Unique agonist-bound cannabinoid CB₁ receptor conformations indicate agonist specificity in signaling

Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB₁ receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB₁ receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (K_D = 0.2 ± 0.03 nM and 2 ± 0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB₁ receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB₁ receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212-bound hCB₁ receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, G_i1 (K_D = 27 ± 8 nM and K_D = 10.7 ± 4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.