Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera.
Solubilization of opioid receptors from rat cortical membranes that retained high-affinity guanine nucleotide-sensitive agonist binding was achieved using 10 mM CHAPS. We report the nature of the interactions of mu and delta opioid receptors with the guanine nucleotide-binding protein G(o) by immunoprecipitation of CHAPS extracts with selective G(o)alpha-subunit protein antisera. Antiserum IM1 raised against amino acids 22-35 of G(o)alpha selectively co-immunoprecipitated G(o)alpha-mu and G(o)alpha-delta opioid receptor complexes detected in the immunoprecipitates by specific [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and [3H][D-Ser2,Leu5,Thr6]enkephalin binding respectively. By contrast, antisera directed against the C-terminal decapeptide (OC2) and the N-terminal hexadecapeptide (ON1) of isoforms of G(o)alpha were unable to immunoprecipitate solubilized opioid receptor-G(o) complexes, although both were able to immunoprecipitate solubilized G(o)alpha and have been shown to reduce the affinity of [D-Ala2,D-Leu5]enkephalin for opioid receptors in rat cortical membranes [Georgoussi, Carr and Milligan (1993) Mol. Pharmacol. 44, 62-69]. These findings demonstrate that CHAPS-solubilized mu and delta opioid receptors from rat cortical membranes form stable complexes with one or more variants of G(o).
Georgoussi
Z
Z
Institute of Biology, National Centre of Scientific Research Demokritos, Athens, Greece.
Milligan
G
G
Zioudrou
C
C
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002540:Cerebral Cortex / Q000737:chemistry*; D002793:Cholic Acids; D004174:Diprenorphine / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis / Q000276:immunology* / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007106:Immune Sera; D007163:Immunosorbent Techniques; D051381:Rats; D011957:Receptors, Opioid / Q000032:analysis / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000032:analysis / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000032:analysis / Q000378:metabolism; D012995:Solubility
D002793:Cholic Acids; D007106:Immune Sera; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D004174:Diprenorphine; D006165:Guanylyl Imidodiphosphate; D016308:Enkephalin, Leucine-2-Alanine; D019204:GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate