Chemically distinct ligands promote differential CB1 cannabinoid receptor-Gi protein interactions

To understand how structurally distinct ligands regulate CB(1) receptor interactions with Gi1, Gi2, and Gi3, we quantified the Galphai and betagamma proteins that coimmunoprecipitate with the CB(1) receptor from a detergent extract of N18TG2 membranes in the presence of ligands. A mixture of A, R, G(GDP) (or G_), and ARG(GDP) (or ARG_) complexes was observed in the presence of aminoalkylindole (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2) for all three RGalphai complexes, cannabinoid desacetyllevonantradol for Galphai1 and Galphai2, and eicosanoid (R)-methanandamide for Galphai3. Desacetyllevonantradol maintained RGalphai3 complexes and (R)-methanandamide maintained RGalphai1 and RGalphai2 complexes even in the presence of a nonhydrolyzable GTP analog. The biaryl pyrazole antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) maintained all three RGalphai complexes. Gbeta proteins, and to a certain extent Ggamma2, exhibited the same association/dissociation pattern as the Galpha proteins. A GDP analog had no influence on any of these association/dissociation reactions and failed to promote sequestration of G proteins. These results can be explained by invoking the existence of an inverse agonist-supported inactive state in the ternary complex equilibrium model. WIN 55,212-2 behaves as an agonist for all three Gi subtypes; SR141716 behaves as an inverse agonist for all three Gi subtypes; desacetyllevonantradol behaves as an agonist for Gi1 and Gi2, and an inverse agonist at Gi3; and (R)-methanandamide behaves as an inverse agonist at Gi1 and Gi2, and an agonist at Gi3. These ligand-selective G protein responses imply that multiple conformations of the receptor could be evoked by ligands to regulate individual G proteins.