Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

Hitoshi Kurose, T. Katada, T. Haga, A. Ichiyama, M. Ui

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

The GTP binding regulatory protein (N(i)) involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding and GTP hydrolyzing activities of reconstituted N(i) were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the V(max) values of these activities, but the K(m) values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of K(m) as that for stimulation of GTPase. The affinity of this binding was reduced by GTPγS, indicating that the high-affinity receptor-N(i) complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of N(i) with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the α-subunit of N(i). The criteria for the receptor-N(i) interaction, i.e. carbachol stimulation of the activities of N(i) and the GTPγS effect on carbachol binding, were no longer observed, when this IAP-treated N(i), instead of the nontreated N(i), was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated N(i) in their basal activities observable without carbachol. N(o), the protein with a character very similar to N(i) in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.

Original languageEnglish
Pages (from-to)6423-6428
Number of pages6
JournalJournal of Biological Chemistry
Volume261
Issue number14
Publication statusPublished - 1986
Externally publishedYes

Fingerprint

Carbachol
Muscarinic Receptors
GTP-Binding Proteins
Pertussis Toxin
Phospholipids
Guanosine Triphosphate
Brain
Adenosine Diphosphate
Rats
Guanosine 5'-O-(3-Thiotriphosphate)
Muscarinic Agonists
Guanosine
GTP Phosphohydrolases
Cholinergic Receptors
Adenylyl Cyclases
NAD
Cholinergic Agents
Proteins
Swine
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. / Kurose, Hitoshi; Katada, T.; Haga, T.; Ichiyama, A.; Ui, M.

In: Journal of Biological Chemistry, Vol. 261, No. 14, 1986, p. 6423-6428.

Research output: Contribution to journalArticle

@article{944f847a5b8f48a2bffe86d0db27bda6,
title = "Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles",
abstract = "The GTP binding regulatory protein (N(i)) involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding and GTP hydrolyzing activities of reconstituted N(i) were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the V(max) values of these activities, but the K(m) values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of K(m) as that for stimulation of GTPase. The affinity of this binding was reduced by GTPγS, indicating that the high-affinity receptor-N(i) complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of N(i) with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the α-subunit of N(i). The criteria for the receptor-N(i) interaction, i.e. carbachol stimulation of the activities of N(i) and the GTPγS effect on carbachol binding, were no longer observed, when this IAP-treated N(i), instead of the nontreated N(i), was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated N(i) in their basal activities observable without carbachol. N(o), the protein with a character very similar to N(i) in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.",
author = "Hitoshi Kurose and T. Katada and T. Haga and A. Ichiyama and M. Ui",
year = "1986",
language = "English",
volume = "261",
pages = "6423--6428",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

AU - Kurose, Hitoshi

AU - Katada, T.

AU - Haga, T.

AU - Ichiyama, A.

AU - Ui, M.

PY - 1986

Y1 - 1986

N2 - The GTP binding regulatory protein (N(i)) involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding and GTP hydrolyzing activities of reconstituted N(i) were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the V(max) values of these activities, but the K(m) values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of K(m) as that for stimulation of GTPase. The affinity of this binding was reduced by GTPγS, indicating that the high-affinity receptor-N(i) complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of N(i) with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the α-subunit of N(i). The criteria for the receptor-N(i) interaction, i.e. carbachol stimulation of the activities of N(i) and the GTPγS effect on carbachol binding, were no longer observed, when this IAP-treated N(i), instead of the nontreated N(i), was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated N(i) in their basal activities observable without carbachol. N(o), the protein with a character very similar to N(i) in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.

AB - The GTP binding regulatory protein (N(i)) involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding and GTP hydrolyzing activities of reconstituted N(i) were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the V(max) values of these activities, but the K(m) values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of K(m) as that for stimulation of GTPase. The affinity of this binding was reduced by GTPγS, indicating that the high-affinity receptor-N(i) complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of N(i) with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the α-subunit of N(i). The criteria for the receptor-N(i) interaction, i.e. carbachol stimulation of the activities of N(i) and the GTPγS effect on carbachol binding, were no longer observed, when this IAP-treated N(i), instead of the nontreated N(i), was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated N(i) in their basal activities observable without carbachol. N(o), the protein with a character very similar to N(i) in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.

UR - http://www.scopus.com/inward/record.url?scp=0023019584&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023019584&partnerID=8YFLogxK

M3 - Article

VL - 261

SP - 6423

EP - 6428

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -