Advertisement

Elucidating Agonist-Selective Mechanisms of G Protein-Coupled Receptor Desensitization

  • Chris P. BaileyEmail author
  • Eamonn Kelly
Protocol
Part of the Neuromethods book series (NM, volume 60)

Abstract

In pharmacology, a central tenet of receptor theory has been that different agonists acting at a particular G protein-coupled receptor subtype produce the same profile of cellular responses. In recent years, advances in molecular pharmacology and the availability of diverse cell signaling assays have indicated that this idea is not sufficient to explain all the data obtained, and that agonists can produce different response profiles ­following binding to a receptor subtype in a cell. This has been termed biased agonism or functional selectivity­, and is thought to be due to the ability of agonists to stabilize different active conformations of the receptor. Logically, there is no reason why this idea cannot also be extended to receptor regulatory mechanisms, since different receptor conformations could exhibit differential affinities for regulatory elements such as the kinases involved in receptor phosphorylation and desensitization. Nevertheless, great care must be taken when analyzing agonist response and regulatory pathways, since other factors such as differences in agonist efficacy need to be considered as contributing factors to agonist-dependent regulation. In the case of the μ-opioid receptor (MOPr), we have shown that two agonists, morphine and the peptide agonist DAMGO, can induce MOPr desensitization by different mechanisms involving largely protein kinase C (PKC) and G protein-coupled receptor kinase/arrestin respectively. This could explain why opioid agonists have variable clinical profiles and liabilities to induce tolerance and dependence. Here we describe the experimental approaches that can be used to investigate mechanisms of MOPr desensitization with a particular focus on endogenous MOPr in neurons. In addition, we discuss the role that agonist efficacy might play in desensitization and describe methods to estimate agonist efficacy for responses downstream of receptor activation, including arrestin recruitment which can be regarded as both a regulatory and a signaling mechanism.

Key words

Desensitization Tolerance Functional selectivity Biased agonists G protein-coupled ­receptors Opioid receptors MOPr Internalization 

References

  1. 1.
    Hausdorff WP, Caron MG, Lefkowitz RJ (1990) Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4:2881–2889PubMedGoogle Scholar
  2. 2.
    Benovic JL, Strasser RH, Caron MG et al (1986) Beta-adrenergic receptor kinase:identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci USA 83:2797–2801PubMedCrossRefGoogle Scholar
  3. 3.
    Lohse MJ, Benovic JL, Codina J et al (1990) Beta-Arrestin:a protein that regulates beta-adrenergic receptor function. Science 248:1547–1550PubMedCrossRefGoogle Scholar
  4. 4.
    Lohse MJ, Benovic JL, Caron MG et al (1990) Multiple pathways of rapid beta 2-adrenergic receptor desensitization: Delineation with specific inhibitors. J Biol Chem 265:3202–3211PubMedGoogle Scholar
  5. 5.
    Pitcher J, Lohse MJ, Codina J et al (1992) Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms. Biochemistry 31:3193–3197PubMedCrossRefGoogle Scholar
  6. 6.
    Kelly E, Bailey CP, Henderson G (2008) Agonist-selective mechanisms of GPCR desensitization. Br J Pharmacol 153 Suppl 1:S379–388PubMedGoogle Scholar
  7. 7.
    Haberstock-Debic H, Kim KA, Yu YJ et al (2005) Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J Neurosci 25:7847–7857PubMedCrossRefGoogle Scholar
  8. 8.
    Raymond JR (1995) Multiple mechanisms of receptor-G protein signaling specificity. Am J Physiol 269:F141–158PubMedGoogle Scholar
  9. 9.
    Urban JD, Clarke WP, von Zastrow M et al (2007) Functional selectivity and classical ­concepts of quantitative pharmacology. J Pharmacol Exp Ther 320:1–13PubMedCrossRefGoogle Scholar
  10. 10.
    Keith DE, Murray SR, Zaki PA et al (1996) Morphine activates opioid receptors without causing their rapid internalization. J Biol Chem 271:19021–19024PubMedCrossRefGoogle Scholar
  11. 11.
    Borgland SL, Connor M, Osborne PB et al (2003) Opioid agonists have a different efficacy profiles for G-protein activation, rapid desensitization, and endocytosis of mu-opioid receptors. J Biol Chem 278:18776–18784PubMedCrossRefGoogle Scholar
  12. 12.
    Schulz S, Mayer D, Pfeiffer M et al (2004) Morphine induces terminal mu-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO J 23:3282–3289PubMedCrossRefGoogle Scholar
  13. 13.
    Koch T, Widera A, Bartzsch K et al (2005) Receptor endocytosis counteracts the development of opioid tolerance. Mol Pharmacol 67:280–287PubMedCrossRefGoogle Scholar
  14. 14.
    Arttamangkul S, Torrecilla M, Kobayashi K et al (2006) Separation of mu-opioid receptor ­desensitization and internalization: ­endogenous receptors in primary neuronal cultures. J Neurosci 26:4118–4125PubMedCrossRefGoogle Scholar
  15. 15.
    Harris GC, Williams JT (1991) Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons. J Neurosci 11:2574–2581PubMedGoogle Scholar
  16. 16.
    Yu Y, Zhang L, Yin X et al (1997) Mu opioid receptor phosphorylation, desensitization, and ligand efficacy. J Biol Chem 272:28869–28874PubMedCrossRefGoogle Scholar
  17. 17.
    Kovoor A, Celver JP, Wu A et al (1998) Agonist induced homologous desensitization of mu-opioid receptors mediated by G protein-­coupled receptor kinases is dependent on agonist efficacy. Mol Pharmacol 54:704–711PubMedGoogle Scholar
  18. 18.
    Whistler JL, von Zastrow M (1998) Morphine-activated opioid receptors elude desensitization by beta-arrestin. Proc Natl Acad Sci USA 95:9914–9919PubMedCrossRefGoogle Scholar
  19. 19.
    Alvarez VA, Arttamangkul S, Dang V et al (2002) Mu-opioid receptors: Ligand-dependent activation of potassium conductance, desensitization, and internalization. J Neurosci 22:5769–5776PubMedGoogle Scholar
  20. 20.
    Blanchet C, Sollini M, Lüscher C (2003) Two distinct forms of desensitization of G-protein coupled inwardly rectifying potassium currents evoked by alkaloid and peptide mu-opioid receptor agonists. Mol Cell Neurosci 24:517–523PubMedCrossRefGoogle Scholar
  21. 21.
    Bailey CP, Couch D, Johnson E et al (2003) Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine. J Neurosci 23:10515–10520PubMedGoogle Scholar
  22. 22.
    Terman GW, Jin W, Cheong YP et al (2004) G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal. Br J Pharmacol 141:55–64PubMedCrossRefGoogle Scholar
  23. 23.
    Clark RB, Knoll BJ, Barber R (1999) Partial agonists and G protein-coupled receptor desensitization. Trends Pharmacol Sci 20:279–286PubMedCrossRefGoogle Scholar
  24. 24.
    Mandyam CD, Thakker DR, Christensen JL et al (2002) Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and mu opioid receptors involves protein kinase C:a molecular mechanism for heterologous cross-talk. J Pharmacol Exp Ther 302:502–9PubMedCrossRefGoogle Scholar
  25. 25.
    Mandyam CD, Thakker DR, Standifer KM (2003) Mu-opioid-induced desensitization of opioid receptor-like 1 and mu-opioid receptors: differential intracellular signaling determines receptor sensitivity. J Pharmacol Exp Ther 306:965–972PubMedCrossRefGoogle Scholar
  26. 26.
    Bailey CP, Kelly E, Henderson G (2004) Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus ceruleus neurons. Mol Pharmacol 66:1592–1598PubMedCrossRefGoogle Scholar
  27. 27.
    Bailey CP, Smith FL, Kelly E et al (2006) How important is protein kinase C in mu-opioid receptor desensitization and morphine tolerance? Trends Pharmacol Sci 27:558–565PubMedCrossRefGoogle Scholar
  28. 28.
    Johnson EA, Oldfield S, Braksator E et al (2006) Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells. Mol Pharmacol 70:676–685PubMedCrossRefGoogle Scholar
  29. 29.
    Bailey CP, Oldfield S, Llorente J et al (2009) Involvement of PKCalpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons. Br J Pharmacol 158:157–164PubMedCrossRefGoogle Scholar
  30. 30.
    Bailey CP, Llorente J, Gabra BH et al (2009) Role of protein kinase C and mu-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons. Eur J Neurosci 29:307–318PubMedCrossRefGoogle Scholar
  31. 31.
    Dang VC, Napier IA, Christie MJ (2009) Two distinct mechanisms mediate acute mu-opioid receptor desensitization in native neurons. J Neurosci 29:3322–3327PubMedCrossRefGoogle Scholar
  32. 32.
    Chu J, Zheng H, Zhang Y et al (2010) Agonist-dependent mu-opioid receptor signaling can lead to heterologous desensitization. Cell Signal 22:684696PubMedCrossRefGoogle Scholar
  33. 33.
    Lewis MM, Watts VJ, Lawler CP et al (1998) Homologous desensitization of the D1A dopamine receptor: efficacy in causing desensitization dissociates from both receptor occupancy and functional potency. J Pharmacol Exp Ther 286:345–353PubMedGoogle Scholar
  34. 34.
    Simmons MA (2006) Functional selectivity of NK1 receptor signaling:peptide agonists can preferentially produce receptor activation or desensitization. J Pharmacol Exp Ther 319:907–913PubMedCrossRefGoogle Scholar
  35. 35.
    Walz W (ed) (2007) Neuromethods, Vol. 38: Patch-clamp analysis: Advanced techniques, Second Edition. Humana, TotowaGoogle Scholar
  36. 36.
    Dang VC, Williams JT (2005) Morphine-induced mu-opioid receptor desensitization. Mol Pharmacol 68:1127–1132PubMedCrossRefGoogle Scholar
  37. 37.
    Ingram S, Wilding TJ, McCleskey EW et al (1997) Efficacy and kinetics of opioid action on acutely dissociated neurons. Mol Pharmacol 52:136–143PubMedGoogle Scholar
  38. 38.
    North RA, Williams JT (1985) On the potassium conductance increased by opioids in rat locus coeruleus neurones. J Physiol 364:265–280PubMedGoogle Scholar
  39. 39.
    Connor M, Vaughan CW, Chieng B et al (1996) Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro. Br J Pharmacol 119:1614–1618PubMedGoogle Scholar
  40. 40.
    Christie MJ, Williams JT, North RA (1987) Cellular mechanisms of opioid tolerance:studies in single brain neurons. Mol Pharmacol 32:633–638PubMedGoogle Scholar
  41. 41.
    Rodriguez-Martin I, Braksator E, Bailey CP et al (2008) Methadone: does it really have low efficacy at mu-opioid receptors? Neuroreport 19:589–593PubMedCrossRefGoogle Scholar
  42. 42.
    Bagley EE, Chieng BC, Christie MJ et al (2005) Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine. Br J Pharmacol 146:68–76PubMedCrossRefGoogle Scholar
  43. 43.
    Johnson EE, Chieng B, Napier I et al (2006) Decreased mu-opioid receptor signalling and a reduction in calcium current density in sensory neurons from chronically morphine-treated mice. Br J Pharmacol 148:947–955PubMedCrossRefGoogle Scholar
  44. 44.
    Li AH, Wang HL (2001) G protein-coupled receptor kinase 2 mediates mu-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus. J Neurochem 77:435–444PubMedCrossRefGoogle Scholar
  45. 45.
    Schechtman D, Mochly-Rosen D (2002) Isozyme-specific inhibitors and activators of protein kinase C. Methods Enzymol 345:470–489PubMedCrossRefGoogle Scholar
  46. 46.
    Koch WJ, Hawes BE, Inglese J et al (1994) Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling. J Biol Chem 269:6193–6197PubMedGoogle Scholar
  47. 47.
    Pusch, M. and Neher, E (1988) Rates of diffusional exchange between small cells and a measuring patch pipette. Pflugers Arch. 411, pp. 204–211PubMedCrossRefGoogle Scholar
  48. 48.
    Hull LC, Llorente J, Gabra BH et al (2010) The effect of PKC and GRK inhibition on tolerance induced by mu-opioid agonists of different efficacy. J Pharmacol Exp Ther 332:1127–1135PubMedCrossRefGoogle Scholar
  49. 49.
    Kong G, Penn R, Benovic JL (1994) A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor. J Biol Chem 269:13084–13087PubMedGoogle Scholar
  50. 50.
    Hwang DY, Carlezon WA, Isacson O et al (2001). A high-efficiency synthetic promoter that drives transgene expression selectively in noradrenergic neurons. Hum Gene Ther 12:1731–1740PubMedCrossRefGoogle Scholar
  51. 51.
    Jaber M, Koch WJ, Rockman H et al (1996) Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function. Proc Natl Acad Sci USA 93:12974–12979PubMedCrossRefGoogle Scholar
  52. 52.
    Bohn LM, Gainetdinov RR, Lin FT et al (2000) Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408:720–723PubMedCrossRefGoogle Scholar
  53. 53.
    Dang VC, Christie MJ (2006) Beta-arrestin-2-independent regulation of mu opioid receptor. Soc Neurosci Abstr 32:426.11Google Scholar
  54. 54.
    Walwyn W, Evans CJ, Hales TG (2007) Beta-arrestin2 and c-Src regulate the constitutive activity and recycling of mu opioid receptors in dorsal root ganglion neurons. J Neurosci 27:5092–5104PubMedCrossRefGoogle Scholar
  55. 55.
    Koch T, Kroslak T, Mayer P et al (1997) Site mutation in the rat mu-opioid receptor demonstrates the involvement of calcium/calmodulin-dependent protein kinase II in agonist-mediated desensitisation. J Neurochem 69:1767–1770PubMedCrossRefGoogle Scholar
  56. 56.
    Connor M, Osborne OB, Christie MJ (2004) Mu-opioid receptor desensitization:is morphine different? Br J Pharmacol 143:685-696PubMedCrossRefGoogle Scholar
  57. 57.
    Black JW, Leff P (1983) Operational models of pharmacological agonism. Proc R Soc Lond B Biol Sci 220:141162.PubMedCrossRefGoogle Scholar
  58. 58.
    Black JW, Leff P, Shankley NP et al (1985) An operational model of pharmacological agonism: the effect of E ⁄ (A) curve shape on agonist dissociation constant estimation. Br J Pharmacol 84:561–571PubMedGoogle Scholar
  59. 59.
    Osborne PB, Williams JT (1995) Characterization of acute homologous desensitization of mu-opioid receptor-induced currents in locus coeruleus neurones. Br J Pharmacol 115:925932PubMedGoogle Scholar
  60. 60.
    Clark MJ, Furman CA, Gilson TD et al (2006) Comparison of the relative efficacy and potency of mu-opioid agonists to activate Galpha i/o proteins containing a pertussis toxin insensitive mutation. J Pharmacol Exp Ther 317:858864PubMedCrossRefGoogle Scholar
  61. 61.
    Clarke WP, Bond RA (1998) The elusive nature of intrinsic efficacy. Trends Pharmacol Sci 19:270276PubMedCrossRefGoogle Scholar
  62. 62.
    Christopoulos A, El-Fakahany EE (1999) Qualitative and quantitative assessment of relative agonist efficacy. Biochem Pharmacol 58:735748PubMedCrossRefGoogle Scholar
  63. 63.
    Selley DE, Liu Q, Childers SR (1998) Signal transduction correlates of mu-opioid agonist intrinsic efficacy: Receptor-stimulated (35S)GTPgammaS binding in mMOR-CHO cells and rat thalamus. J Pharmacol Exp Ther 285:496505PubMedGoogle Scholar
  64. 64.
    Ehlert FJ (1985) The relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium. Mol Pharmacol 28:410421PubMedGoogle Scholar
  65. 65.
    Furchgott RF, and Bursztyn P (1967) Comparison of dissociation constants and of relative efficacies of selected agonists acting on parasympathomimetic receptors. Ann NY Acad Sci 144:882893CrossRefGoogle Scholar
  66. 66.
    Law P-Y, Erickson LJ, El-Kouhen R et al (2000) Receptor density and recycling affect the rate of agonist-induced desensitization of mu-opioid receptor. Mol Pharmacol 58:388398PubMedGoogle Scholar
  67. 67.
    Zheng H, Loh HH, Law PY (2008) Beta-arrestin-dependent mu-opioid receptor-activated extracellular signal-regulated kinases (ERKs) translocate to nucleus in contrast to G protein-dependent ERK activation. Mol Pharmacol 73:178–190PubMedCrossRefGoogle Scholar
  68. 68.
    Harrison C, Traynor JR (2003) The (35S)GTPgammaS binding assay: approaches and applications in pharmacology. Life Sci 12:489–508CrossRefGoogle Scholar
  69. 69.
    Van Koppen CJ, Jakobs KH (2004) Arrestin-independent internalization of G protein-coupled receptors. Mol Pharmacol 66:365–367PubMedCrossRefGoogle Scholar
  70. 70.
    Kallal L, Benovic JL (2000) Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking. Trends Pharmacol Sci 21:175–180PubMedCrossRefGoogle Scholar
  71. 71.
    Mundell SJ, Matharu AL, Pula G et al (2001) Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent. J Neurochem 78:546–551PubMedCrossRefGoogle Scholar
  72. 72.
    Mundell SJ, Pula G, McIlhinney RA et al (2004) Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors. Biochemistry 43:7541–7551PubMedCrossRefGoogle Scholar
  73. 73.
    Krasel C, Bunemann M, Lorenz K et al (2005) Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation. J Biol Chem 280:9528–9535PubMedCrossRefGoogle Scholar
  74. 74.
    van Der Lee MM, Bras M, van Koppen CJ et al (2008) Beta-arrestin recruitment assay for the identification of agonists of the sphingosine 1-phosphate receptor EDG1. J Biomol Screen 13:986–998CrossRefGoogle Scholar
  75. 75.
    Johnson EE, Christie MJ, Connor M (2005) The role of opioid receptor phosphorylation and trafficking in adaptations to persistent opioid treatment. Neurosignals 14:290–302PubMedCrossRefGoogle Scholar
  76. 76.
    Busillo JM, Armando S, Sengupta R et al (2010) Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling. J Biol Chem 285:7805–7817PubMedCrossRefGoogle Scholar
  77. 77.
    Kobilka BK, Gether U (2002) Use of fluorescence spectroscopy to study conformational changes in the beta 2-adrenoceptor. Methods Enzymol 343:170–182PubMedCrossRefGoogle Scholar
  78. 78.
    Zurn A, Zabel U, Vilardaga JP et al (2009) Fluorescence resonance energy transfer ­analysis of alpha 2a-adrenergic receptor activation reveals distinct agonist-specific conformational changes. Mol Pharmacol 75:534–541PubMedCrossRefGoogle Scholar
  79. 79.
    McPherson J, Rivero G, Baptist M et al (2010) mu-Opioid receptor internalization: correlation of agonist operational efficacy for G protein activation with ability to activate processes leading to internalization. Mol Pharmacol 78: 756–766PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Pharmacy and PharmacologyUniversity of BathBathUK

Personalised recommendations