TY - JOUR
T1 - Opioid Receptors in Magnesium‐Digitonin‐Solubilized Rat Brain Membranes Are Tightly Coupled to a Pertussis Toxin‐Sensitive Guanine Nucleotide‐Binding Protein
AU - Wong, Yung H.
AU - Demoliou‐Mason, Catherine D.
AU - Barnard, Eric A.
PY - 1989
Y1 - 1989
N2 - Abstract 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 Goα subunits, respectively.
AB - Abstract 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 Goα subunits, respectively.
KW - G proteins
KW - Opioid receptors
KW - Pertussis toxin
UR - http://www.scopus.com/inward/record.url?scp=0024583845&partnerID=8YFLogxK
U2 - 10.1111/j.1471-4159.1989.tb01840.x
DO - 10.1111/j.1471-4159.1989.tb01840.x
M3 - Article
C2 - 2538569
AN - SCOPUS:0024583845
SN - 0022-3042
VL - 52
SP - 999
EP - 1009
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 4
ER -