Structural domains of the CB1 cannabinoid receptor that contribute to constitutive activity and G-protein sequestration

Abstract

The CB1 cannabinoid receptor is a constitutively active receptor that can sequester Gi/o-proteins and prevent other Gi/ocoupled receptors from signaling (Bouaboula et al., 1997; Pan et al., 1998; Vasquez and Lewis, 1999). G-protein sequestration occurs because the population of CB1 cannabinoid receptors exists in both an inactive G-protein-precoupled RGGDP state and a constitutively active R*GGTP state. We tested the hypothesis that the distal C-terminal tail acts to prevent G-protein activation. We found that truncation of the distal C-terminal tail of the CB1 receptor (CB1-417) enhanced both the constitutive activity and the ability of the receptor to sequester G-proteins. In addition, we tested the hypothesis that the conserved aspartate (D2.50) in the second transmembrane domain of the CB1 cannabinoid receptor is crucial for constitutive activity and G-protein sequestration. We found that the mutation of aspartate to asparagine (CB1-D164N) abolished G-protein sequestration and constitutive receptor activity without disrupting agonist-stimulated activity. We conclude that the CB1-D164N mutation and the C-terminal truncation shift the population of receptors in opposite directions. The CB1-D164N mutation shifts the receptor into an inactive R state upcoupled from G-proteins, whereas the C-terminal truncation (CB1-417) shifts the receptor into the active R*GGTP state. Thus the distal C-terminal tail acts to constrain the receptor from activating G-proteins, whereas the aspartate (D2.50) in the second transmembrane domain stabilizes the receptor in both the inactive RGGDP state and the active R*GGTP state.