Interaction of atrial muscarinic receptors with three kinds of GTP-binding proteins.
Purified porcine atrial muscarinic acetylcholine receptors were reconstituted into lipid vesicles with three different G proteins (Gi, Go and Gn)1 purified from porcine cerebrum. All the G proteins interacted with the receptor as evidenced by GTP-sensitive high affinity binding with acetylcholine, and stimulation by acetylcholine of GTP gamma S binding and GTPase activities. The curves of displacement by acetylcholine of [3H]QNB binding were explained by assuming two sites with the same affinity for [3H]QNB but different affinities for acetylcholine. The proportion of the high affinity site increased from 3 to 7% up to 82 to 83% of total binding sites with increasing G protein concentration, and essentially the same results were obtained with the three G proteins. The GTPase activities of Gi, Go and Gn in the reconstituted vesicles were 2.7-, 1.7- and 1.6-times higher, respectively, in the presence of 1 mM acetylcholine than those in the presence of 10 microM atropine. An obvious enhancement by acetylcholine of the GTP gamma S binding was observed in the presence of 10 to 100 microM GDP, while the enhancement was minimal, if at all, in the absence of GDP. When the molar ratios of reconstituted Gi, Go and Gn to muscarinic receptors were 54, 84 and 107, respectively, the acetylcholine-induced increase in the [35S]GTP gamma S binding was as much as 12, 35 and 27 mol with Gi, Go and Gn, respectively, per mole of the receptor molecule, indicating that the muscarinic receptors interact with G proteins catalytically.(ABSTRACT TRUNCATED AT 250 WORDS)
Ikegaya
T
T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Nishiyama
T
T
Haga
K
K
Haga
T
T
Ichiyama
A
A
Kobayashi
A
A
Yamazaki
N
N
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006325:Heart Atria / Q000378:metabolism / Q000648:ultrastructure*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013552:Swine
D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins; D000109:Acetylcholine