Received for publication October 18, 2007.
Revised December 14, 2007.
Accepted for publication December 31, 2007.

Mapping the Structural Requirements in the CB₁ Cannabinoid Receptor Transmembrane Helix II for Signal Transduction

Abstract

Amino acid residues in the transmembrane domains of the CB₁ receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the TMH II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB₁) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant, I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands were investigated. Recombinant human CB₁ receptors, stably expressed in HEK-293 cells, were assayed for ligand affinity and agonist-stimulated GTPS binding. The charge conserved mutant, D2.63E, behaved similar to wild-type. The charge-neutralization mutation, D2.63N, attenuated the potency of CP55,940, WIN55,212-2, AM4056, and HU210 for the stimulation of GTPS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. Strikingly, the double mutant (I2.62T-D2.63N) displayed a drastic and a synergistic increase (by ~50-fold) in the EC₅₀ for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.

Key words: binding, cannabinoid, cannabinoid receptor, cell lines, mutagenesis, signal transduction