al (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377(6549):532–535. CrossRefGoogle Scholar
  76. Miller RL, Thompson AA, Trapella C, Guerrini R, Malfacini D, Patel N, Han GW, Cherezov V, Calo G, Katritch V, Stevens RC (2015) The importance of ligand-receptor conformational pairs in stabilization: spotlight on the N/OFQ G protein-coupled receptor. Structure 23(12):2291–2299. CrossRefPubMedPubMedCentralGoogle Scholar
  77. Miyakawa K, Uchida A, Shiraki T, Teshima K, Takeshima H, Shibata S (2007) ORL1 receptor-mediated down-regulation of mPER2 in the suprachiasmatic nucleus accelerates re-entrainment of the circadian clock following a shift in the environmental light/dark cycle. Neuropharmacology 52(3):1055–1064. CrossRefPubMedGoogle Scholar
  78. Mollereau C, Moisand C, Butour JL, Parmentier M, Meunier JC (1996) Replacement of Gln280 by his in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids. FEBS Lett 395(1):17–21CrossRefGoogle Scholar
  79. Mouledous L, Topham CM, Moisand C, Mollereau C, Meunier JC (2000) Functional inactivation of the nociceptin receptor by alanine substitution of glutamine 286 at the C terminus of transmembrane segment VI: evidence from a site-directed mutagenesis study of the ORL1 receptor transmembrane-binding domain. Mol Pharmacol 57(3):495–502CrossRefGoogle Scholar
  80. Musheshe N, Schmidt M, Zaccolo M (2018) cAMP: from long-range second messenger to nanodomain signalling. Trends Pharmacol Sci 39(2):209–222. CrossRefPubMedGoogle Scholar
  81. Okawa H, Nicol B, Bigoni R, Hirst RA, Calo G, Guerrini R, Rowbotham DJ, Smart D, McKnight AT, Lambert DG (1999) Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors. Br J Pharmacol 127(1):123–130. CrossRefPubMedGoogle Scholar
  82. Onaran HO, Ambrosio C, Ugur O, Madaras Koncz E, Gro MC, Vezzi V, Rajagopal S, Costa T (2017) Systematic errors in detecting biased agonism: analysis of current methods and development of a new model-free approach. Sci Rep 7:44247. CrossRefPubMedGoogle Scholar
  83. Ozawa A, Brunori G, Mercatelli D, Wu J, Cippitelli A, Zou B, Xie XS, Williams M, Zaveri NT, Low S, Scherrer G, Kieffer BL, Toll L (2015) Knock-in mice with NOP-eGFP receptors identify receptor cellular and regional localization. J Neurosci Off J Soc Neurosci 35(33):11682–11693. CrossRefGoogle Scholar
  84. Peters MF, Knappenberger KS, Wilkins D, Sygowski LA, Lazor LA, Liu J, Scott CW (2007) Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs. J Biomol Screen 12(3):312–319. CrossRefPubMedGoogle Scholar
  85. Pheng LH, Regoli D (1998) Bioassays for NPY receptors: old and new. Regul Pept 75-76:79–87CrossRefGoogle Scholar
  86. Rajagopal S, Ahn S, Rominger DH, Gowen-MacDonald W, Lam CM, Dewire SM, Violin JD, Lefkowitz RJ (2011) Quantifying ligand bias at seven-transmembrane receptors. Mol Pharmacol 80(3):367–377. CrossRefPubMedPubMedCentralGoogle Scholar
  87. Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ Jr, Civelli O (1995) Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270(5237):792–794CrossRefGoogle Scholar
  88. Reinscheid RK, Ardati A, Monsma FJ Jr, Civelli O (1996) Structure-activity relationship studies on the novel neuropeptide orphanin FQ. J Biol Chem 271(24):14163–14168CrossRefGoogle Scholar
  89. Rinken A, Lavogina D, Kopanchuk S (2018) Assays with detection of fluorescence anisotropy: challenges and possibilities for characterizing ligand binding to GPCRs. Trends Pharmacol Sci 39(2):187–199. CrossRefPubMedGoogle Scholar
  90. Rizzi D, Bigoni R, Rizzi A, Jenck F, Wichmann J, Guerrini R, Regoli D, Calo G (2001) Effects of Ro 64-6198 in nociceptin/orphanin FQ-sensitive isolated tissues. Naunyn Schmiedebergs Arch Pharmacol 363(5):551–555CrossRefGoogle Scholar
  91. Rizzi A, Rizzi D, Marzola G, Regoli D, Larsen BD, Petersen JS, Calo G (2002) Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice. Br J Pharmacol 137(3):369–374. CrossRefPubMedGoogle Scholar
  92. Rizzi A, Spagnolo B, Wainford RD, Fischetti C, Guerrini R, Marzola G, Baldisserotto A, Salvadori S, Regoli D, Kapusta DR, Calo G (2007) In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor. Peptides 28(6):1240–1251. CrossRefPubMedGoogle Scholar
  93. Rizzi A, Malfacini D, Cerlesi MC, Ruzza C, Marzola E, Bird MF, Rowbotham DJ, Salvadori S, Guerrini R, Lambert DG, Calo G (2014) In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives. Br J Pharmacol 171(17):4138–4153. CrossRefPubMedGoogle Scholar
  94. Rizzi A, Cerlesi MC, Ruzza C, Malfacini D, Ferrari F, Bianco S, Costa T, Guerrini R, Trapella C, Calo G (2016) Pharmacological characterization of cebranopadol a novel analgesic acting as mixed nociceptin/orphanin FQ and opioid receptor agonist. Pharmacol Res Perspect 4(4):e00247. CrossRefPubMedGoogle Scholar
  95. Schrage R, Schmitz AL, Gaffal E, Annala S, Kehraus S, Wenzel D, Bullesbach KM, Bald T, Inoue A, Shinjo Y, Galandrin S, Shridhar N, Hesse M, Grundmann M, Merten N, Charpentier TH, Martz M, Butcher AJ, Slodczyk T, Armando S, Effern M, Namkung Y, Jenkins L, Horn V, Stossel A, Dargatz H, Tietze D, Imhof D, Gales C, Drewke C, Muller CE, Holzel M, Milligan G, Tobin AB, Gomeza J, Dohlman HG, Sondek J, Harden TK, Bouvier M, Laporte SA, Aoki J, Fleischmann BK, Mohr K, Konig GM, Tuting T, Kostenis E (2015) The experimental power of FR900359 to study Gq-regulated biological processes. Nat Commun 6:10156. CrossRefPubMedGoogle Scholar
  96. Schroder R, Janssen N, Schmidt J, Kebig A, Merten N, Hennen S, Muller A, Blattermann S, Mohr-Andra M, Zahn S, Wenzel J, Smith NJ, Gomeza J, Drewke C, Milligan G, Mohr K, Kostenis E (2010) Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol 28(9):943–949. CrossRefPubMedGoogle Scholar
  97. Schroder R, Schmidt J, Blattermann S, Peters L, Janssen N, Grundmann M, Seemann W, Kaufel D, Merten N, Drewke C, Gomeza J, Milligan G, Mohr K, Kostenis E (2011) Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells. Nat Protoc 6(11):1748–1760. CrossRefPubMedGoogle Scholar
  98. Scott CW, Peters MF (2010) Label-free whole-cell assays: expanding the scope of GPCR screening. Drug Discov Today 15(17–18):704–716. CrossRefPubMedGoogle Scholar
  99. Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, Reiter E, Premont RT, Lichtarge O, Lefkowitz RJ (2006) Beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem 281(2):1261–1273. CrossRefPubMedGoogle Scholar
  100. Sim LJ, Xiao R, Childers SR (1996) Identification of opioid receptor-like (ORL1) peptide-stimulated [35S]GTP gamma S binding in rat brain. Neuroreport 7(3):729–733CrossRefGoogle Scholar
  101. Spagnolo B, Carra G, Fantin M, Fischetti C, Hebbes C, McDonald J, Barnes TA, Rizzi A, Trapella C, Fanton G, Morari M, Lambert DG, Regoli D, Calo G (2007) Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrah ydro-5H-benzocyclohepten-5-ol]: in vitro studies. J Pharmacol Exp Ther 321(3):961–967. CrossRefPubMedGoogle Scholar
  102. Spampinato S, Baiula M (2006) Agonist-regulated endocytosis and desensitization of the human nociceptin receptor. Neuroreport 17(2):173–177CrossRefGoogle Scholar
  103. Spampinato S, Di Toro R, Qasem AR (2001) Nociceptin-induced internalization of the ORL1 receptor in human neuroblastoma cells. Neuroreport 12(14):3159–3163CrossRefGoogle Scholar
  104. Spampinato S, Di Toro R, Alessandri M, Murari G (2002) Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells. Cell Mol Life Sci 59(12):2172–2183CrossRefGoogle Scholar
  105. Spampinato S, Baiula M, Calienni M (2007) Agonist-regulated internalization and desensitization of the human nociceptin receptor expressed in CHO cells. Curr Drug Targets 8(1):137–146CrossRefGoogle Scholar
  106. Strasser A, Wittmann HJ, Seifert R (2017) Binding kinetics and pathways of ligands to GPCRs. Trends Pharmacol Sci 38(8):717–732. CrossRefPubMedGoogle Scholar
  107. Thompson AA, Liu W, Chun E, Katritch V, Wu H, Vardy E, Huang XP, Trapella C, Guerrini R, Calo G, Roth BL, Cherezov V, Stevens RC (2012) Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485(7398):395–399. CrossRefPubMedPubMedCentralGoogle Scholar
  108. Toll L, Bruchas MR, Calo G, Cox BM, Zaveri NT (2016) Nociceptin/orphanin FQ receptor structure, signaling, ligands, functions, and interactions with opioid systems. Pharmacol Rev 68(2):419–457. CrossRefPubMedPubMedCentralGoogle Scholar
  109. Trapella C, Guerrini R, Piccagli L, Calo G, Carra G, Spagnolo B, Rubini S, Fanton G, Hebbes C, McDonald J, Lambert DG, Regoli D, Salvadori S (2006) Identification of an achiral analogue of J-113397 as potent nociceptin/orphanin FQ receptor antagonist. Bioorg Med Chem 14(3):692–704. CrossRefPubMedGoogle Scholar
  110. Trombella S, Vergura R, Falzarano S, Guerrini R, Calo G, Spisani S (2005) Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation. Peptides 26(8):1497–1502. CrossRefPubMedGoogle Scholar
  111. Vezzi V, Onaran HO, Molinari P, Guerrini R, Balboni G, Calo G, Costa T (2013) Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors. J Biol Chem 288(33):23964–23978. CrossRefPubMedPubMedCentralGoogle Scholar
  112. Wang T, Li Z, Cvijic ME, Zhang L, Sum CS (2004) Measurement of cAMP for galphas- and galphai protein-coupled receptors (GPCRs). In: Sittampalam GS, Coussens NP, Brimacombe K et al (eds) Assay guidance manual, vol 11(5). National Institutes of Health, Bethesda, pp 461–470Google Scholar
  113. Wang HL, Kuo YL, Hsu CY, Huang PC, Li AH, Chou AH, Yeh TH, Chen YL (2006) Two C-terminal amino acids, Ser(334) and Ser(335), are required for homologous desensitization and agonist-induced phosphorylation of opioid receptor-like 1 receptors. Cell Signal 18(5):670–678. CrossRefPubMedGoogle Scholar
  114. Wnendt S, Kruger T, Janocha E, Hildebrandt D, Englberger W (1999) Agonistic effect of buprenorphine in a nociceptin/OFQ receptor-triggered reporter gene assay. Mol Pharmacol 56(2):334–338CrossRefGoogle Scholar
  115. Wright KE, McDonald J, Barnes TA, Rowbotham DJ, Guerrini R, Calo G, Lambert DG (2003) Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells. Neurosci Lett 346(3):145–148CrossRefGoogle Scholar
  116. Wu YL, Pu L, Ling K, Zhao J, Cheng ZJ, Ma L, Pei G (1997) Molecular characterization and functional expression of opioid receptor-like1 receptor. Cell Res 7(1):69–77. CrossRefPubMedGoogle Scholar
  117. Yang Z, Yang F, Zhang D, Liu Z, Lin A, Liu C, Xiao P, Yu X, Sun JP (2017) Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model. Mol Pharmacol 92(3):201–210. CrossRefPubMedGoogle Scholar
  118. Yung LY, Joshi SA, Chan RY, Chan JS, Pei G, Wong YH (1999) GalphaL1 (Galpha14) couples the opioid receptor-like1 receptor to stimulation of phospholipase C. J Pharmacol Exp Ther 288(1):232–238PubMedGoogle Scholar
  119. Zhang G, Murray TF, Grandy DK (1997) Orphanin FQ has an inhibitory effect on the guinea pig ileum and the mouse vas deferens. Brain Res 772(1–2):102–106CrossRefGoogle Scholar
  120. Zhang NR, Planer W, Siuda ER, Zhao HC, Stickler L, Chang SD, Baird MA, Cao YQ, Bruchas MR (2012) Serine 363 is required for nociceptin/orphanin FQ opioid receptor (NOPR) desensitization, internalization, and arrestin signaling. J Biol Chem 287(50):42019–42030. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Molecular, Cellular and Pharmacobiology SectionInstitute for Pharmaceutical Biology, University of BonnBonnGermany
  2. 2.Section of Pharmacology, Department of Medical SciencesNational Institute of Neurosciences, University of FerraraFerraraItaly

Personalised recommendations