Abstract
Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the μ-opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to δ-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), α2-adrenergic (α2AR), cannabinoid 1, and serotonin 1A receptors. We further demonstrate that all receptor pairs share a common pool of AC. The MOR agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and α2AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal.
Footnotes
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants R01-DA04087, F31-DA023339, T32-DA07267]; and the National Institutes of Health National Institute of General Medical Sciences [Grant T32-GM07767].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064816.
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- SNC80
- (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide
- DPDPE
- [d-Pen2,5]-enkephalin
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
- OFQ
- orphanin FQ
- 8-OH-DPAT
- 8-hydroxy-2-(di-n-propylamino)tetralin hydrochloride
- CTAP
- d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2
- ICI 174,864
- N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH
- J113397
- 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one
- [35S]GTPγS
- guanosine-5′-O-(3-[35S]thio)triphosphate
- MOR
- μ-opioid receptor
- DOR
- δ-opioid receptor
- NOPr
- nociceptin/orphanin FQ peptide receptor
- α2AR
- α2-adrenergic receptor
- CB1
- cannabinoid receptor 1
- 5-HT1A
- 5-hydroxytryptamine-1A receptor
- AC
- adenylyl cyclase
- PTX
- pertussis toxin
- IBMX
- 3-isobutyl-1-methylxanthine
- ANOVA
- analysis of variance
- LC
- locus ceruleus
- UK14,304
- 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline
- CP 55,940
- 5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol
- WIN 55212-2
- (2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl)methanone monomethanesulfonate
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- Ro 64-6198
- (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one
- BW373U86
- (±)-[1(S*),2α,5β]-4-[[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-hydroxyphenyl)methyl]-N,N-diethylbenzamide dihydrobromide.
- Received March 17, 2010.
- Accepted November 22, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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