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 [3H]propylbenzilylcholine mustard [( 3H]PrBCM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]PrBCM-labeled receptors showed that the endoglycosidase F treatment caused a decrease in apparent Mr from 70 to 51 kDa, the Mr 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.
Ohara
K
K
Department of Biochemistry, Hamamatsu University School of Medicine, Japan.
Uchiyama
H
H
Ohara
K
K
Haga
T
T
Ichiyama
A
A
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001921:Brain / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000502:physiology*; D006026:Glycoside Hydrolases; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010095:Oxotremorine / Q000494:pharmacology; D011438:Propylbenzilylcholine Mustard; D011813:Quinuclidinyl Benzilate; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
D008024:Ligands; D011976:Receptors, Muscarinic; D011438:Propylbenzilylcholine Mustard; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D019204:GTP-Binding Proteins; D005947:Glucose