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Differentiation of agonist conformation and antagonist conformation in multiple opioid receptors
Neuroscience Letters, 27 (1981) 205-209
205
Elsevier/North-Holland Scientific Publishers Ltd.
D I F F E R E N T I A T I O N OF A G O N I S T C O N F O R M A T I O N A N D A N T A G O N I S T C O N F O R M A T I O N IN M U L T I P L E O P I O I D R E C E P T O R S
NORIO OGAWA, YASUHIDEYAMAWAKI*,HIROO KURODA*, ITARU NUKINA* and TADASHI OFUJI* Department of Neurochemistry, Institute for Neurobiology, and *Third Department of Internal Medicine, Okayama University Medical School, Okayama 700 (Japan)
(Received August 24th, 1981; Revised version received and accepted September 24th, 1981)
To differentiate the opiate (naloxone) receptor and the enkephalin receptor in rat brain, we solubilized the receptor molecules by detergent and determined the molecular weights by gel filtration. The receptor preparation was bound to [3H]naloxoneor [3H]MetS-enkephalin, and was solubilized by Triton X-100. On gel chromatographywith a Sepharose 6B column, the agonist and the antagonist conformation of opioid receptors eluted as molecules with the molecularweights of 240,000 and 120,000, and with Stokes' radii of 5.5 nm and 4.3 nm, respectively. Further, it was also disclosed that Na ÷ was bound to the antagonist conformation of opioid receptors but not to the agonist conformation.
Since the discovery of specific binding sites for opiates in the animal brain [5, 7, 10], there have been many reports about binding characteristics using crude synaptic membrane fragments. Although it has been widely accepted that opiate agonists and antagonists competitively bind to the same receptor site but in different conformational states, several reports suggest that there are differences between enkephalin receptor binding and naloxone receptor binding. That is, several opiates are less potent displacers of [3H]enkephalin binding than of [3H]opiate binding [1-3, 6, 11]. In the study reported here, we attempt to clarify the differences between the agonist conformation and the antagonist conformation of solubilized opioid receptors (OpR; authentic opiate receptor and enkephalin receptor taken together) using [3H]MetS-enkephalin and [3H]naloxone as ligands. Male Sprague-Dawley rats (200-250 g) were decapitated and the brains were immediately removed and the cerebellum discarded. Crude synaptic membranes were obtained as described previously [4]. Synaptic membranes of rat brain (15 mg protein) were incubated in a glass flask with 3.6 × 10 -8 M [3H]naloxone (spec. act. 18.5 Ci/mmol; New England Nuclear) or 6.8 × 10 -8 M [3H]MetS-enkephalin (spec. act. 38 Ci/mmol; Amersham) in the presence or absence of unlabeled naloxone (10 #M) or unlabeled MetS-enkephalin (5 #M) in a total volume of 5 ml Tris buffer (50 mM Tris-HCl buffer containing 50/~g/ml of bacitracin, pH 7.6). After incubation for 2 h in ice, 50 #1 of Triton X-100 was added to the mixture and solubilized with a 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
206 magnetic stirrer for 30 min at 4 °C. The mixture was then centrifuged at 200,000 × g for 60 min at 4 °C. Approximately 97°7o of the ligand-receptor complex of synaptic membranes was recovered in the supernatant. Three ml of the supernatant was placed on top of a Sepharose 6B column (1 × 70 cm), and the elution was carried out with cold Tris buffer supplemented by 0.1o70 Triton X-100 for protecting aggregation. One-ml fractions were collected, and the radioactivity of an aliquot of each fraction was measured on an automatic beta counter. Fig. 1A shows the gel filtration pattern of the solubilized opiate (naloxone) receptor in the absence of NaCI. The solubilized [3H]naloxone-receptor complex had three peaks (peak I, I1 and III), and was separated from free [3H]naloxone. As compared with the elution volumes of 7 marker proteins, the apparent molecular weights of 3 solubilized naloxone-receptor complex fractions were 440,000 (peak I), 240,000 (peak II) and 120,000 (peak III), respectively. The Stokes' radii of the solubilized naloxone-receptor complex fractions were estimated to be 6.3 nm for peak I, 5.5. nm for peak 11 and 4.3 nm for peak III. In the absence of Na ÷, all opiate (naloxone) receptor peaks were displaced by an excess amount of unlabeled naloxone, whereas only peak II of naloxone receptors was displaced by unlabeled MetS-enkephalin. In the presence of 100 mM NaCI, on the other hand, peak 11 showed a remarkable decrease while the peak III showed a marked increase (Fig. 1B). In the presence of Na ~, all 3 peaks of the opiate (naloxone) receptors were displaced by a large amount of unlabeled naloxone, but peak I1 was the only peak which was displaced by unlabeled MetS-enkephalin. On the other hand, the gel filtration pattern of the solubilized [3H]MetSenkephalin-receptor complex showed only a single peak with an apparent molecular weight of 240,000 with a Stokes' radius of 5.5 nm, which coincides with peak II of the naloxone-bound receptor complex (Fig. 1C). From these observations, peak II of naloxone receptors may be regarded as the agonist conformation of Op-R, and peak III, the antagonist conformation. Peak I, on the other hand, is assumed to be a receptor having nothing to do with opiates or MetS-enkephalin, since it was not affected by sodium and was not displaced by enkephalin. In order to explain the effects of Na + on Op-R binding, the allosteric model of conformational change in the opiate receptor was proposed [8, 9]. According to the model, the binding of Na ~ to its own allosteric site results in a conformational change in the receptor molecule which alters the shape of the binding site. The Na ÷altered site exhibits greater affinity towards antagonists and lower affinity towards agonists than the conformation that exists in the absence of Na + . In order to clarify whether Na ÷ is actually bound to the antagonist conformation of Op-R, the following experiment was conducted. [3H]Naloxone was allowed to combine with the receptor preparation in the presence of 100 mM NaC1 and was solubilized by Triton X-100. Gel filtration was carried out with cold Tris buffer without Na÷. The upper panel of Fig. 2 shows the elution patterns of this solubi-
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Fig. 1. Sepharose 6B gel filtration pattern of solubilized opiate (naloxone)-receptor complex (A and B) and MetS-enkephalin-receptor complex (C). Synaptic membranes were incubated in glass flasks with tritiated ligands in the absence ( - - - ) or presence of 10 #M unlabeled naloxone (----) or 5 #M unlabeled MetS-enkephalin (. . . . . ). After incubation in ice, Triton X-100 was added to the mixture and solubilized with a magnetic stirrer. In the experiment B, incubation buffer and elution buffer contained 100 mM NaCI. All other details as in the text. Results are the mean values of 3 independent experiments with less than 14% variation.
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Fig. 2. Distribution of solubilized opiate (naloxone)-receptor complex in the presence of 100 mM NaCI (upper panel) and concentration of Na in each fraction (lower panel). [3H]Naloxone was allowed to bind with the receptor preparation in the presence of 100 mM NaCI and solubilized with Triton X-100. Gel filtration was carried out with Na ' -free Tris buffer supplemented with 0. I o7o Triton X-100. All other methods and symbols are outlined in the legend to Fig. I.
lized naloxone-receptor complex and the lower panel shows the concentration of Na ÷ in each fraction. Na + was found in the same fraction as peak III while Na ÷ was not detected in the fraction o f peak II, and unbound Na ÷ was detected in the free [3H]naloxone fraction. These findings provide evidence that, like the model proposed previously [8, 9], Na + is bound to the antagonist conformation o f Op-R. As stated above, the apparent molecular size of the agonist conformation o f Op-R is bigger than that o f the antagonist conformation. This fact suggests that the binding of Na + to the agonist conformation causes the dissociation of a macromolecular subunit from Op-R, which results in Op-R changing to the antagonist conformation. It is not clear, however, whether the change of Op-R between the agonist conformation and the antagonist conformation is reversible in vivo. Further work is
209
required to clarify the nature of the agonist and antagonist conformation of Op-R molecules. This work was supported in part by grants from the Ministry of Education and from the Ministry of Health and Welfare, Japan.
1 Audigier, Y., Malforoy-Camine, B. and Schwartz, J.C., Binding of [3H]Leu-enkephalin in rat striatum: partial inhibition by morphine or naloxone, Europ. J. Pharmacol., 41 (1977) 247-248. 2 Jacquet, Y.F., Klee, W.A., Rice, K.C., Iijima, I. and Minikawa, J., Stereospecific and nonstereospecific effects of (+)- and (-)-morphine: evidence for a new class of receptors? Science, 198 (1977) 842-845. 3 Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.A., Endogenous opioid peptides: multiple agonists and receptors, Nature (Lond.), 267 (1977) 495-499. 4 0 g a w a , N., Yamawaki, Y., Kuroda, H. and Ofuji, T., Effects of bromocriptine on receptor binding of methionine-enkepalin, Neurosci. Lett., 23 (1981) 215-218. 5 Pert, C.B. and Snyder, S.H., Opiate receptor: demonstration in nervous tissue, Science, 179 (1973) 1011-1014. 6 Simantov, R., Childers, S.R. and Snyder, S.H., The opiate receptor binding interactions of [3H]methionine enkephalin, an opioid peptide, Europ. J. Pharmacol., 47 (1978) 319-331. 7 Simon, E.J., Hiller, J.M. and Edelman, 1., Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat-brain homogenate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1947-1949. 8 Simon, E.J. and Hiller, J.M., In vitro studies on opiate receptors and their ligands. Fed. Proc., 37 (1978) 141-146. 9 Snyder, S.H., Neurotransmitter and drug receptors in the brain, Biochem. Pharmacol., 24 (1975) 1371-1374. 10 Terenius, L., Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex, Acta pharmacol, toxicol., 32 (1973) 317-320. 11 Terenius, L., Opioid peptides and opiates differ in receptor selectivity, Psychoneuroendocrinology, 2 (1977) 53-58.
205
Elsevier/North-Holland Scientific Publishers Ltd.
D I F F E R E N T I A T I O N OF A G O N I S T C O N F O R M A T I O N A N D A N T A G O N I S T C O N F O R M A T I O N IN M U L T I P L E O P I O I D R E C E P T O R S
NORIO OGAWA, YASUHIDEYAMAWAKI*,HIROO KURODA*, ITARU NUKINA* and TADASHI OFUJI* Department of Neurochemistry, Institute for Neurobiology, and *Third Department of Internal Medicine, Okayama University Medical School, Okayama 700 (Japan)
(Received August 24th, 1981; Revised version received and accepted September 24th, 1981)
To differentiate the opiate (naloxone) receptor and the enkephalin receptor in rat brain, we solubilized the receptor molecules by detergent and determined the molecular weights by gel filtration. The receptor preparation was bound to [3H]naloxoneor [3H]MetS-enkephalin, and was solubilized by Triton X-100. On gel chromatographywith a Sepharose 6B column, the agonist and the antagonist conformation of opioid receptors eluted as molecules with the molecularweights of 240,000 and 120,000, and with Stokes' radii of 5.5 nm and 4.3 nm, respectively. Further, it was also disclosed that Na ÷ was bound to the antagonist conformation of opioid receptors but not to the agonist conformation.
Since the discovery of specific binding sites for opiates in the animal brain [5, 7, 10], there have been many reports about binding characteristics using crude synaptic membrane fragments. Although it has been widely accepted that opiate agonists and antagonists competitively bind to the same receptor site but in different conformational states, several reports suggest that there are differences between enkephalin receptor binding and naloxone receptor binding. That is, several opiates are less potent displacers of [3H]enkephalin binding than of [3H]opiate binding [1-3, 6, 11]. In the study reported here, we attempt to clarify the differences between the agonist conformation and the antagonist conformation of solubilized opioid receptors (OpR; authentic opiate receptor and enkephalin receptor taken together) using [3H]MetS-enkephalin and [3H]naloxone as ligands. Male Sprague-Dawley rats (200-250 g) were decapitated and the brains were immediately removed and the cerebellum discarded. Crude synaptic membranes were obtained as described previously [4]. Synaptic membranes of rat brain (15 mg protein) were incubated in a glass flask with 3.6 × 10 -8 M [3H]naloxone (spec. act. 18.5 Ci/mmol; New England Nuclear) or 6.8 × 10 -8 M [3H]MetS-enkephalin (spec. act. 38 Ci/mmol; Amersham) in the presence or absence of unlabeled naloxone (10 #M) or unlabeled MetS-enkephalin (5 #M) in a total volume of 5 ml Tris buffer (50 mM Tris-HCl buffer containing 50/~g/ml of bacitracin, pH 7.6). After incubation for 2 h in ice, 50 #1 of Triton X-100 was added to the mixture and solubilized with a 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
206 magnetic stirrer for 30 min at 4 °C. The mixture was then centrifuged at 200,000 × g for 60 min at 4 °C. Approximately 97°7o of the ligand-receptor complex of synaptic membranes was recovered in the supernatant. Three ml of the supernatant was placed on top of a Sepharose 6B column (1 × 70 cm), and the elution was carried out with cold Tris buffer supplemented by 0.1o70 Triton X-100 for protecting aggregation. One-ml fractions were collected, and the radioactivity of an aliquot of each fraction was measured on an automatic beta counter. Fig. 1A shows the gel filtration pattern of the solubilized opiate (naloxone) receptor in the absence of NaCI. The solubilized [3H]naloxone-receptor complex had three peaks (peak I, I1 and III), and was separated from free [3H]naloxone. As compared with the elution volumes of 7 marker proteins, the apparent molecular weights of 3 solubilized naloxone-receptor complex fractions were 440,000 (peak I), 240,000 (peak II) and 120,000 (peak III), respectively. The Stokes' radii of the solubilized naloxone-receptor complex fractions were estimated to be 6.3 nm for peak I, 5.5. nm for peak 11 and 4.3 nm for peak III. In the absence of Na ÷, all opiate (naloxone) receptor peaks were displaced by an excess amount of unlabeled naloxone, whereas only peak II of naloxone receptors was displaced by unlabeled MetS-enkephalin. In the presence of 100 mM NaCI, on the other hand, peak 11 showed a remarkable decrease while the peak III showed a marked increase (Fig. 1B). In the presence of Na ~, all 3 peaks of the opiate (naloxone) receptors were displaced by a large amount of unlabeled naloxone, but peak I1 was the only peak which was displaced by unlabeled MetS-enkephalin. On the other hand, the gel filtration pattern of the solubilized [3H]MetSenkephalin-receptor complex showed only a single peak with an apparent molecular weight of 240,000 with a Stokes' radius of 5.5 nm, which coincides with peak II of the naloxone-bound receptor complex (Fig. 1C). From these observations, peak II of naloxone receptors may be regarded as the agonist conformation of Op-R, and peak III, the antagonist conformation. Peak I, on the other hand, is assumed to be a receptor having nothing to do with opiates or MetS-enkephalin, since it was not affected by sodium and was not displaced by enkephalin. In order to explain the effects of Na + on Op-R binding, the allosteric model of conformational change in the opiate receptor was proposed [8, 9]. According to the model, the binding of Na ~ to its own allosteric site results in a conformational change in the receptor molecule which alters the shape of the binding site. The Na ÷altered site exhibits greater affinity towards antagonists and lower affinity towards agonists than the conformation that exists in the absence of Na + . In order to clarify whether Na ÷ is actually bound to the antagonist conformation of Op-R, the following experiment was conducted. [3H]Naloxone was allowed to combine with the receptor preparation in the presence of 100 mM NaC1 and was solubilized by Triton X-100. Gel filtration was carried out with cold Tris buffer without Na÷. The upper panel of Fig. 2 shows the elution patterns of this solubi-
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Fig. 1. Sepharose 6B gel filtration pattern of solubilized opiate (naloxone)-receptor complex (A and B) and MetS-enkephalin-receptor complex (C). Synaptic membranes were incubated in glass flasks with tritiated ligands in the absence ( - - - ) or presence of 10 #M unlabeled naloxone (----) or 5 #M unlabeled MetS-enkephalin (. . . . . ). After incubation in ice, Triton X-100 was added to the mixture and solubilized with a magnetic stirrer. In the experiment B, incubation buffer and elution buffer contained 100 mM NaCI. All other details as in the text. Results are the mean values of 3 independent experiments with less than 14% variation.
208
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Fig. 2. Distribution of solubilized opiate (naloxone)-receptor complex in the presence of 100 mM NaCI (upper panel) and concentration of Na in each fraction (lower panel). [3H]Naloxone was allowed to bind with the receptor preparation in the presence of 100 mM NaCI and solubilized with Triton X-100. Gel filtration was carried out with Na ' -free Tris buffer supplemented with 0. I o7o Triton X-100. All other methods and symbols are outlined in the legend to Fig. I.
lized naloxone-receptor complex and the lower panel shows the concentration of Na ÷ in each fraction. Na + was found in the same fraction as peak III while Na ÷ was not detected in the fraction o f peak II, and unbound Na ÷ was detected in the free [3H]naloxone fraction. These findings provide evidence that, like the model proposed previously [8, 9], Na + is bound to the antagonist conformation o f Op-R. As stated above, the apparent molecular size of the agonist conformation o f Op-R is bigger than that o f the antagonist conformation. This fact suggests that the binding of Na + to the agonist conformation causes the dissociation of a macromolecular subunit from Op-R, which results in Op-R changing to the antagonist conformation. It is not clear, however, whether the change of Op-R between the agonist conformation and the antagonist conformation is reversible in vivo. Further work is
209
required to clarify the nature of the agonist and antagonist conformation of Op-R molecules. This work was supported in part by grants from the Ministry of Education and from the Ministry of Health and Welfare, Japan.
1 Audigier, Y., Malforoy-Camine, B. and Schwartz, J.C., Binding of [3H]Leu-enkephalin in rat striatum: partial inhibition by morphine or naloxone, Europ. J. Pharmacol., 41 (1977) 247-248. 2 Jacquet, Y.F., Klee, W.A., Rice, K.C., Iijima, I. and Minikawa, J., Stereospecific and nonstereospecific effects of (+)- and (-)-morphine: evidence for a new class of receptors? Science, 198 (1977) 842-845. 3 Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.A., Endogenous opioid peptides: multiple agonists and receptors, Nature (Lond.), 267 (1977) 495-499. 4 0 g a w a , N., Yamawaki, Y., Kuroda, H. and Ofuji, T., Effects of bromocriptine on receptor binding of methionine-enkepalin, Neurosci. Lett., 23 (1981) 215-218. 5 Pert, C.B. and Snyder, S.H., Opiate receptor: demonstration in nervous tissue, Science, 179 (1973) 1011-1014. 6 Simantov, R., Childers, S.R. and Snyder, S.H., The opiate receptor binding interactions of [3H]methionine enkephalin, an opioid peptide, Europ. J. Pharmacol., 47 (1978) 319-331. 7 Simon, E.J., Hiller, J.M. and Edelman, 1., Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat-brain homogenate, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 1947-1949. 8 Simon, E.J. and Hiller, J.M., In vitro studies on opiate receptors and their ligands. Fed. Proc., 37 (1978) 141-146. 9 Snyder, S.H., Neurotransmitter and drug receptors in the brain, Biochem. Pharmacol., 24 (1975) 1371-1374. 10 Terenius, L., Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex, Acta pharmacol, toxicol., 32 (1973) 317-320. 11 Terenius, L., Opioid peptides and opiates differ in receptor selectivity, Psychoneuroendocrinology, 2 (1977) 53-58.