Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components.
A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.
Haga
K
K
Department of Biochemistry, Hamamatsu University School of Medicine, Japan.
Uchiyama
H
H
Haga
T
T
Ichiyama
A
A
Kangawa
K
K
Matsuo
H
H
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D037342:Pertussis Toxin; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013552:Swine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D011813:Quinuclidinyl Benzilate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins