title: Chemically distinct ligands promote differential CB1 cannabinoid receptor-Gi protein interactions.
abstract: 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.
authors.affiliation: Neuroscience of Drug Abuse Research Program, J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 700 George Street, Durham, NC 27707, USA. smukhopadhyay@nccu.edu
authors.forename: Somnath
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Allyn C
authors.initials: AC
authors.lastname: Howlett
mesh_terms: D000818:Animals; D048588:Benzoxazines; D002186:Cannabinoids / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D008024:Ligands; D009025:Morpholines; D009281:Naphthalenes; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000378:metabolism*
chemical_list: D048588:Benzoxazines; D002186:Cannabinoids; D008024:Ligands; D009025:Morpholines; D009281:Naphthalenes; D043884:Receptor, Cannabinoid, CB1; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; D019206:GTP-Binding Protein alpha Subunits, Gi-Go