Agonist Selective Regulation of G Proteins by Cannabinoid CB1 and CB2 Receptors
- Section on Signal Transduction, National Institutes of Health, National Institute on Deafness and Other Communication Disorders, Rockville, Maryland
Abstract
We have examined the ligand regulation and G protein selectivity of the human cannabinoid CB1 and CB2 receptors by an in situ reconstitution technique directly measuring G protein activation. Membranes from Spodoptera frugiperda cells expressing CB1 and CB2 receptors were chaotrope extracted to denature endogenous GTP-binding proteins. The ability of the receptors to catalyze the GDP-GTP exchange of each G protein was then examined with purified bovine brain Gi and Go. Activation of CB1 receptors produced a high-affinity saturable interaction for both Gi and Go. Agonist stimulation of CB2 receptors also resulted in a high-affinity saturable interaction with Gi. In contrast, CB2 receptors did not interact efficiently with Go. G protein activation was then examined with a diverse group of ligands. For the interaction of CB2receptors with Gi, HU210 was the only compound tested that demonstrated maximal activation. In contrast, WIN55,212 (64%), anandamide (42%), and Δ9-tetrahydrocannabinol (Δ9-THC) (44%) all initiated submaximal levels of G protein activation. For CB1 receptor-catalyzed activation of Gi, HU210, WIN55,212, and anandamide all elicited maximal activation, whereas Δ9-THC (56 ± 6%) caused only partial Gi activation. In contrast, only HU210 effected maximal CB1 stimulation of Go, with anandamide, WIN55,212, and Δ9-THC all stimulating between 60 and 75% compared with HU210. These data demonstrate that different agonists induce different conformations of the CB1receptor, which in turn can distinguish between different G proteins. Our data thus demonstrate agonist-selective G protein signaling by the CB1 receptor and suggest that therapeutic agents may be designed to regulate individual G protein-signaling pathways selectively.
Footnotes
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Send reprint requests to: Dr. John K. Northup, Section on Signal Transduction, National Institute on Deafness and Other Communication Disorders, 5 Research Court, Rockville, MD 20850. E-mail:drjohn{at}codon.nih.gov
- Abbreviations:
- GTPγS
- guanosine-5′-O-(3-thio)-triphosphate
- Sf9
- Spodoptera frugiperda cells
- 5-HT
- 5-hydroxytryptamine
- HU210
- (−)-11-hydroxy-Δ8-tetrahydrocannabinol-dimethylheptyl
- WIN55,212
- (R)-(+)-(2,3-dihydro-5-methyl-3-[(morphonolinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl)(1-napthalenyl)methanone mesylate
- SR141716A
- N-(peperidino-1-yl)-5-(4-chloropheyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide, hydrochloride
- SR144528
- N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide
- Δ9-THC
- Δ9-tetrahydrocannabinol
- MOPS
- 4-morpholinepropanesulfonic acid
- P2
- postnuclear fraction
- CHO
- Chinese hamster ovary
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- Received June 15, 1999.
- Accepted September 14, 1999.
- U.S. Government
