Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera

Abstract

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).

Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames

“…Although receptors can often be resolved from their cognate G proteins by detergent solubilization of membranes, in a number of examples such solubilization of membranes can result in the maintenance of receptor-G protein contacts (15)(16)(17). The term "precoupled" is often used to describe such avid interactions between receptors and their cognate G protein.…”

Activation of μ-Opioid Receptors Transfers Control of Gα Subunits to the Regulator of G-protein Signaling RGS9-2

“…3 and references therein) through a direct interaction (4,5). In the central nervous system, MORs play an important role in the antinociceptive action of opioids, but they become desensitized after repeated administration.…”

Pancreastatin receptor is coupled to a guanosine triphosphate-binding protein of the Gg/11α family in rat liver membranes

“…In fact, binding of C-terminal antibodies seems to uncouple receptors and G proteins only functionally rather than physically, as previously described in other systems. 56 On the other hand, the amino termini of G proteins have recently been shown to have a functional importance for interactions with both effector and receptor, at least in the particular case of G q ␣ . .…”

“…56 Aliquots of solubilized membranes (1 mg of protein) were diluted five times in the same buffer lacking Triton X-100 and incubated with appropriate antisera (1:50 final dilution) at 4°C under constant rotation. After 2 hours, 100 µL of 50% (wt/vol) Protein A-agarose beads were added and the mixture further incubated for 1 hour.…”