The turkey β-adrenergic receptor (β-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 β-AR was glycosylated; electrophoretic behavior of the two MAChRs also suggested glycosylation. The β-AR stimulated endogenous adenylyl cyclase in response to β-adrenergic agonists. The β-AR and both MAChRs were purified and coreconstituted with various purified G proteins in phospholipid vesicles. The recombinant β-AR catalyzed the agonist-dependent activation of Gs by guanosine 5'-0-(thiotriphosphate) (GTPγS) with the same efficiency as did the natural β-AR. The m2 MAChR efficiently catalyzed GTPγS binding to Go and to the recently identified G protein Gz (Gx). The m2 MAChR also catalyzed the activation of Gi,1 and Gi,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 β-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.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jan 5 1991|
ASJC Scopus subject areas