INTERACTION OF DEGLYCOSYLATED MUSCARINIC RECEPTORS WITH LIGANDS AND G-PROTEINS

Endoglycosidase F was used to investigate the role of the carbohydrate moiety of muscarinic acetylcholine receptors in antagonist and agonist binding, and the interaction with G proteins. The receptors were purified from porcine cerebrum, treated with endoglycosidase F and then covalently labeled with [H-3]propylbenzilylcholine mustard ([H-3]PrBCM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [H-3]PrBCM-labeled receptors showed that the endoglycosidase F treatment caused a decrease in apparent M(r) from 70 to 51 kDa, the M(r) predicted for the peptide portions of M1, M2 and M4 subtypes. Endoglycosidase F-treated receptors had essentially the same affinities for both agonists and antagonists as those of control receptors. In addition, treated receptors that had been reconstituted in lipid vesicles with G proteins showed guanine nucleotide-sensitive high affinity for agonists. These results suggest that the carbohydrate moiety of muscarinic acetylcholine receptors is not involved in their interaction with muscarinic ligands and G proteins.