Different molecular weight forms of opioid receptors revealed by polyclonal antibodies

Polyclonal antibodies were raised against a purified opioid receptor from bovine brain (Cho, et. al., 1986), and shown to inhibit 3H-diprenorphine binding to this receptor in a dose-dependent fashion. These antibodies were then used to characterize opioid-binding material present in rat brain and in NG108-15 neuroblastoma-glioma hybrid cells. Western blot analysis revealed that the antibodies reacted with a single species of 58,000 molecular weight in rat brain membranes; this closely corresponds in size to the bovine opioid receptor used to raise the antibodies. In contrast, the polyclonal antibodies reacted with a 45,000 molecular weight species in NG108-15 neuroblastoma-glioma hybrid cells; moreover, this band was specifically reduced in NG108-15 cells in which opioid receptors had been down-regulated by incubation with D-ala2-D-leu5-enkephalin for 24 hours. Thus at least two distinct opioid receptor molecules have been identified, which have antigenic similarities.     

Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

Identification and characterization of the beta-adrenergic receptor on neuroblastoma × glioma hybrid NG108-15 cells

1. Using [3H]DHA and unlabeled L-alprenolol, a substantial amount of over 64% specific binding of beta-adrenergic receptor has been identified on the neuroblastoma x glioma hybrid NG108-15 cell, which has been proven to display numerous functional characteristics of intact neurons. 2. Beta-adrenergic receptor binding on intact NG108-15 cells does not change significantly upon morphological differentiation, induced by 1 mM dibutyryl cyclic AMP (dBcAMP). 3. The [3H]DHA binding on intact NG108-15 cells is rapid, saturable, and reversible, having a t1/2 of 1.0 min for association and 3.5 min for dissociation. 4. The affinity constant (Kd) and maximum binding capacity (Bmax) for binding of [3H]DHA to beta-adrenergic receptors on NG108-15 cells have been estimated by Scatchard plot analysis to be 2.5 and 0.23 nM, respectively. Further analysis indicates a single class of receptors for [3HDHA binding on NG108-15 cells. 5. Studies on kinetic properties have revealed on-rate (K + 1) and off-rate (K - 1) constants of 0.7 X 10(-9) M min-1 and 0.19 min-1, respectively. Further, the IC50 value and inhibition constant (Ki) for unlabeled L-alprenolol to inhibit [3HDHA binding on NG108-15 cells have been estimated to be 10(-5) and 8.9 X 10(-6) M, respectively. 6. The rank-order potency of catecholamine agonists, (-)ISO greater than (+)ISO greater than EPI greater than NE, reveals the presence of type 2 receptor for the beta-adrenergic binding on both differentiated and undifferentiated NG108-15 cells. 7. The present study indicates that the clonal neuroblastoma x glioma hybrid NG108-15 cell line possesses substantial amounts of beta-adrenergic receptors with characteristics similar to those on neuronal cells.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Comparison of the butyrate effects on neurotransmitter receptors in neurohybrids NG108-15 and NCB-20 cells

Our previous study demonstrated that long term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the density of delta-opioid receptors with a much lesser effect on muscarinic cholinergic and no effect on alpha 2-adrenergic receptors. In the present study we investigated the effect of sodium butyrate on these three types of receptors in NG108-15 cells whose neuroblastoma parent is the same as that of NCB-20 cells. Long term treatment of NG108-15 cells with sodium butyrate (0.5 mM) induced a 2-fold increase in the density of the specific binding of 3H-clonidine. A comparable increase in the number of binding sites was detected when 3H-yohimbine was used as the receptor ligand. The butyrate-induced increase in the alpha 2-adrenergic receptor binding could be totally abolished by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that synthesis of receptor protein is involved. The same butyrate treatment had no significant effect on opioid and muscarinic cholinergic receptor bindings. Thus, butyrate effects on the expression of these three types of receptors in NG108-15 and NCB-20 cells are dramatically different. These data suggest that induction by butyrate of neurotransmitter receptors requires concerted action of genetic factors of both parents of the neurohybrids.     

Sodium regulation of opioid agonist binding is potentiated by pertussis toxin

Pretreatment of intact NG108-15 cells with pertussis toxin suppresses opioid inhibition of cyclic AMP accumulation mediated by the inhibitory guanine nucleotide-binding regulatory protein, Ni, which apparently also mediates the inhibitory nucleotide effects on opioid against binding. The toxin treatment had no effect on opioid agonist binding measured in NG108-15 cell membranes without sodium present. However, the toxin potentiated the inhibitory effect of sodium on agonist binding, leading to an agonist-specific reduction of opioid receptor affinity in the presence of sodium in the binding reaction. The potency of the stable GTP analog, GTP gamma S, to reduce agonist binding in the presence of sodium was little changed in membranes prepared from pertussis toxin-treated cells compared to control membranes, whereas the potency of the stable GDP analog, GDP beta S, was magnified. The data indicate that ADP-ribosylation of Ni by pertussis toxin potentiates sodium regulation of opioid agonist binding and that the communication between Ni and opioid receptors is not lost by the covalent modification of Ni.     

Characterization of beta-125I-endorphin cross-linked proteins in NG108-15 cell membranes. A 25-kilodalton protein with properties of delta-opioid-binding site.

Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.     

Multiple receptors linked to inhibition of adenylate cyclase accelerate Na+/H+ exchange in neuroblastoma x glioma cells via a mechanism other than decreased cAMP accumulation

alpha 2-Adrenergic receptors, a population of receptors linked to inhibition of adenylate cyclase, accelerate Na+/H+ exchange in NG108-15 neuroblastoma x glioma cells (Isom, L. L., Cragoe, E. J., Jr., and Limbird, L. E. (1987) J. Biol. Chem. 262, 6750-6757). We now report that two other receptor populations linked to inhibition of adenylate cyclase, muscarinic cholinergic and delta-opiate receptors, also alkalinize the interior of NG108-15 cells, as measured with the pH-sensitive fluorescent probe, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein. Manipulations that block Na+/H+ exchange, i.e. removal of extracellular Na+, reduction of extracellular pH to equal that of intracellular pH, and addition of 5-amino-substituted analogs of amiloride, all block alpha 2-adrenergic, delta-opiate, or muscarinic cholinergic receptor-induced alkalinization in a parallel fashion. These data suggest that all three populations of receptors alkalinize NG108-15 cells by acceleration of Na+/H+ exchange and do so via a shared or similar mechanism. Although these three receptor populations are linked to inhibition of adenylate cyclase, decreased production of cAMP does not appear to be the mechanism responsible for receptor-accelerated Na+/H+ exchange. Thus, ADP-ribosylation of intact NG108-15 cells with Bordetella pertussis islet-activating protein prevents attenuation of prostaglandin E1-stimulated cAMP accumulation by alpha 2-adrenergic, muscarinic, and delta-opiate agonists but has no measurable effect on the ability of these agonists to accelerate Na+/H+ exchange. Similarly, manipulations that block receptor-accelerated Na+/H+ exchange influence but do not block receptor-mediated attenuation of cAMP accumulation. Thus, the present data suggest that these two receptor-mediated biochemical events, acceleration of Na+/H+ exchange and attenuation of cAMP accumulation, occur through divergent mechanisms in NG108-15 cells.     

125I-beta-endorphin binding to neuroblastoma X glioma NG108-15 cells: distribution of delta opioid receptors.

The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)). Silver grains resulting from 125I-beta E binding to the opioid sites occurred in diffuse patches over several cells, with preferential location in dense cell patches. Pretreatment of NG108-15 with the delta agonist DADLE, previously shown to decrease beta E binding to delta sites on intact cells, also reduced silver grain density; however, some cells located in dense cell clusters were resistant to substantial agonist induced loss of labeling. These results suggest that delta opioid binding has a heterogenous cellular distribution in NG108.     

Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.     

Enhanced acetylcholine secretion in neuroblastoma x glioma hybrid NG108-15 cells transfected with rat choline acetyltransferase cDNA.

Neuroblastoma x glioma hybrid NG108-15 cells and mouse neuroblastoma N18TG-2 and N1E-115 cells were transiently transfected with the sense cDNA coding for rat choline acetyltransferase (ChAT). All transfected cell lines showed a high level of ChAT activity. ACh secretion was monitored by recording miniature end-plate potentials (MEPPs) in striated muscle cells that had been co-cultured with transfected cells. The number of muscle cells with synaptic responses and the MEPP frequency were higher in co-culture with transfected NG108-15 cells than with control or mock cells. No synaptic response was detected in muscle cells co-cultured with transfected N18TG-2 or N1E-115 cells. The results show that ACh secretion into the synaptic cleft was enhanced due to ChAT overexpression in NG108-15 hybrid cells but not in neuroblastoma cells.     

Rapid agonist-induced loss of 125I-beta-endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells.

We have measured mu and delta opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of 125I-beta-endorphin (beta E) as a tracer, together with beta E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface mu and delta opioid receptor sites. Labeling was at delta sites in NG108-15 cells and predominantly at mu sites in SK-N-SH cells. Pretreatment with the mu and delta agonist, DADLE, caused a rapid loss of cell surface delta receptor sites in NG108-15 cells, but failed to reduce significantly mu receptor density in SK-N-SH cells.     

2-Arachidonoylglycerol and the cannabinoid receptors

2-Arachidonoylglycerol (2-AG) is a unique molecular species of monoacylglycerol isolated from rat brain and canine gut as an endogenous cannabinoid receptor ligand (Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A., Waku, K., 1995. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem. Biophys. Res. Commun. 215, 89-97; Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N. E., Schatz, A.R., Gopher, A., Almog, S., Martin, B.R., Compton, D.R., Pertwee, R.G., Giffin, G., Bayewitch, M., Brag, J., Vogel, Z., 1995. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83-90). 2-AG binds to the cannabinoid receptors (CB1 and CB2) and exhibits a variety of cannabimimetic activities in vitro and in vivo. Recently, we found that 2-AG induces Ca(2+) transients in NG108-15 cells, which express the CB1 receptor, and in HL-60 cells, which express the CB2 receptor, through a cannabinoid receptor- and Gi/Go-dependent mechanism. Based on the results of structure-activity relationship experiments, we concluded that 2-AG but not anandamide is the natural ligand for both the CB1 and the CB2 receptors and both receptors are primarily 2-AG receptors. Evidences are gradually accumulating that 2-AG is a physiologically essential molecule, although further detailed studies appear to be necessary to determine relative importance of 2-AG and anandamide in various animal tissues. In this review, we described mainly our previous and current experimental results, as well as those of others, concerning the tissue levels, bioactions and metabolism of 2-AG.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor

Recently, we have described a COOH-terminal deletion mutation of the human insulin receptor (HIR delta CT) that exhibits normal insulin-mediated kinase activity and endocytosis, but is inefficient in stimulating glucose transport and glycogen synthase (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J.M. (1988) J. Biol. Chem. 263, 8904-8911; Maegawa, H., McClain, D. A., Freidenberg, G., Olefsky, J. M., Napier, M., Lipari, T., Dull, T. J., Lee, J., and Ullrich, A. (1988) J. Biol. Chem. 263, 8912-8917). In this paper, we report that despite this defect in metabolic signaling, the truncated receptor exhibits augmented mitogenic activity compared to normal receptors. These results were verified in three independently isolated clones of Rat 1 fibroblasts transfected with the HIR delta CT cDNA. The increase in insulin sensitivity of mitogenic stimulation was proportional to the number of HIR delta CT receptors expressed on the cells. By contrast, only the cells with normal receptors and none of the HIR delta CT clones exhibit increased sensitivity for a metabolic action of insulin, the stimulation of glucose uptake. Stimulation of cells by other mitogens and autoradiographic analysis confirm that the enhanced mitogenic effects seen in HIR delta CT cells are attributable only to the presence of the truncated insulin receptors. These receptors mediate the tyrosine phosphorylation of a number of cellular proteins, and the pattern of these phosphorylations differs quantitatively from that seen in cells with normal receptors. We conclude: 1) The COOH terminus plays a role in signaling metabolic actions of insulin, perhaps through its recognition of substrates for the receptor kinase. 2) By contrast, the COOH terminus is an inhibitory regulator of mitogenesis, and removal of the terminal 43 amino acids converts the receptor from a moderately active growth signaler to a very active one. 3) The changes seen in biologic activities of the HIR delta CT receptor are associated with quantitative changes in substrate phosphorylation by the receptor kinase.