Residues proximal to the DRY motif are involved in cannabinoid receptor 1 activation and G protein specificity

Date of Completion

January 2006


Biology, Molecular




The cannabinoid receptor CB1 is a member of the G protein-coupled receptor superfamily. Signal transduction via CB1 is initiated by the binding of an extracellular ligand, followed by a conformational change, and activation of an intracellular G protein. However, the molecular-level changes associated with CB1 activation are unknown. This work contains the first examination of the role of residues N-terminal to the transmembrane-segment 3 Asp-Arg-Tyr (DRY) motif in the resting and activated states of CB1 , and how perturbations of this region effect specificity for either the inhibitory or the stimulatory G protein (Gi or Gs, respectively). ^ Leu207 of CB1 is on the same helical face as Arg 214 of the DRY motif. Its replacement by Ala and the expression of the mutant receptor in HEK293 cells resulted in increased affinity for agonists and decreased affinity for inverse agonists, with the latter partially sensitive to GTPγS, an outcome consistent with a constitutively active receptor. Measurement of cAMP accumulation in L207A receptor-expressing cells revealed an increase in ligand-independent, inverse-agonist sensitive, Gs signaling. However, agonist treatment activated Gi in a dose-dependent manner. The L207A mutant receptor exhibited distinguishable ligand-dependent and ligand-independent G protein associations, consistent with the prediction of multiple active states for the receptor. The replacement of Thr210, located one helical turn closer to the DRY motif, with Ile also increased agonist affinity and decreased inverse agonist affinity, while an Ala substitution resulted in the opposite pattern of affinity changes; the former consistent with constitutive activity and the latter with an increase in the resting state receptor population. Confocal microscopy demonstrated that the localization of the corresponding GFP-CB, fusion proteins correlated with changes in receptor activity; T210I internalized whereas T210A receptors were expressed predominantly on the cell surface. A correlation between receptor activity and thermal stability was also evident for the T210I and the T210A receptors. ^ This structural and functional analysis of CB1 provides an increased understanding of this receptor and GPCRs in general. The data show differences in the active and inactive receptor forms and this work has broad applications for the development of therapeutics which target these receptor states.^