Reconstitution of high-affinity opioid agonist binding in brain membranes.

Abstract

In synaptosomal membranes from rat brain (12,13).Although the results outlined above have implicated modulationt of ligand-receptor interaction by G proteins, they did not establish the molecular identity of the high-and lowaffinity components of opioid binding. In this study, we have directly linked high-affinity opioid agonist binding and opioid-sensitive GTPase activity to membrane G protein. Inactivation of G protein at high pH abolished high-affinity agonist, but not antagonist, binding. Guanine nucleotidesensitive high-affinity opioid binding and opioid-sensitive GTPase activity were restored after fusion of the alkalitreated synaptosomal brain membranes with membranes from opioid receptor-deficient, but inhibitory G proteincontaining, C6 glioma cells. The results describe high-affinity opioid agonist binding as a ternary complex composed of ligand, receptor, and G protein. Guanine nucleotidedependent dissociation of the complex then yields the lowaffinity form of the ligand-occupied receptor. A preliminary report on these findings was presented recently (14). Isolated Membranes and Cell Culture. The synaptosomal preparation was isolated from brain cortices ofmale SpragueDawley rats as described (15). Prior to their freezing at -80'C, the synaptosomes were disrupted in a Dounce homogenizer. MATERIALS AND METHODSTo reach confluence, the C6 glioma cells were grown as a monolayer in a minimal essential medium supplemented with 10% fetal bovine serum and 1% pyruvate for 8-9 days at 370C in 95% air/5% CO2. After detachment from the flask wall by incubation at 370C in saline, the cells were disrupted in hypotonic buffer with a Dounce homogenizer. The pelleted membranes were resuspended in 50 mM Tris HCl (pH 7.4) to a protein concentration of 1 mg/ml and were stored at -800C.Protein was determined according to Bradford (16)

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

“…We have previously described the functional coupling of A, 8, and K opioid receptors to G proteins in brain membranes (23,24) and have subsequently characterized the ,u opioid receptor-G protein complex formed upon receptor occupancy by agonists (25). Recently, we have quantified the functional responses of G protein and adenylyl cyclase in normal (26) and opioid-tolerant (27) SH-SY5Y neural cells.…”

Stabilization of high-affinity opioid agonist binding in neural cell membranes rigidified by cholesterol

“…Regulation of ligand binding to G protein-coupled receptors, including the opioid, occurs through receptor-transducer association and dissociation, a process modulated by guanine nucleotides and ions. The high-affinity binding state, representing the ternary complex of agonist-occupied receptor and G protein (4), converts in the presence of GTP or its stable analogues into the low-affinity, G proteinuncoupled, form of the receptor. Previous studies with several receptors, including txl-and 13adrenergic and dopamine have described the formation of a high-affinity agonist state of receptor at low (2-4 °C) temperatures (5-7) or by a combination of deoxycholate and low temperature (8).…”

Membrane Microviscosity Modulates μ‐Opioid Receptor Conformational Transitions and Agonist Efficacy

“…The molecular consequence of sodium binding to the opioid receptor was suggested to be a conformational change from an “agonist‐favoring” to an “antagonist‐favoring” state (Simon and Groth, 1975; Simon and Hiller, 1981), although in the present study this simple conformational model does not fully explain the observed changes in receptor binding because agonist binding was strongly modulated by sodium, whereas antagonist binding was unaffected. The presence of a G protein has been shown to be required for the stabilization of the high‐affinity opioid agonist binding (Remmers and Medzihradsky, 1991 a ). In the absence of G protein, the receptor exists in a conformation with low affinity for ligand.…”

“…The binding of opioid ligands is modulated by addition of sodium and guanine nucleotides (Blume, 1978; Ott and Costa, 1988). High‐affinity binding of agonists to the receptor requires the association of receptor with G protein (Remmers and Medzihradsky, 1991 a ), and addition of sodium or guanine nucleotides dissociates this complex to convert the receptor to a conformation with low affinity for agonist (Blume, 1978; Childers and Snyder, 1980; Ott and Costa, 1988). In contrast, antagonist binding is increased by sodium and unchanged by further addition of guanine nucleotides, indicating an insensitivity to the presence of G protein (Simantov et al, 1976; Childers and Snyder, 1980; Nijssen and Childers, 1987).…”