Functional reconstitution in situ of 5-hydroxytryptamine2c (5HT2c) receptors with alphaq and inverse agonism of 5HT2c receptor antagonists.

Membranes prepared after infection of Sf9 cells with recombinant baculovirus containing the rat 5HT2c receptor DNA, but not after infection with wild-type virus, expressed high affinity binding sites for 125I-lysergic acid diethylamide and [3H]mesulergine. The receptor site density reached an optimum of 50-70 pmol/mg membrane protein at 60 h postinfection. Extraction of peripheral membrane proteins from the postnuclear membrane fraction with 6 M urea depleted GTPgammaS-binding 4-fold without decreasing 5HT2c receptor binding activity. Urea-extracted Sf9 membranes expressing the 5HT2c receptor catalyzed the activation of squid retinal alphaq but not bovine retinal alphat or bovine alphao/alphai. Productive interaction of 5HT2c receptors with squid alphaq was enhanced by the addition of betagamma dimers prepared from either bovine brain or bovine rod outer segment discs. While the addition of serotonin increased 5HT2c receptor-catalyzed GTPgammaS binding to alphaq, the unoccupied receptor was also catalytically active. The 5HT2c receptor antagonists, mesulergine, mianserin, and ketanserin competitively inhibited 5HT activation of the receptor with predicted rank-order affinities; and mianserin and ketanserin markedly inhibited basal 5HT2c receptor activity. Interestingly, this "inverse agonist" efficacy did not correlate with antagonist affinity for the 5HT2c receptor. Baculoviral expression of the 5HT2c receptor and urea extraction of postnuclear Sf9 cell membranes have provided a high density of in situ, uncoupled, G-protein-linked receptor useful for reconstitution with purified G-protein subunits. This has allowed for independent manipulation of receptor and G-protein chemical concentrations and has revealed that a G-protein-linked receptor can possess a significant basal catalytic activity and that antagonist compounds can act as inverse agonists of this basal activity at the level of receptor activation of G-proteins.