Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling
- PMID: 9580609
Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling
Abstract
The cannabinoid receptors, CB1 and CB2, are members of the G-protein coupled receptor family and share many of this family's structural features. A highly conserved aspartic acid residue in the second transmembrane domain of G-protein coupled receptors has been shown for many of these receptors to be functionally important for agonist binding and/or G-protein coupling. To determine whether this residue is involved in cannabinoid receptor function, we used site-directed mutagenesis of receptor cDNA followed by expression of the mutant receptor in HEK 293 cells. Aspartate 163 (in CB1) and aspartate 80 (in CB2) were substituted with either asparagine or glutamate. Stably transfected cell lines were tested for radioligand binding and inhibition of cAMP accumulation. Binding of the cannabinoid receptor agonist [3H]CP-55,940 was not affected by either mutation in either the CB1 or CB2 receptor, nor were the affinities of anandamide or (-)-delta 9-tetrahydrocannabinol. Binding of the CB1-selective receptor antagonist SR141716A also was unaltered. However, the affinity of WIN 55,212-2 was attenuated significantly in the CB1, but not the CB2, mutant receptors. Studies examining inhibition of cAMP accumulation showed reduced effects of cannabinoid agonists in the mutated receptors. Our data suggest that this aspartate residue is not generally important for ligand recognition in the cannabinoid receptors; however, it is required for communication with G proteins and signal transduction.
Similar articles
-
Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands.J Pharmacol Exp Ther. 1996 Sep;278(3):989-99. J Pharmacol Exp Ther. 1996. PMID: 8819477
-
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. J Pharmacol Exp Ther. 1998. PMID: 9435190
-
Characterization of two cloned human CB1 cannabinoid receptor isoforms.J Pharmacol Exp Ther. 1996 Aug;278(2):871-8. J Pharmacol Exp Ther. 1996. PMID: 8768742
-
The pharmacology of cannabinoid receptors and their ligands: an overview.Int J Obes (Lond). 2006 Apr;30 Suppl 1:S13-8. doi: 10.1038/sj.ijo.0803272. Int J Obes (Lond). 2006. PMID: 16570099 Review.
-
The neurobiology and evolution of cannabinoid signalling.Philos Trans R Soc Lond B Biol Sci. 2001 Mar 29;356(1407):381-408. doi: 10.1098/rstb.2000.0787. Philos Trans R Soc Lond B Biol Sci. 2001. PMID: 11316486 Free PMC article. Review.
Cited by
-
Targeting the cannabinoid CB2 receptor: modelling and structural determinants of CB2 selective ligands.Br J Pharmacol. 2008 Jan;153(2):335-46. doi: 10.1038/sj.bjp.0707567. Epub 2007 Nov 5. Br J Pharmacol. 2008. PMID: 17982473 Free PMC article. Review.
-
Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of Cannabinoid 1 Receptor Positive Allosteric Modulators.J Med Chem. 2020 Jan 23;63(2):542-568. doi: 10.1021/acs.jmedchem.9b01142. Epub 2020 Jan 3. J Med Chem. 2020. PMID: 31756109 Free PMC article.
-
Molecular mechanism of allosteric modulation for the cannabinoid receptor CB1.Nat Chem Biol. 2022 Aug;18(8):831-840. doi: 10.1038/s41589-022-01038-y. Epub 2022 May 30. Nat Chem Biol. 2022. PMID: 35637350
-
The NPXXY Motif Regulates β-Arrestin Recruitment by the CB1 Cannabinoid Receptor.Cannabis Cannabinoid Res. 2023 Oct;8(5):731-748. doi: 10.1089/can.2021.0223. Epub 2022 Jul 6. Cannabis Cannabinoid Res. 2023. PMID: 35792570 Free PMC article.
-
Cannabinoids and neuroinflammation.Br J Pharmacol. 2004 Mar;141(5):775-85. doi: 10.1038/sj.bjp.0705667. Epub 2004 Feb 2. Br J Pharmacol. 2004. PMID: 14757702 Free PMC article. Review.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Other Literature Sources
Miscellaneous