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• W2022595536 abstract "We have examined the binding of two radioligands ([3H]spiperone and [3H]raclopride) to D2 dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites, whereas in the absence of sodium ions [3H]raclopride labeled about half the number of sites labeled by [3H]spiperone. In competition experiments in the absence of sodium ions, however, raclopride was able to inhibit [3H]spiperone binding fully. In saturation analyses with [3H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a dimer, and in the absence of sodium ions, raclopride exerts negative cooperativity across the dimer both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here. We have examined the binding of two radioligands ([3H]spiperone and [3H]raclopride) to D2 dopamine receptors expressed in Chinese hamster ovary cells. In saturation binding experiments in the presence of sodium ions, both radioligands labeled a similar number of sites, whereas in the absence of sodium ions [3H]raclopride labeled about half the number of sites labeled by [3H]spiperone. In competition experiments in the absence of sodium ions, however, raclopride was able to inhibit [3H]spiperone binding fully. In saturation analyses with [3H]spiperone in the absence of sodium ions raclopride exerted noncompetitive effects, decreasing the number of sites labeled by the radioligand. These data are interpreted in terms of a model where the receptor exists as a dimer, and in the absence of sodium ions, raclopride exerts negative cooperativity across the dimer both for its own binding and the binding of spiperone. A model of the receptor has been produced that provides a good description of the experimental phenomena described here. G-protein-coupled receptor N-methyl-d-glucamine analysis of variance Chinese hamster ovary The G-protein-coupled receptors (GPCRs)1 constitute a large family of proteins responsible for the transduction of a wide range of signals (e.g. hormones, neurotransmitters, odorants, light, etc.) via G-proteins (1Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). GPCRs possess a common structural motif of seven α-helical membrane-spanning domains, and it is often assumed that the functional unit (i.e. the ligand binding and G-protein interaction domains) of the GPCR is wholly contained in a single polypeptide. Indeed, most models of GPCR function assume a monomeric receptor interacting with the G-protein (see, for example, Ref. 2Samama O. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar). Several lines of evidence, however, suggest that the some GPCRs may exist in dimeric or oligomeric forms. Immunoblotting has in several cases revealed species corresponding not only to the predicted molecular weight of the receptor but also to multiples of the molecular weight. Bands corresponding to approximately twice the predicted molecular weight of the receptor have been interpreted as homodimers for several receptors including D2 dopamine (3Ng G.Y.K. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (240) Google Scholar, 4Zawarynski P. Tallerico T. Seeman P. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1998; 441: 383-386Crossref PubMed Scopus (115) Google Scholar), D3 dopamine (5Nimchinsky E.A. Hof P.R. Janssen W.G.M. Morrison J.H. Schmauss C. J. Biol. Chem. 1997; 272: 29229-29237Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), β2-adrenergic (6Hebert T. Moffett S. Morello J.-P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar), substance P (7Schreurs J. Yamamoto R. Lyons J. Munemitsu S. Conroy L. Clark R. Takeda Y. Krause J.E. Innis M. J. Neurochem. 1995; 64: 1622-1631Crossref PubMed Scopus (16) Google Scholar), opiate (8Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar) and M1 and M2 muscarinic acetylcholine receptors (9Parker E.M. Kameyama K. Higashijima T. Ross E.M. J. Biol. Chem. 1991; 266: 519-527Abstract Full Text PDF PubMed Google Scholar). Co-immunoprecipitation has also been used to demonstrate homodimer formation for the β2-adrenergic receptor (6Hebert T. Moffett S. Morello J.-P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar), opiate receptor (8Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar), and somatostatin SSTR5 receptor (10Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (734) Google Scholar). In some cases, formation of heterodimers of GPCRs has been reported with differences in the pharmacological properties of the receptors in the heterodimer (e.g. GABAB receptor isoforms (11Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (906) Google Scholar, 12Kaupmann K. Malitschek B. Schuler B. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shiegemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (998) Google Scholar, 13White J.H. Wise A. Main M.J. Green A. Fraiser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (992) Google Scholar), δ and κ opiate receptors (14Jordan B.A. Devi L.A. Nature. 1999; 396: 697-700Crossref Scopus (958) Google Scholar), dopamine, and somatostatin receptors (10Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (734) Google Scholar)). Further evidence for interaction of GPCRs was provided by Maggioet al. (15Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (295) Google Scholar), who created two chimeric receptors α2/M3 and M3/α2, in which the C-terminal regions (transmembrane domains VI and VII) were exchanged between the α2C adrenergic and M3muscarinic receptors. Expression of either chimera alone did not result in any detectable binding of typical radiolabeled muscarinic or adrenergic ligands. However, cotransfection of COS7 cells with both chimeras resulted in the appearance of binding activity corresponding to both native receptors. This has lead to the proposal that some GPCRs might form domain-swapped dimers (16Gouldson P.R. Snell C.R. Bywater R.P. Higgs C. Reynolds C.A. Protein Eng. 1998; 1: 1181-1193Crossref Scopus (112) Google Scholar). Evidence for GPCR interaction in cells has been obtained by expressing GPCRs fused to different chromophores. Transfer of energy between the chromophores has been shown for the β2-adrenergic receptor (17Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar) and somatostatin SSTR5 receptor (10Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (734) Google Scholar) and provides good evidence for the close proximity of the two molecules of GPCRs. Some radioligand binding studies suggest differences in the number of binding sites labeled by different radioligands. At M2muscarinic receptors, the antagonist [3H]QNB labeled twice as many sites as did [3H]AF-DX 384 orN-[3H]methylscopolamine under certain conditions (18Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). These data were interpreted in terms of a model where the receptor exists as a tetramer. The D2 dopamine receptor is of interest in this regard. Several studies suggest that the substituted benzamide radioligand [3H]nemonapride can label more D2 receptor sites in radioligand binding studies than the butyrophenone [3H]spiperone (3Ng G.Y.K. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (240) Google Scholar, 19Niznik H.B. Grigoriadis D.E. Pri-Bar I. Buchman O. Seeman P. Naunyn-Schmiedeberg's Arch. Pharmacol. 1985; 329: 333-343Crossref PubMed Scopus (100) Google Scholar, 20Terai M. Hidaka K. Nakamura Y. Eur. J. Pharmacol. 1989; 173: 177-182Crossref PubMed Scopus (128) Google Scholar, 21Seeman P. Guan H.-C. Civelli O. van Tol H.H.M. Sunahara R.K. Niznik H.B. Eur. J. Pharmacol. 1992; 227: 139-146Crossref PubMed Scopus (62) Google Scholar), although this was not seen in all reports (22Vile J.M. D'Souza U.M. Strange P.G. J. Neurochem. 1995; 64: 940-943Crossref PubMed Scopus (22) Google Scholar, 23Malmberg A. Jerning E. Mohell N. Eur. J. Pharmacol. 1996; 303: 123-128Crossref PubMed Scopus (28) Google Scholar). For D2dopamine receptors expressed in recombinant cells, Seeman et al. (21Seeman P. Guan H.-C. Civelli O. van Tol H.H.M. Sunahara R.K. Niznik H.B. Eur. J. Pharmacol. 1992; 227: 139-146Crossref PubMed Scopus (62) Google Scholar) reported that [3H]raclopride labeled more D2 dopamine receptor sites than did [3H]spiperone, although Malmberg et al. (23Malmberg A. Jerning E. Mohell N. Eur. J. Pharmacol. 1996; 303: 123-128Crossref PubMed Scopus (28) Google Scholar) were unable to replicate these findings. Interestingly, Hall et al. (24Hall H. Wedel I. Halldin C. Kopp J. Farde L. J. Neurochem. 1990; 55: 2048-2057Crossref PubMed Scopus (98) Google Scholar) found that the number of sites labeled by [3H]raclopride in rat striatal membranes was dependent on the conditions used. [3H]Raclopride labeled more sites in the presence of sodium ions than in their absence. The number of sites labeled by [3H]spiperone was, however, unaffected by sodium ions and was similar to the number of sites labeled by [3H]raclopride in the presence of sodium ions. Theodorouet al. (25Theodorou A.E. Jenner P. Marsden C.D. Life Sci. 1983; 32: 1243-1254Crossref PubMed Scopus (20) Google Scholar) found that another substituted benzamide, [3H]sulpiride, also detected more D2 receptor binding sites in rat striatum in the presence of sodium ions than it did in their absence, and similar observations have been made for [3H]raclopride binding to the related D3dopamine receptor expressed in recombinant cells (26Malmberg A. Jackson D.M. Eriksson A. Mohell N. Mol. Pharmacol. 1993; 43: 749-754PubMed Google Scholar). These observations are not consistent with the labeling by these radioligands of single populations of independent D2dopamine receptors. They are more consistent with the labeling of oligomeric arrays with different degrees of cooperativity between the monomeric units, depending on assay conditions (18Wreggett K.A. Wells J.W. J. Biol. Chem. 1995; 270: 22488-22499Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 27Strange P.G. Trends Pharmacol. Sci. 1994; 15: 317-319Abstract Full Text PDF PubMed Scopus (32) Google Scholar). Because of the importance of the D2 receptor in the actions of the antipsychotic drugs, we have examined this phenomenon in more detail. In this paper, therefore, we have studied the binding of two radioligands ([3H]spiperone and [3H]raclopride) to D2 dopamine receptors expressed in CHO cells and provide evidence for the formation of homodimers for this receptor. [3H]Spiperone (15–30 Ci/mmol) was purchased from Amersham Pharmacia Biotech, and [3H]raclopride (60–86 Ci/mmol) was purchased from PerkinElmer Life Sciences. S-(−)-Sulpiride, haloperidol, and butaclamol were purchased from RBI (Natick, MA). All other materials were obtained from commercial sources and were of the highest available purity. CHO cells expressing the human D2(short) dopamine receptor (28Gardner B.R. Hall D.A. Strange P.G. J. Neurochem. 1997; 69: 2589-2598Crossref PubMed Scopus (36) Google Scholar) were grown as monolayers in RPMI medium supplemented with 2 mm glutamine, 200 μg/ml active Geneticin, and 5% fetal calf serum at 37 °C in a moist, 5% CO2 atmosphere. Cells were washed with 5 ml of ice-cold buffer A (20 mm HEPES, 1 mm EDTA (free acid) 1 mm EGTA, pH 7.4, with KOH), removed from the flask by gentle shaking with 2-mm diameter glass beads in 5 ml of buffer A, and homogenized with 30 strokes of a Dounce homogenizer. The homogenate was centrifuged at 260 × g for 10 min, and the resulting supernatant centrifuged at 48,000 × g for 1 h at 4 °C. The pellet was resuspended in ice-cold buffer A to ∼5–10 mg/ml, and aliquots were stored at −70 °C. Protein concentration was determined by the method of Lowry et al.(29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 263-273Abstract Full Text PDF Google Scholar). Control experiments were performed in buffer A, while 100 mm NaCl or 100 mm N-methyl-d-glucamine (NMDG) was included in the buffer in order to determine the effects of sodium ions or ionic strength. Total and nonspecific binding were defined in the presence of 3 μm (−)-butaclamol and 3 μm (+)-butaclamol, respectively. [3H]raclopride saturation binding experiments were performed in a total volume of 0.5 ml using 3–15 μg of membrane protein per tube and 10 concentrations of [3H]raclopride typically ranging between 20 pm and 10 nm. [3H]Spiperone saturation binding experiments were performed in total volumes of either 1 or 10 ml, both using 10–30 μg of membrane protein/tube and 10 concentrations of the radioligand, typically between 10 pm and 5 nm for 1-ml saturations, or 16 concentrations between 1 pm and 1 nm for 10-ml experiments. In experiments that included raclopride or haloperidol, the range of [3H]spiperone concentrations was varied according to the apparentKd values obtained in initial experiments. Each experiment was performed in triplicate and incubated at 25 °C for 3 h ([3H]raclopride and 1-ml [3H]spiperone experiments) or 7 h (10-ml [3H]spiperone experiments), by which time the radioligands had reached equilibrium. Experiments were terminated by rapid filtration through Whatman GF/C glass fiber filters using a Brandel cell harvester followed with four washes of 3 ml of ice-cold phosphate-buffered saline (140 mm NaCl, 10 mmKCl, 1.5 mm KH2PO4, 8 mm Na2HPO4). Filter discs were soaked in 2 ml each of Optiphase Hi-Safe 3 (Wallac) for at least 6 h before radioactivity was determined by liquid scintillation spectroscopy. A range of concentrations of the competing ligand were incubated with 10–30 μg of membranes and a fixed concentration of [3H]spiperone in triplicate for 3 h at 25 °C before harvesting as described above. Total and nonspecific binding were defined in the presence of 3 μm (−)-butaclamol and 3 μm (+)-butaclamol, respectively. Data were analyzed using Prizm (GraphPad, San Diego CA). In saturation experiments, specifically and nonspecifically bound [3H]spiperone were calculated from saturation data using the method of Golds et al. (30Golds P.R. Przylo F.R. Strange P.G. Br. J. Pharmacol. 1980; 68: 541-549Crossref PubMed Scopus (62) Google Scholar), which makes a correction for the depletion of the radioligand. Data were fitted to equations describing one- or two-site binding models, and the best fit was determined using an F-test. Competition experiments were fitted to four-parameter logistic equations, and the best fit between a variable Hill coefficient and a Hill coefficient fixed to unity was determined using an F-test. In the analysis of the competition data, the free radioligand concentration was taken as the added minus total bound in the absence of competitor. The amount bound will be in fact be different at the top and bottom of the competition curve. The total bound was, however, ∼12 and ∼1% of the added radioligand in the absence and presence, respectively, of saturating concentrations of competitor ([3H]spiperone, ∼0.25 nm). The total bound radioligand in the absence of competitor was <10% for the higher radioligand concentrations used. The effect of this correction on estimates of Ki is slight (∼5% at 0.25 nm radioligand). The statistical significance of difference between data was determined at the 0.05 level, using ANOVA or Student's t test, as appropriate. Ki and Kd values were first converted to the respective normally distributed negative logarithm (pKi or pKd). Mean values are quoted with the respective S.E. Saturation binding studies were performed on human D2(short) dopamine receptors expressed in membrane preparations from recombinant CHO cells. [3H]raclopride binding in the absence of sodium ions (“control” conditions) revealed a single population of binding sites with a Kd of 1.1 nm and Bmax of 0.84 pmol/mg of membrane protein (Fig. 1 and Table I). In the presence of 100 mm NaCl, the data were also best described by a single population of binding sites in which theKd for [3H]raclopride was decreased significantly to 0.23 nm and Bmaxsignificantly increased to 1.63 pmol/mg. 100 mm NMDG was used as a control for changes in ionic strength, and experiments performed under these conditions gave values for Kdof 1.2 nm and Bmax of 0.89 pmol/mg, which did not differ significantly from the control values (ANOVA,p > 0.05).Table IThe binding of [3 H]spiperone and [3 H]raclopride to D2 dopamine receptorsRadioligandControlNa+NMDGControl (1 ml spiperone)Spiperone (10 ml)pKd10.84 ± 0.11 (7)10.87 ± 0.05 (4)10.76 ± 0.13 (4)10.20 ± 0.04 (52)Kd(nm)0.0150.0130.0170.063Bmax (pmol/mg)1.21 ± 0.11 (3)1.99 ± 0.13 (4)1.61 ± 0.06 (4)1.56 ± 0.15 (3)[Receptor] (pm)3.66.04.846.8RaclopridepKd8.97 ± 0.11 (7)9.63 ± 0.06 (7)8.92 ± 0.09 (6)Kd(nm)1.10.231.2Bmax (pmol/mg)0.84 ± 0.16 (4)1.63 ± 0.27 (4)0.89 ± 0.18 (3)[Receptor] (pm)25.248.926.7The binding of [3H]spiperone and [3H]raclopride to D2(short) dopamine receptors expressed in CHO cells was determined as described under “Experimental Procedures.”Kd and Bmax values were determined for both radioligands, and pKd andBmax values are given as the mean ± S.E. with the number of experiments in parentheses. The correspondingKd value is given as nm. [3H]Spiperone saturation assays were performed in a total volume of 10 ml in the presence of 100 mm NaCl or 100 mm NMDG or in the absence of either ion and were also performed in a total volume of 1 ml in the absence of ions. [3H]Raclopride saturation assays were performed in 0.5 ml volumes under the same ionic conditions as [3H]spiperone.Bmax determinations were all performed on the same preparation of membranes in order to allow comparisons, whereasKd values are from different preparations. The concentration of receptor binding sites in the assays is given in pm. Open table in a new tab The binding of [3H]spiperone and [3H]raclopride to D2(short) dopamine receptors expressed in CHO cells was determined as described under “Experimental Procedures.”Kd and Bmax values were determined for both radioligands, and pKd andBmax values are given as the mean ± S.E. with the number of experiments in parentheses. The correspondingKd value is given as nm. [3H]Spiperone saturation assays were performed in a total volume of 10 ml in the presence of 100 mm NaCl or 100 mm NMDG or in the absence of either ion and were also performed in a total volume of 1 ml in the absence of ions. [3H]Raclopride saturation assays were performed in 0.5 ml volumes under the same ionic conditions as [3H]spiperone.Bmax determinations were all performed on the same preparation of membranes in order to allow comparisons, whereasKd values are from different preparations. The concentration of receptor binding sites in the assays is given in pm. Saturation binding experiments with [3H]spiperone were performed under two conditions: large volume (10-ml) assays to minimize the extent of ligand depletion and, more routinely, smaller volume (1-ml) assays. In control conditions, in both assay volumes a single population of binding sites was found, with Bmaxvalues of 1.2 pmol/mg and 1.6 pmol/mg for 10-ml and 1-ml volume assays, respectively (Table I). These values are not significantly different (ANOVA, p > 0.05). The dissociation constant of [3H]spiperone, 15 pm, was significantly lower when determined in 10-ml assays, compared with the value, 63 pm, found in 1-ml assays (Table I). In [3H]spiperone saturation assays performed in a 10-ml volume, the Bmax value found in the presence of 100 mm NaCl, 2 pmol/mg, was not significantly different from that found in the presence of 100 mm NMDG, 1.6 pmol/mg (Table I) (ANOVA, p > 0.05). In the absence of monovalent cations (control conditions), theBmax value of [3H]spiperone was 1.2 pmol/mg, which, while significantly less than the value in the presence of sodium ions, was not different from the value in the presence of NMDG (ANOVA). The dissociation constant of [3H]spiperone was unaffected by either sodium ions or NMDG (Table I). Parallel [3H]spiperone saturation binding experiments (1-ml volume) were performed in the absence of sodium ions and in the absence and presence of raclopride (10 μm). In the absence of raclopride, theBmax value for [3H]spiperone was 1.7 ± 0.1 pmol/mg (n = 3). Including 10 μm raclopride in the [3H]spiperone saturation assays increased the apparent Kd value ∼116-fold and significantly reduced the Bmaxto 1.3 ± 0.1 pmol/mg (n = 3) (Fig.2 A). A range of raclopride concentrations was included in 1-ml volume [3H]spiperone saturation binding experiments. Analysis of the data according to the method of Schild gave rise to the plot in Fig. 2 b. The pA2 value for raclopride derived from these data was 7.12, corresponding to 76 nm. The pA2 value predicted from a simple competitive model, using the affinity of [3H]raclopride given in Table I, was 8.97, corresponding to 1.1 nm, a difference of 71-fold from the measured value. Similar experiments were performed, in the absence of sodium ions or NMDG, using haloperidol as the competing ligand. Fig.3 A shows that haloperidol had no significant effect on the Bmax of [3H]spiperone, while the pA2 value of 9.32 (0.48 nm) from the Schild plot (Fig. 3 B) agreed very well with the value predicted by a simple competitive model: pA2 = 9.45 (0.36 nm) (31Hoare S.R.J. Strange P.G. Mol. Pharmacol. 1996; 50: 1295-1308PubMed Google Scholar). 2D. Armstrong and P. G. Strange, unpublished results. In the presence of 100 mm NaCl, no significant effect of raclopride on [3H]spiperone Bmaxwas found (Fig. 4 A). Analysis of the data according to the method of Schild (Fig. 4 B) resulted in a pA2 value for raclopride in the presence of sodium ions of 8.79 (1.6 nm). This is 6.9-fold greater than the value of 9.63 (0.23 nm) predicted by a simple competitive model. Raclopride was competed against different [3H]spiperone concentrations between 0.2 and 4.3 nm in the absence of sodium ions, and representative competition curves are shown in Fig. 5. Raclopride inhibited [3H]spiperone binding to within 5% of the level of nonspecific binding defined in the presence of 3 μm (+)-butaclamol. The data from 14 experiments (at different [3H]spiperone concentrations) were fitted well by a sigmoidal competition curve with a Hill coefficient of unity. The mean Ki of raclopride derived from all of these data was 19.5 nm (7.71 ± 0.06, pKi ± S.E., n = 14). Competition binding experiments with raclopride and 0.25 nm [3H]spiperone were also performed in the presence of 100 mm NaCl, yielding aKi of 0.95 nm (9.02 ± 0.05, pKi ± S.E., n = 3) or in the presence of 100 mm NMDG, resulting in aKi of 11.3 nm (7.95 ± 0.08, pKi ± S.E., n = 3). [3H]spiperone binding was inhibited by raclopride to the level of nonspecific binding under both of these conditions. In this study, we have examined the binding of two ligands (raclopride and spiperone) to human D2(short) dopamine receptors expressed in CHO cells. Saturation and competition experiments have been used to show that the D2 receptor functions as an oligomer and that the properties of this oligomeric receptor may be modulated by the ionic conditions. In saturation binding assays, sodium ions were found to exert allosteric effects on [3H]raclopride binding to recombinant human D2(short) dopamine receptors. The presence of sodium ions was found to increase the affinity of [3H]raclopride for the D2 receptor, and this phenomenon has been described extensively before for drugs of the substituted benzamide class (25Theodorou A.E. Jenner P. Marsden C.D. Life Sci. 1983; 32: 1243-1254Crossref PubMed Scopus (20) Google Scholar, 32Steffanini E. Marchisio A.M. Devoto P. Vernaleone F. Collu R. Spano P.F. Brain Res. 1980; 198: 229-233Crossref PubMed Scopus (65) Google Scholar, 33Imafuku J. Brain Res. 1987; 402: 331-338Crossref PubMed Scopus (40) Google Scholar, 34Reader T.A. Boulianne S. Molina-Holgado E. Dewar K.M. Biochem. Pharmacol. 1990; 40: 1739-1746Crossref PubMed Scopus (15) Google Scholar, 35Neve K.A. Mol. Pharmacol. 1991; 39: 570-578PubMed Google Scholar, 36Neve K.A. Cox B.A. Henningsen R.A. Spanoyannis A. Neve R.L. Mol. Pharmacol. 1991; 39: 737-739Google Scholar, 37Neve K.A. Henningsen R.A. Kinzie J.M. De Paulis T. Schmidt D.E. Kessler R.M. Janowsky A. J. Pharmacol. Exp. Ther. 1990; 252: 1108-1116PubMed Google Scholar, 38Schetz J.A. Chu A. Sibley D.R. J. Pharmacol. Exp. Ther. 1999; 289: 956-964PubMed Google Scholar). In the present study, the dissociation constant decreased from 1.1 nm approximately 5-fold to 0.23 nm. The effect was specific to sodium ions, since the presence of an equal concentration (100 mm) of NMDG as a control for changes in ionic strength had no appreciable effect on the Kd of [3H]raclopride. Sodium ions also exerted a second effect on [3H]raclopride binding to D2 receptors by changing the number of binding sites that were labeled (Bmax). A 2-fold increase inBmax was found in the presence of sodium ions as compared with the absence of sodium ions. Again, there was no appreciable effect of NMDG compared with the absence of ions. This effect of sodium ions on the number of sites labeled by [3H]raclopride has been noticed before in studies of D2 dopamine receptors in the brain and in recombinant cells (24Hall H. Wedel I. Halldin C. Kopp J. Farde L. J. Neurochem. 1990; 55: 2048-2057Crossref PubMed Scopus (98) Google Scholar, 38Schetz J.A. Chu A. Sibley D.R. J. Pharmacol. Exp. Ther. 1999; 289: 956-964PubMed Google Scholar) but not analyzed further. In contrast to [3H]raclopride, the Kdof [3H]spiperone for binding to D2 receptors was unaffected by the presence or absence of sodium ions, in agreement with many other reports (19Niznik H.B. Grigoriadis D.E. Pri-Bar I. Buchman O. Seeman P. Naunyn-Schmiedeberg's Arch. Pharmacol. 1985; 329: 333-343Crossref PubMed Scopus (100) Google Scholar, 24Hall H. Wedel I. Halldin C. Kopp J. Farde L. J. Neurochem. 1990; 55: 2048-2057Crossref PubMed Scopus (98) Google Scholar, 35Neve K.A. Mol. Pharmacol. 1991; 39: 570-578PubMed Google Scholar, 39Schetz J.A. Sibley D.R. J. Neurochem. 1997; 68: 1990-1997Crossref PubMed Scopus (66) Google Scholar). It should be noted that experimental design can be important in determining the affinity of some high affinity radioligands. Here an assay volume of 1 ml gave a value for the Kd of [3H]spiperone of ∼63 pm. When assays were performed to minimize ligand depletion via binding to receptor and nonspecifically to tissue (i.e. use of a 10-ml total volume, in which there is 10-fold more radioligand present but the same amount of receptor), this experimental protocol provided an estimate of Kd for [3H]spiperone of 15 pm. Even under these conditions, there is some depletion at the lower concentrations of radioligand, but the estimate of Kd agrees well with determinations of Kd for spiperone designed to eliminate depletion artifacts (23Malmberg A. Jerning E. Mohell N. Eur. J. Pharmacol. 1996; 303: 123-128Crossref PubMed Scopus (28) Google Scholar). The effects of such artifacts on determination of Kd values have been discussed in detail elsewhere (23Malmberg A. Jerning E. Mohell N. Eur. J. Pharmacol. 1996; 303: 123-128Crossref PubMed Scopus (28) Google Scholar, 40Hulme E.C. Birdsall N.J.M. Hulme E.C. Receptor-Ligand Interactions: A Practical Approach. Oxford University Press, Oxford1992: 63-176Google Scholar, 41Strange P.G. Neuropsychopharmacology. 1997; 16: 122-166Google Scholar, 42Zahniser N.R. Dubocovich M.L. J. Pharmacol. Exp. Ther. 1983; 227: 592-599PubMed Google Scholar, 43Seeman P. Ulpian C. Wreggett K.A. Wells J.A. J. Neurochem. 1984; 43: 221-235Crossref PubMed Scopus (160) Google Scholar). The effect of sodium ions on the number of binding sites labeled by [3H]spiperone was less than for [3H]raclopride. Bmax values for [3H]spiperone were similar in the presence of sodium ions, in the presence of NMDG, and in the absence of monovalent cations (in 1-ml assays) and similar to the Bmax seen for [3H]raclopride in the presence of sodium ions. In 10-ml assays in the absence of monovalent cations, some reduction inBmax was seen for [3H]spiperone. These data suggest that the Bmax of [3H]spiperone is less sensitive to monovalent cations than the Bmax of [3H]raclopride, but from the data of Table I it appears that the binding of [3H]spiperone is not completely insensitive to the effects of sodium ions. An important comparison can be made between theBmax values for the two ra" @default.
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• W2022595536 title "Dopamine D2 Receptor Dimer Formation" @default.
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