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

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

Research output: Contribution to journalArticlepeer-review

108 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

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles'. Together they form a unique fingerprint.

Cite this