Abstract
Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB1 and CB2 cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB2 to different extents. Because CB2 is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB1 and CB2 trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB2 receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB2 receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB2 internalization. Despite these differences in internalization, both compounds activated CB2 receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin2 to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB2 receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB2 ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB2 receptors and suggest that different classes of CB2 ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA011322, DA021696, DA009158]; and the National Institutes of Health National Institute for Research Resources [Grant RR025761].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- 2-AG
- 2-arachidonoylglycerol
- A-836339
- N-[3-(2-methoxyethyl)-4,5-dimethyl-1,3-thiazol-2-ylidene]-2,2,3,3-tetramethylcyclopropane-1-carboxamide
- AEA
- anandamide
- AM1241
- (2-iodo-5-nitrophenyl)-[1-[(1-methylpiperidin-2-yl)methyl]indol-3-yl]methanone
- AM1710
- 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one
- AM630
- iodopravadoline
- BSA
- bovine serum albumin
- CB1
- cannabinoid receptor subtype 1
- CB2
- cannabinoid receptor subtype 2
- CP47,497
- 2-[(1R,3S)-3-hydroxycyclohexyl]- 5-(2-methyloctan-2-yl)phenol
- CP55,940
- 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol
- ECS
- extracellular solution
- ERK
- extracellular signal-regulated kinase
- FAAH
- fatty acid amide hydrolase
- GPCR
- G-protein-coupled receptor
- GW405833
- 1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole
- HA
- hemagglutinin 11
- HBS
- HEPES-buffered saline
- h
- human
- HEK
- human embryonic kidney
- HU-210
- Δ8-tetrahydrocannabinol dimethyl heptyl
- HU-308
- [(1R,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7-dimethyl-4-bicyclo[3.1.1]hept-3-enyl] methanol
- JWH015
- (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone
- JWH018
- naphthalen-1-yl-(1-pentylindol-3-yl)methanone
- JWH133
- (6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro -6,6,9-trimethyl-6H-dibenzo[b,d]pyran
- JZL184
- 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate
- m
- mouse
- MAPK
- mitogen-activated protein kinase
- MGL
- monoacylglycerol lipase
- mRFP
- modified red fluorescent protein
- PBS
- phosphate-buffered saline
- PFA
- paraformaldehyde
- pplss
- preprolactin signal sequence
- PTX
- pertussis toxin
- r
- rat
- SR141716
- rimonabant
- SR144528
- N-((1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide)
- SR144528
- N-[(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-pyrazole-3-carboxamide
- TBS
- Tris-buffered saline
- THC
- Δ9-tetrahydrocannabinol
- THCV
- tetrahydrocannabivarin
- URB597
- [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate
- VGCC
- voltage-gated calcium channel
- WIN55,212-2
- (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone.
- Received June 8, 2011.
- Accepted November 7, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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