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 G alpha i and beta gamma proteins that coimmunoprecipitate with the CB1 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 RG alpha i complexes, cannabinoid desacetyllevonantradol for G alpha i1 and G alpha i2, and eicosanoid (R)-methanandamide for G alpha i3. Desacetyllevonantradol maintained RG alpha i3 complexes and (R)-methanandamide maintained RG alpha i1 and RG alpha i2 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 RG alpha i complexes. G beta proteins, and to a certain extent G gamma 2, exhibited the same association/dissociation pattern as the G alpha 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.