Abstract
Under reducing conditions of SDS-polyacrylamide gel electrophoresis, the CB1 receptor exists in its monomeric form as well as in an SDS-resistant high molecular weight form that appears to be devoid of G proteins. The CB1 cannabinoid receptor was immunoprecipitated from 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate-solubilized rat brain membranes using an antibody against the CB1receptor N terminus. The CB1 receptor was coimmunoprecipitated with its associated G proteins, specifically those of the Gαi/o family, but not Gαs, Gαq, or Gαz. The CB1receptor-Gαi/o complex existed in the absence of exogenous agonists, and the cannabinoid receptor agonist desacetyllevonantradol failed to alter the stoichiometry of the receptor-Gαi/o interaction. Guanosine-5′-O-(3-thio)triphosphate could disrupt the interaction. A peptide derived from the CB1 receptor juxtamembrane C-terminal domain, peptide CB1401-417, autonomously activates Gi/o proteins. Peptide CB1401-417 competitively disrupted the CB1receptor association with Gαo and Gαi3 but not Gαi1 or Gαi2. This G protein specificity was also observed in detergent extracts from membranes of the frontal cortex, striatum, and cerebellum. Alternative peptides, including peptides from the CB1 receptor third intracellular loop and the G protein activating peptide mastoparan-7, failed to promote uncoupling from Gαo. A CB2receptor juxtamembrane C-terminal peptide failed to disrupt the CB1 receptor-Gαo complex. These studies illustrate that the CB1 receptor can exist as an SDS-resistant multimer. In 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate detergent, the CB1 receptor exists in a complex with G proteins of the Gi/o family in the absence of exogenous agonists. Furthermore, this study provides the first description of domain specificity for interaction with a selective set of G proteins.
Footnotes
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Send reprint requests to: Dr. Allyn C. Howlett, Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. E-mail: howletta{at}slu.edu
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↵1 Present address: National Enzyme Company, Forsyth, MO 65653.
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This work was supported in part by NIDA Grants R01-DA03690 and K05-DA00182.
- Abbreviations:
- MAPK
- mitogen-activated protein kinase
- CAPS
- 3-[cyclohexylamino]-1-propanesulfonic acid
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
- DALN
- desacetyllevonantradol
- ECL
- enhanced chemiluminescence
- GTPγS
- guanosine-5′-O-(3-thio)- triphosphate
- PAGE
- polyacrylamide gel electrophoresis
- Received June 4, 1999.
- Accepted October 8, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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