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PMID: 20735408 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to G(s) and G(i).

British journal of pharmacology ·Vol. 161 ·No. 8 ·2010-12-00 ·Pages 1817-34

Chen XP, Yang W, Fan Y, Luo JS, Hong K, Wang Z, Yan JF, Chen X, Lu JX, Benovic JL, Zhou NM

Abstract

The cannabinoid CB(1) receptor is primarily thought to be functionally coupled to the G(i) form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB(1) receptors and characterized the structural determinants that mediate selective coupling to G(s) and G(i). A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB(1)/CB(2) receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G(s) and G(i). CB(1) receptors could couple to both G(s)-mediated cAMP accumulation and G(i)-induced activation of ERK1/2 and Ca(2+) mobilization, whereas CB(2) receptors selectively coupled to G(i) and inhibited cAMP production. Using CB(1)/CB(2) chimeric receptors, the second intracellular loop (ICL2) of the CB(1) receptor was identified as primarily responsible for mediating G(s) and G(i) coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G(s) to G(i), while to Ile or Val led to balanced coupling of the mutant receptor with G(s) and G(i) . The ICL2 of CB(1) receptors and in particular Leu-222, which resides within a highly conserved DRY(X)(5) PL motif, played a critical role in G(s) and G(i) protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB(1) receptors to G proteins and cannabinoid-induced tolerance.

MeSH Terms
3T3 Cells Adenylyl Cyclases/drug effects Animals CHO Cells COS Cells Calcium/metabolism Cell Line, Transformed Chlorocebus aethiops Cricetinae Cricetulus Cyclic AMP/metabolism Extracellular Signal-Regulated MAP Kinases/metabolism GTP-Binding Protein alpha Subunits, Gi-Go/metabolism GTP-Binding Protein alpha Subunits, Gs/metabolism HEK293 Cells Humans Mice Mutagenesis, Site-Directed/methods Protein Binding/genetics Protein Interaction Domains and Motifs Receptor, Cannabinoid, CB1/genetics,metabolism Receptor, Cannabinoid, CB2/genetics,metabolism
Chemicals
Receptor, Cannabinoid, CB1 Receptor, Cannabinoid, CB2 Cyclic AMP Extracellular Signal-Regulated MAP Kinases GTP-Binding Protein alpha Subunits, Gi-Go GTP-Binding Protein alpha Subunits, Gs Adenylyl Cyclases Calcium
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Chen X P
Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
Yang W
Fan Y
Luo J S
Hong K
Wang Z
Yan J F
Chen X
Lu J X
Benovic J L
Zhou N M
References (61)
61 references, click to expand
  1. Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+,K+-ATPase.
    Clin Exp Hypertens. 1998 May;20(4):465-80 PMID: 9607407
  2. LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation.
    J Pharmacol Exp Ther. 1998 Jan;284(1):291-7 PMID: 9435190
  3. The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling.
    FEBS Lett. 1995 Nov 13;375(1-2):143-7 PMID: 7498464
  4. Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.
    J Biol Chem. 2008 Nov 7;283(45):31068-78 PMID: 18772143
  5. 2-Arachidonoylglycerol, a putative endogenous cannabinoid receptor ligand, induces rapid, transient elevation of intracellular free Ca2+ in neuroblastoma x glioma hybrid NG108-15 cells.
    Biochem Biophys Res Commun. 1996 Dec 4;229(1):58-64 PMID: 8954083
  6. Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes.
    Mol Pharmacol. 1984 Nov;26(3):532-8 PMID: 6092901
  7. Structural basis for nucleotide exchange on G alpha i subunits and receptor coupling specificity.
    Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):2001-6 PMID: 17264214
  8. Cannabis: pharmacology and toxicology in animals and humans.
    Addiction. 1996 Nov;91(11):1585-614 PMID: 8972919
  9. Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity.
    J Biomol Screen. 2009 Mar;14(3):246-55 PMID: 19211780
  10. Selective coupling of prostaglandin E receptor EP3D to Gi and Gs through interaction of alpha-carboxylic acid of agonist and arginine residue of seventh transmembrane domain.
    J Biol Chem. 1995 Jul 7;270(27):16122-7 PMID: 7608175
  11. Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1).
    J Pharmacol Exp Ther. 1999 Jun;289(3):1427-33 PMID: 10336536
  12. Multiple G-protein coupling of the dog thyrotropin receptor.
    Mol Cell Endocrinol. 1997 Mar 14;127(1):81-90 PMID: 9099903
  13. D2 dopamine receptors modulate Galpha-subunit coupling of the CB1 cannabinoid receptor.
    J Pharmacol Exp Ther. 2004 Mar;308(3):880-6 PMID: 14634050
  14. Cannabinoid signalling.
    Life Sci. 2006 Jan 2;78(6):549-63 PMID: 16109430
  15. Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase.
    J Neurochem. 1993 Jul;61(1):352-5 PMID: 8515284
  16. Dual activation and inhibition of adenylyl cyclase by cannabinoid receptor agonists: evidence for agonist-specific trafficking of intracellular responses.
    J Pharmacol Exp Ther. 1998 Dec;287(3):884-8 PMID: 9864268
  17. Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor.
    J Biol Chem. 1995 Sep 29;270(39):22820-6 PMID: 7559413
  18. Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7656-60 PMID: 8395053
  19. Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.
    J Biol Chem. 1992 Aug 5;267(22):15795-801 PMID: 1322406
  20. An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion.
    Proc Natl Acad Sci U S A. 2003 Mar 4;100(5):2969-74 PMID: 12591945
  21. Paradoxical action of the cannabinoid WIN 55,212-2 in stimulated and basal cyclic AMP accumulation in rat globus pallidus slices.
    Br J Pharmacol. 1997 Apr;120(8):1397-8 PMID: 9113356
  22. Cannabinoid receptor binding and agonist activity of amides and esters of arachidonic acid.
    Mol Pharmacol. 1994 Sep;46(3):516-22 PMID: 7935333
  23. Crystal structure of rhodopsin: A G protein-coupled receptor.
    Science. 2000 Aug 4;289(5480):739-45 PMID: 10926528
  24. Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.
    J Biol Chem. 1993 Oct 25;268(30):22273-6 PMID: 8226735
  25. Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs.
    Mol Pharmacol. 1986 Mar;29(3):307-13 PMID: 2869405
  26. Cannabinoid-induced tolerance is associated with a CB1 receptor G protein coupling switch that is prevented by ultra-low dose rimonabant.
    Behav Pharmacol. 2007 Dec;18(8):767-76 PMID: 17989514
  27. Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor.
    J Neurosci. 1997 Jul 15;17(14):5327-33 PMID: 9204917
  28. Molecular characterization of a peripheral receptor for cannabinoids.
    Nature. 1993 Sep 2;365(6441):61-5 PMID: 7689702
  29. gpDB: a database of GPCRs, G-proteins, effectors and their interactions.
    Bioinformatics. 2008 Jun 15;24(12):1471-2 PMID: 18441001
  30. Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.
    Cell Mol Life Sci. 2003 Jun;60(6):1200-8 PMID: 12861385
  31. Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A.
    Nature. 1997 Nov 6;390(6655):88-91 PMID: 9363896
  32. A novel mechanism for coupling of m4 muscarinic acetylcholine receptors to calmodulin-sensitive adenylyl cyclases: crossover from G protein-coupled inhibition to stimulation.
    Biochemistry. 1994 Feb 1;33(4):943-51 PMID: 8305442
  33. Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms.
    J Biol Chem. 2007 Aug 24;282(34):25100-13 PMID: 17595161
  34. Guide to Receptors and Channels (GRAC), 4th Edition.
    Br J Pharmacol. 2009 Nov;158 Suppl 1:S1-254 PMID: 19883461
  35. Src phosphorylation of micro-receptor is responsible for the receptor switching from an inhibitory to a stimulatory signal.
    J Biol Chem. 2009 Jan 23;284(4):1990-2000 PMID: 19029294
  36. Coupling of the alpha 2A-adrenergic receptor to multiple G-proteins. A simple approach for estimating receptor-G-protein coupling efficiency in a transient expression system.
    J Biol Chem. 1994 Feb 25;269(8):5730-4 PMID: 7907086
  37. Conserved helix 7 tyrosine acts as a multistate conformational switch in the 5HT2C receptor. Identification of a novel "locked-on" phenotype and double revertant mutations.
    J Biol Chem. 2002 Sep 27;277(39):36577-84 PMID: 12145300
  38. The carboxyl terminus of the Galpha-subunit is the latch for triggered activation of heterotrimeric G proteins.
    Mol Pharmacol. 2006 Jan;69(1):397-405 PMID: 16210429
  39. [Medical use of cannabis through history].
    J Pharm Belg. 2001 Sep-Oct;56(5):111-8 PMID: 11759149
  40. WebLogo: a sequence logo generator.
    Genome Res. 2004 Jun;14(6):1188-90 PMID: 15173120
  41. Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain.
    J Neurochem. 1990 Jul;55(1):21-6 PMID: 2162376
  42. Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.
    J Biol Chem. 2001 Sep 7;276(36):33596-607 PMID: 11443126
  43. Cannabinoid receptor-G protein interactions: G(alphai1)-bound structures of IC3 and a mutant with altered G protein specificity.
    Protein Sci. 2002 Oct;11(10):2526-31 PMID: 12237474
  44. Dual intracellular signaling pathways mediated by the human cannabinoid CB1 receptor.
    Eur J Pharmacol. 1999 Jun 25;374(3):445-55 PMID: 10422789
  45. Coupling of the human A1 adenosine receptor to different heterotrimeric G proteins: evidence for agonist-specific G protein activation.
    Br J Pharmacol. 2004 Nov;143(6):705-14 PMID: 15302686
  46. Sequence logos: a new way to display consensus sequences.
    Nucleic Acids Res. 1990 Oct 25;18(20):6097-100 PMID: 2172928
  47. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.
    Pharmacol Rev. 2002 Jun;54(2):161-202 PMID: 12037135
  48. Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: use of a chemically preactivated reagent.
    Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4883-7 PMID: 11320238
  49. A conservative, single-amino acid substitution in the second cytoplasmic domain of the human Serotonin2C receptor alters both ligand-dependent and -independent receptor signaling.
    J Pharmacol Exp Ther. 2008 Mar;324(3):1084-92 PMID: 18065501
  50. Cannabinoids reduce cAMP levels in the striatum of freely moving rats: an in vivo microdialysis study.
    Brain Res. 2004 Apr 16;1005(1-2):117-23 PMID: 15044071
  51. Is the cannabinoid CB1 receptor a 2-arachidonoylglycerol receptor? Structural requirements for triggering a Ca2+ transient in NG108-15 cells.
    J Biochem. 1997 Oct;122(4):890-5 PMID: 9399597
  52. Identification of cannabinoid receptors in cultures of rat cerebellar granule cells.
    Brain Res. 1993 Feb 12;603(1):102-10 PMID: 8384043
  53. Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.
    J Neurochem. 1999 May;72(5):2032-8 PMID: 10217281
  54. The stimulatory effect of cannabinoids on calcium uptake is mediated by Gs GTP-binding proteins and cAMP formation.
    Neurosignals. 2003 Jan-Feb;12(1):39-44 PMID: 12624527
  55. A cluster of aromatic amino acids in the i2 loop plays a key role for Gs coupling in prostaglandin EP2 and EP3 receptors.
    J Biol Chem. 2004 Mar 19;279(12):11016-26 PMID: 14699136
  56. Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression.
    Eur J Biochem. 1996 May 1;237(3):704-11 PMID: 8647116
  57. M3 muscarinic acetylcholine receptor-mediated signaling is regulated by distinct mechanisms.
    Mol Pharmacol. 2008 Aug;74(2):338-47 PMID: 18388243
  58. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
    Nucleic Acids Res. 1994 Nov 11;22(22):4673-80 PMID: 7984417
  59. Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model.
    Mol Pharmacol. 1988 Mar;33(3):297-302 PMID: 3352594
  60. Effects of anandamide on cannabinoid receptors in rat brain membranes.
    Biochem Pharmacol. 1994 Feb 11;47(4):711-5 PMID: 8129747
  61. Structure of a cannabinoid receptor and functional expression of the cloned cDNA.
    Nature. 1990 Aug 9;346(6284):561-4 PMID: 2165569
Article Info
Journal
British journal of pharmacology
Abbr.
Br J Pharmacol
ISSN
1476-5381
DOI
10.1111/j.1476-5381.2010.01006.x
Published
2010-12-00
Pages
1817-34
Language
English
Region
England
NLM ID
7502536
PMCID
PMC3010585
Subset
IM
Grants
NIGMS NIH HHS · GM47417 · United States
NIGMS NIH HHS · R01 GM044944 · United States
NIGMS NIH HHS · GM44944 · United States
NIGMS NIH HHS · R01 GM047417 · United States
NIGMS NIH HHS · R37 GM047417 · United States
External Links
PubMed source DOI: 10.1111/j.1476-5381.2010.01006.x PMC Full Text View journal details
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