Functional reconstitution of purified Gi and Go with μ-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide

Hiroshi Ueda, Hidemi Misawa, Toshiaki Katada, Michio Ui, Hiroshi Takagi, Masamichi Satoh

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

Functional coupling between μ-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na+-dependent manner. The same μ-opioid agonist {[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephaIin (DAGO)} caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi( (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in mem-branes. The μ-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The μ-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ-opioid receptor may exist in at least two different forms, separately coupled to G1 or Go.

Original languageEnglish
Pages (from-to)841-848
Number of pages8
JournalJournal of Neurochemistry
Volume54
Issue number3
Publication statusPublished - 1990
Externally publishedYes

Fingerprint

Corpus Striatum
Ethylmaleimide
Opioid Receptors
GTP-Binding Proteins
Guinea Pigs
Guanosine Triphosphate
GTP Phosphohydrolases
Membranes
Ala(2)-MePhe(4)-Gly(5)-enkephalin
Pertussis Toxin
Opioid Analgesics
Proteins
Adenylyl Cyclases
Adenosine Diphosphate
Assays
Brain
Recovery
Experiments

Keywords

  • μ-opioid receptors
  • G
  • G
  • GTP-binding regulatory proteins
  • Islet-activating protein
  • N-ethylmaleimide
  • Pertussis toxin
  • Striatal membranes-[D-Ala,N-Me-Phe,Gly-ol]enkephalin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Functional reconstitution of purified Gi and Go with μ-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide. / Ueda, Hiroshi; Misawa, Hidemi; Katada, Toshiaki; Ui, Michio; Takagi, Hiroshi; Satoh, Masamichi.

In: Journal of Neurochemistry, Vol. 54, No. 3, 1990, p. 841-848.

Research output: Contribution to journalArticle

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abstract = "Functional coupling between μ-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na+-dependent manner. The same μ-opioid agonist {[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephaIin (DAGO)} caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi( (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in mem-branes. The μ-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The μ-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ-opioid receptor may exist in at least two different forms, separately coupled to G1 or Go.",
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AU - Takagi, Hiroshi

AU - Satoh, Masamichi

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N2 - Functional coupling between μ-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na+-dependent manner. The same μ-opioid agonist {[D-Ala2,N-Me-Phe4,Gly5-ol]-enkephaIin (DAGO)} caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi( (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in mem-branes. The μ-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The μ-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ-opioid receptor may exist in at least two different forms, separately coupled to G1 or Go.

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