PT  - JOURNAL ARTICLE
AU  - Tao, Qing
AU  - Abood, Mary E.
TI  - Mutation of a Highly Conserved Aspartate Residue in the Second Transmembrane Domain of the Cannabinoid Receptors, CB1 and CB2, Disrupts G-Protein Coupling
DP  - 1998 May 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 651--658
VI  - 285
IP  - 2
4099  - http://jpet.aspetjournals.org/content/285/2/651.short
4100  - http://jpet.aspetjournals.org/content/285/2/651.full
SO  - J Pharmacol Exp Ther1998 May 01; 285
AB  - 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 (−)-Δ9-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. The American Society for Pharmacology and Experimental Therapeutics