Opioid receptors in magnesium-digitonin-solubilized rat brain membranes are tightly coupled to a pertussis toxin-sensitive guanine nucleotide-binding protein.
Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively.
Wong
Y H
YH
MRC Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge, England.
Demoliou-Mason
C D
CD
Barnard
E A
EA
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D004072:Digitonin; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008274:Magnesium; D008566:Membranes / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D012995:Solubility; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*; D014909:Wheat Germ Agglutinins
D011957:Receptors, Opioid; D010566:Virulence Factors, Bordetella; D014909:Wheat Germ Agglutinins; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D008274:Magnesium; D004072:Digitonin