title: Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling.
abstract: 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 (KD=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 hCB(1) 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, Gi1 (KD=27+/-8 nM and KD=10.7+/-4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.
authors.affiliation: Department of Medical Pharmacology, The University of Arizona, Tucson, Arizona, 85721, United States.
authors.forename: Teodora
authors.initials: T
authors.lastname: Georgieva
authors.forename: Savitha
authors.initials: S
authors.lastname: Devanathan
authors.forename: Dagmar
authors.initials: D
authors.lastname: Stropova
authors.forename: Chad K
authors.initials: CK
authors.lastname: Park
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Salamon
authors.forename: Gordon
authors.initials: G
authors.lastname: Tollin
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Hruby
authors.forename: William R
authors.initials: WR
authors.lastname: Roeske
authors.forename: Henry I
authors.initials: HI
authors.lastname: Yamamura
authors.forename: Eva
authors.initials: E
authors.lastname: Varga
mesh_terms: D000818:Animals; D048588:Benzoxazines / Q000378:metabolism; D002478:Cells, Cultured; D003511:Cyclohexanols / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D009025:Morpholines / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D011487:Protein Conformation; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists* / Q000737:chemistry* / Q000502:physiology; D015398:Signal Transduction; D018411:Spodoptera
chemical_list: D048588:Benzoxazines; D003511:Cyclohexanols; D009025:Morpholines; D009281:Naphthalenes; D043884:Receptor, Cannabinoid, CB1; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D019204:GTP-Binding Proteins