Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor: BINDING SITE ELUCIDATION AND RELATIONSHIP TO G PROTEIN SIGNALING*

The cannabinoid 1 (CB₁) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid 2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) ({"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569), has the paradoxical effect of increasing the equilibrium binding of ³H](−)-3-2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 can displace the CB₁ antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 at CB₁. To this end, we used computation, synthesis, mutation, and functional studies to identify the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site in the CB₁ TMH3-6-7 region. This site is consistent with the results of K3.28¹⁹²A, F3.36²⁰⁰A, W5.43²⁷⁹A, W6.48³⁵⁶A, and F3.25¹⁸⁹A mutation studies, which revealed the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 piperidine ring nitrogen and K3.28¹⁹² that is important for {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 to act as an inverse agonist. At this allosteric site, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 promotes an intermediate conformation of the CB₁ receptor, explaining {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to increase equilibrium binding of CP55,940. This site also explains {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63¹⁷⁶. 3) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.