Thomas E. Côté

Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized μ-opioid receptors from rat brain : Coimmunoprecipitation of the G proteins Gαo, Gαi1, and Gαi3

Abstract: Antibodies directed against the C‐terminal and the N‐terminal regions of the μ‐opioid receptor were generated to identify the G proteins that coimmunoprecipitate with the μ receptor. Two fusion proteins were constructed: One contained the 50 C‐terminal amino acids of the μ receptor, and the other contained 61 amino acids near the N terminus of the receptor. Antisera directed against both fusion proteins were capable of immunoprecipitating ∼70% of solubilized rat brain μ receptors as determined by [3H][D‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO) saturation binding. The material immunoprecipitated with both of the antisera was recognized as a broad band with a molecular mass between 60 and 75 kDa when screened in a western blot. Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) had an EC50 of 0.4 nM in diminishing [3H]DAMGO binding to the immunoprecipitated pellet. The ratio of G proteins to μ receptors in the immunoprecipitated material was 1:1. When the material immunoprecipitated with affinity‐purified antibody was screened for the presence of G protein α subunits, it was determined that Gαo, Gαi1, Gαi3, and to a lesser extent Gαi2, but not Gαs or Gαq/11, were coimmunoprecipitated with the μ receptor. Inclusion of GTPγS during the immunoprecipitation process abolished the coimmunoprecipitation of G proteins.

Solubilization of High-Affinity, Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes†

Abstract: High‐affinity μ‐opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane‐associated receptors with morphine during solubilization in the detergent 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate appeared to stabilize the μ‐opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3‐[(3‐cholamidopropyl)dimethyl]‐1‐propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][d‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin ([3H]DAMGO), a μ‐selective opioid agonist, with high affinity (KD = 1.90 ± 0.93 nM; Bmax = 629 ± 162 fmol/mg of protein). Of the membrane‐associated [3H]‐DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 ± 0.39 nM, Bmax = 920 ± 154 fmol/mg of protein). Guanosine 5′‐O‐(3‐thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [d‐Pen2,d‐Pen5]‐enkephalin, a δ‐selective opioid agonist, and U50488H, a κ‐selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two‐site than a one‐site model with KDhigh = 2.17 ± 1.5 nM, Bmax = 648 ± 110 fmol/mg of protein and KDlow = 468 ± 63 nM, Bmax = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′‐O‐(3‐thiotriphosphate), the DAMGO displacement curve better fit a one‐ than a two‐site model with KD = 815 ± 33 nM, Bmax = 965 ± 124 fmol/mg of protein.

Opioid‐Induced Increase in [Ca2+]i in ND8‐47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein‐Coupled δ‐Opioid Receptors and Desensitized by Chronic Exposure to Opioid

Abstract: δ‐Receptor agonists induce a concentration‐dependent increase in intracellular calcium concentration ([Ca2+]i) in ND8‐47 cells by activating dihydropyridine‐sensitive Ca2+ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d‐Ser2,Leu5]enkephalin‐Thr (DSLET)‐induced increase in [Ca2+]i. Cholera toxin (10 nM, 24 h) had no effect on DSLET‐induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET‐induced increase in [Ca2+]i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [3H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits αi2, αi3, αs, and αq were detected in ND8‐47 cell membranes by western blot; αo and αi1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α‐subunits. These results suggest that the DSLET‐induced increase in [Ca2+]i is mediated through pertussis toxin‐sensitive G proteins (probably Gi2 or Gi3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.

Reconstitution of the solubilized μ-opioid receptor coupled to a GTP-binding protein

Abstract A μ-opioid receptor-GTP binding protein (μ-opioid receptor-G-protein) complex from the 7315c cell was solubilized with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) and reconstituted into phospholipid vesicles. Pretreatment of the tissue with either [3H]etorphine or morphine greatly improved recovery of the receptor and maintained it in a GTP-sensitive state. GTP sensitivity was consistent with the hypothesis that a receptor-G-protein complex had been obtained. Other evidence consistent with this hypothesis was that recovery of the solubilized, prelabelled receptor was decreased by approximately 70% by pretreatment of 7315c cells with pertussis toxin. The reconstituted receptor was μ-selective: DAGO (Tyr-D-Ala-Gly-Met-Phe-NH(CH2)2OH), but not ICI 174864 or U50488-H, displaced [3H]etorphine binding with high affinity. The affinity of the reconstituted receptor for [3H]etorphine (1.25 ± 0.20 nM) was similar to that observed for the membrane-associated receptor (0.53 ± 0.25 nM). GTPγS decreased this affinity 3-fold without changing the number of binding sites. The potencies of GTPγS and GTP in diminishing [3H]etorphine binding were similar in the membrane and vesicle preparations, but were 10-fold lower than the potencies observed in diminishing binding to the solubilized receptor. The ability to reconstitute a functional μ-opioid receptor-G-protein complex will facilitate further study of the structure and function of the receptor and the specific identification of the associated GTP-binding protein(s).

Inhibition of Adenylyl Cyclase Activity by a Homogeneous Population of Dopamine Receptors: Selective Blockade by Antisera Directed Against Gi1 and/or Gi2

Abstract: The 7315c pituitary tumor cell expresses a homogeneous population of dopamine receptors that are functionally similar to brain dopamine D2 receptors. [3H]‐Sulpiride binding to 7315c cell homogenates was specific and saturable, and Ki values for compounds to compete for these sites were highly correlated with values for the same compounds at D2 receptors in brain. Dopamine maximally inhibited ∼65% of forskolin‐stimulated cyclase activity in cell membranes. Some D2 agonists had lower efficacies, suggesting that some compounds are partial agonists at this receptor. Removal of GTP from the assay buffer or pretreatment of the tissue with pertussis toxin abolished the inhibition of adenylyl cyclase by dopamine. Immunodetection of most of the known Gα subunits revealed that Gi1, Gi2, Gi3, Go, Gq, and Gs are present in the 7315c membrane. Pretreatment with the AS antibody (which recognizes the C‐terminal regions of Gαi1 and Gαi2) significantly attenuated the inhibition of adenylyl cyclase activity by dopamine, whereas antibodies to C‐terminal regions of the other Gα subunits had no effect. These findings suggest that the dopamine D2 receptor regulates cyclase inhibition predominantly via Gi1 and/or Gi2 and that the 7315c tumor cells provide a useful model for studying naturally expressed dopamine D2 receptors in the absence of other dopamine receptor subtypes.

Mu opioid receptor activation enhances regulator of G protein signaling 4 association with the mu opioid receptor/G protein complex in a GTP‐dependent manner

The interaction of Regulator of G protein Signaling 4 (RGS4) with the rat mu opioid receptor (MOR)/G protein complex was investigated. Solubilized MOR from rat brain membranes was immunoprecipitated in the presence of RGS4 with antibodies against the N‐terminus of MOR (anti‐MOR10–70). Activation of MOR with [D‐Ala2, N‐Me‐Phe4, Gly5‐ol] enkephalin (DAMGO) during immunoprecipitation caused a 150% increase in Goα and a 50% increase in RGS4 in the pellet. When 10 μM GTP was included with DAMGO, there was an additional 72% increase in RGS4 co‐immunoprecipitating with MOR (p = 0.003). Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) increased the amount of co‐precipitating RGS4 by 93% (compared to DAMGO alone, p = 0.008), and the inclusion of GTPγS caused the ratio of MOR to RGS4 to be 1 : 1 (31 fmoles : 28 fmoles, respectively). GTPγS also increased the association of endogenous RGS4 with MOR. In His6RGS4/Ni2+‐NTA agarose pull down experiments, 0.3 μM GTPγS tripled the binding of Goα to His6RGS4, whereas the addition of 100 μM GDP blocked this effect. Importantly, activation of solubilized MOR with DAMGO in the presence of 100 μM GDP and 0.3 μM GTPγS increased Goα binding to His6RGS4/Ni2+‐NTA agarose (p = 0.001).

Increased opioid efficacy for inhibition of adenylyl cyclase in rat brain and 7315c cell membranes induced by chronic naltrexone treatment

Long term administration of the opioid antagonist naltrexone results in an increase in μ-opioid receptor density in brain (1,2). Chronic naltrexone treatment also produces an increase in the analgesic potency of morphine (3) and this may be related to the increase in opioid receptors. It is unknown, however, whether the upregulation of the μ-opioid receptor following naltrexone treatment results in an enhanced regulation of second messenger systems by opioid agonists. In the current study, the effects of chronic naltrexone have been examined on the ability of μ-opioid receptors on brain membranes and on a non-neuronal tissue, the transplantable 7315c tumor cell that expresses μ- but not δ- or κ- opioid receptors (4,5), to bind [ 3 H]DAMGO and to respond to μ-opioid agonists and GTPγS in an adenylyl cyclase assay