Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells.
The turkey beta-adrenergic receptor (beta-AR), the m1 and m2 forms of the human muscarinic cholingeric receptor (MAChR) and several other mutant and wild-type G protein-coupled receptors were produced in insect Sf9 cells by infection with recombinant baculoviruses. Maximal expression for most receptors was 5-30 pmol receptor/mg protein (2-15 nmol/liter culture). The receptors displayed typical ligand binding characteristics. The beta-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The beta-AR stimulated endogenous adenylyl cyclase in response to beta-adrenergic agonists. The beta-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant beta-AR catalyzed the agonist-dependent activation of Gs by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with the same efficiency as did the natural beta-AR. The m2 MAChR efficiently catalyzed GTP gamma S binding to Go and to the recently identified G protein Gz (Gx). The m2 MAChR also catalyzed the activation of Gj,1 and Gj,3 weakly. Activation of these same G proteins by the ml MAChR was much less efficient, consistent with its known selectivity for pertussis toxin-insensitive G proteins ("Gp") that have not yet been isolated. The beta-AR and m2 MAChR were characteristically stimulated by reduction of disulfides. These results demonstrate the general utility of the baculovirus system for production of large quantities of native G protein-coupled receptors.
Parker
E M
EM
Department of Pharmacology, Southwestern Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Kameyama
K
K
Higashijima
T
T
Ross
E M
EM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004082:Dihydroalprenolol / Q000378:metabolism; D004837:Epinephrine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D020120:Iodocyanopindolol; D007545:Isoproterenol / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009638:Norepinephrine / Q000378:metabolism; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014422:Turkeys
D008024:Ligands; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D004082:Dihydroalprenolol; D020120:Iodocyanopindolol; D010869:Pindolol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D009638:Norepinephrine; D004837:Epinephrine