Life Sciences, Vol. Printed in the USA
51, pp.
1509-1516
Pergamon
Press
AN ANTIBODYTO DOPAMINED2 RECEPTORINHIBITSDOPAMINEANTAGONISTAND AGONIST BINDINGTO D O P A M I D2 RECEPTORc D N A TRANSFECTEDMOUSEFIBROBLASTCELLS. Shakeel M. Farooqui* and Chandan Prasad**.
Laboratory ofNeurosciences Pennington Biomedical Research Center. Baton Rouge, LA 70808 and **Department o f Medicine, LSUMC, New Orleans, L A 70112 (Received
in final
form September
4, 1992)
SUMMARY A polyclonal antibody to dopamine D2 receptor (D2-rece~otor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [aH]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM091512 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 + 5.26 and 76.20 + 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltkand Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB ceils with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor
The nucleotide sequence for the dopamine D2 receptor (D2-receptor) gene suggests that molecular weight of the core D2-receptor protein should be around 46 kDa (1, 2). However, past studies devoted to purification of the functional D2-receptor (the receptor capable of binding antagonists or agonists) suggest an apparent molecular weight of 71 kDa to 220 kDa (3, 4, 5, 6, 7, 8). To facilitate the examination of the chemical basis for the apparent differences in the molecular weights of "core" and "functional" D2receptors, we have raised and characterized both monoclonal and polyclonal antibodies to D2 receptors (7, 8). The polyclonal antibody to an amino terminal (amino acid 23-34) D2-receptor peptide was found to selectively inhibit the binding of [3H]YM-09151-2 or 125I-azido-NAPS to striatai membrane (7); furthermore both 125I-azido NAPS and one of the polyclonal antibody label a protein of the same molecular weight (7). Although the above data suggest a specificity of the antibody to D2-receptor, it is * Correspondance and request for reprints to: Shakeel M. Farooqui, Laboratory of Neurosciences, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1510
Antibody to Dopamine D2 Receptor
Vol. 51, No. 19, 1992
conceivable that the antibody may also label some non-D2-receptor protein(s). For example, both [3H]YM09151-2 and 125I-azido-NAPS are known to label 5-HT as well as other dopamine receptor subtypes (9, 10, 11); in addition, both monoclonal and polyclonal antibodies recognize multiple striatal proteins ranging in molecular weights between 46kDa to 220 kDa (7,8). Therefore, it was of interest to examine the specificity of D2-receptor antibody using a system devoid of these interfering receptors. To this end, we examined the inhibition of the binding of a D2receptor antagonist, (+)-cis-N-(1-benzyi-2-[3H] -methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4methylamino-benzamide, [3H]YM09151-2 (12) and an agonist, (+)-2-(N-phenylethyl-N-porpyl)amino-5hydroxytetralin fluorescien, PPHT-fluorescein (13) to Ltk- cells (normally devoid of dopaminergic and serotonergic receptors) transfected with dopamine receptor DNA. The results of this study show that D2receptor antibody inhibits both the antagonist and agonist binding to the D2-receptor expressing cells and therefore, we conclude that the polyclonal peptide antibody (amino acid 23-34) is highly specific for D2receptor. Since PPHT-fluorescein binds only to the transfected cells, a relatively pure population of cells expressing D2-receptor can be obtained by sorting the cells for PPHT-fluorescence in a fluorescence activated cell sorter (FACS).
Materials and Methods Polyclonal antibody: The properties and characterization of the polyclonal D2-receptor antibody (pep I) has been described elsewhere (7, 8, 14). The pep I refers to the peptide containing amino acid 23 to 34 in rat D2-receptor sequence. The antibody was reactive to homologous peptide antigen (titer, 1:105 on ELISA) but failed to show cross reactivity to peptides from other regions of the D2-receptor and D1receptor (7, and unpublished observations). The amino acid sequence of 23-34 of the rat D2 receptor, and the oligopeptide antigen used for the production of peptide I antibody, share little sequence homology with other dopamine receptors subtypes or with other kno~aa receptor sequences: human D1 9%; rat D1 9%; human D2 27%; rat D3 18%; human D4 27%; and human D5 9% (14). This will give an inherent selectivity for the D2 receptor protein over other G-protein coupled receptors. Cell culture and receptor binding: The mouse fibroblasts (Ltk- cells) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat denatured fetal calf serum under humidified 90% 02 and 10% CO2 atmosphere at 37°C and were fed every 3-4 days. The Ltk- cells were transfected with dopanfine receptor cDNA plasimd pZem3 and neomyacin resistant plasmid pRSVneo by standard CaPO 4 precipitation technique (1). The transfectants were isolated in 350 ~tg/ml of G418. The D2-receptor expressing Ltk- cells were screened by receptor binding in intact cells using a specific D2-receptor antagonist, [3H]YM-09151-2. Briefly, the cells [(1.2-1.4) X 106 cells/well] were washed 3 times in 10 mM phosphate buffer, pH 7.4 and 150 mM NaCI (PBS) and incubated in 2 ml of binding buffer (25 mM Hepes, pH 7.4; 0.2% bovine serum albumin in Hanks balanced salt solution) containing 300 pM [3H]YM-09151-2 (NEN, specific activity 87 Ci/mmoles) at 37°C for 30 minutes. The cells were washed three times in 2 ml PBS on ice, lysed in 10% KOH, and the radioactivity was measured. The non-specific binding was defined in the presence of 2 p.M (-)sulpride and it was 23-35% of the total binding. The specific binding of the [3H]YM09151-2 to Ltk-RGB cell membranes was carried out as described earlier for rat striatal membranes (7,15). Briefly, the cells were washed in PBS and homogenized in buffer A (50 mM Tris, pH 7.4; 8 mM MgC12; 5 mM EDTA) containing protease inhibitors (10 p.g/ml leupeptin; 5 gg/ml pepstatin; 5 p.g/ml aprotinin; 1 mM PMSF) and centrifuged at 65,000 xg for 30 minutes at 4°C. The membrane pellet was reconstituted in buffer B (50 mM Tris, pH 7.4; 120 mM NaCI; 5 mM KC1; 1.5 mM CaCI2; 1 mM EDTA and 0.1% ascorbic acid) containing protease inhibitors. The partially purified membranes were incubated with increasing concentration (10200 pM) of [3H]YM0915 I-2 in the binding buffer B under reduced light for 60 minutes at 25°C in a final volume of 500 /al. The binding was terminated by rapid filtration on a GF/B filters followed by three washes with ice cold buffer A and the radioactivity on the filters was counted. The non-specific binding was determined in the presence of 2 ~tM (-)sulpride and constituted less than 18% of the total bound
VOI. 51, NO. 19, 1992
Antibody to Dopamine D2 Receptor
1511
radioactivity. The saturation kinetics of the [3H]YM09151-2 binding was analyzed by non linear regression analyses. The equilibrium binding parameters were calculated after obtaining the best fit curves for single site analysis of the specific binding and transfroming the data for Scatchard plot analyses using the computer program "Graph Pad" (ISI, Philadelphia, PA). Flow cvtomet~ and data collection: For flow cytometric analysis, cells (2.5-3.2 X 106) were washed with PBS at 37oc and incubated with either preimmune or immune sera at 1:250 dilutions in DMEM for 30 minutes at 37°C. After briefly washing the cells with PBS at 25°C, 5 ml of binding buffer containing 300 pM PPHT-fluorescein was added and the cells were further incubated for an additional 30 minutes at 25°C. The cells were washed once with ice cold PBS and suspended in DMEM at 1-2 X 106 cells/ml on ice. PPHT-fluorescein fluorescence was analyzed in a FACS-440 (Becton-Dickinson, San Jose, CA) utilizing the 488 nm line from a 2-W argon laser (Coherent, Inc., Palo Alto, CA) operating at 200 mW. Data collection through the FACS-440 was triggered using only forward light scatter but list mode data were collected for forward light scatter, 90 ° light scatter and green fluorescence emission parameters (530/30 band pass filters). The data were analyzed using a DEC micro-VAX II workstation (Digital Equipment corporation, Westminster, MA) and the Consort-40 flow cytometry program package (BectonDickinson); histograms were composed of 104 cells per sample.
Results and Discussion The expression of D2-receptor in the transfected and untransfeeted mouse fibroblast cells was examined by measuring the specific binding of [3H]YM-09151-2 in these cells. The specific binding of [3H]YM-0915 I-2 was 4-5 fold higher in the Ltk-RGB cells compared with the recipient Ltk- cells (mean + SEM; transfected, 435 + 37, untransfected Ltk- cells 58 + 18 fmoles/mg protein, p< 0.05). The specific binding of [3H]YM-09151-2 was of 2-3 orders of magnitude higher compared to the spiperone binding in these cell membranes [1]. This could probably be due to marked differences in the affinity and specificity of [3H]YM09151-2 for D2 receptors compared with spiperone (13). In order to ascertain that dopamine receptor antibody interacts with the cloned D2-receptor similar to striatal D2 receptors, the equilibrium kinetics of the [3H]YM09151-2 binding to the membranes from the Ltk-RGB cells was studied at increasing ligand (10-200 pM) concentrations in the absence and presence of immune or preimmune sera. The specific binding of [3H]YM09151-2 was significantly reduced in the presence of D2-receptor antibody at all concentrations of ligand studied (Figure 1). The Scatchard analysis of the specific binding data indicated a single class of [3H]YM09151-2 binding sites in these membranes. The presence of immune serum led to a 30-35% decrease in the total number of [3H]YM09151-2 binding sites with no apparent change in the receptor affinity (Kd). In contrast, the addition of preimmune serum did not alter either the Bmax or the Kd of the D2-receptor (Table I). These results are in agreement with the antibody dependent inhibition of the [3H]YM09151-2 binding to rat striatal membranes (7), suggesting a common putative antibody binding site at or close to the ligand interaction motif on the cloned or striatal D2-receptors. Although the exact molecular nature of the D2receptor observed in these cells is unknown, the transfection of dopamine D2 receptor DNA in Chinese hamster ovary cells suggested DR to be a 71 kDa protein (3). In contrast, the DR in the brain is a 95-220 kDa protein (4, 5, 6, 7, 8). These observations suggest possible differences in the post-translational processing of the receptor core protein in cultured cells. In spite of the differences in the molecular weights of D2-receptor, the antibody recognized both the cloned and striatal receptor forms, suggesting that the epitope may reside on the receptor core protein. Next we studied the antibody dependent inhibition in the binding of D2-receptor agonist PPHTfluorescein to Ltk-RGB cells in a fluorescence activated cell sorter (FACS). Figure 2 shows the forward scatter, mean fluorescence channel (MFC) values and the 90° scatter of Ltk- and Ltk-RGB cells preincubated with preimmune or immune sera followed by PPHT-fluorescein. The PPHT-fluorescein labeled Ltk- cells exhibited a single peak for forward scatter, 90° scatter and a MFC value of 55 + 9 (Figure 2; Panel A, B and C). Similar values for the forward scatter, 90 ° scatter and MFC value were
1512
Antibody to Dopamine D2 Receptor
Vol. 51, No. 19, 1992
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(PM) Fig. 1. The saturation curves of the [3H]YM09151-2 binding to dopamine receptor DNA transfected Ltk-RGB ccll membranes. The cell membranes were incubated with increasing concentration of [3H]YM09151-2 in the binding buffer B at 25°C for 60 minutes in the absence of serum ( A ) or presence of preimmune ( O ) or immune ((~)) sera. The Scatchard plot of the of the specific binding data from preimmune and immune sera treated samples are shown in the inset.
T A B L E I. Equilibrium Kinetic Parameters o f Antibody Dependent Inhibition o f [3H]YM09151-2 Binding to the Ltk-RGB cells.
Additions
Bmax (fmol/mg protein)
Kd (pM)
None (control)
120.26 + 5.26
27.49 + 3.95
Preimmune serum
110.56 + 9.30
28.39 + 7.75
Immune serum
76.20 + 5.18"
20.12 + 5.14
The binding of [3H]YM09151-2 to Ltk-RGB cell membranes in the absence and presence of preimmune or immune sera (2 ~tl serum/assay tube). The results are expressed as mean + S.E.M. * p < 0.05 as compared to preimmune group.
VOI. 51, No. 19, 1992
Antibody to Dopamine D2 Receptor
1513
obtained when untreated Ltk- cells were used in the FACS assay (data not shown). The forward and 90 ° scattering of light depends on the cell size and shape; the larger cells will scatter more light than the smaller ones. Thus these results suggest that untransfeeted Ltk- cells constitute a homogeneous population of cells exhibiting intense auto-fluorescence. When PPHT-fluorescein treated Ltk-RGB cells were analyzed in the FACS assay (figure not shown), the forward scatter (peak 1 = 45; peak 2 = 105) and MFC (MFC 1 = 48. + 6 and MFC 2 = 79 + 8) analyses yielded two peaks each. However, washed, but PPHT-fluorescein untreated Ltk-RGB cells exhibited similar forward scatter and 90 ° scatter values, but showed only one MFC peak (MFC value 51 + 3; data not shown). In sum, these results suggest that LtkRGB cells constitute large (55%) and small (45%) cells and the MFC2 peak is associated with the PPHTfluorecein fluorescence. In a separate set of experiments, transfeeted cells were incubated with preimmune or immune serum before labeling with the PPHT-fluorescein and were used for FACS analysis.
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Mean Fluorescence intensity Fig. 2. Flow cytometric analysis of PPHT-Fluorescein binding to Ltk- and Ltk-RGB cells. Subce.,tfluent cultures (3x106 ceils/flask) were washed with PBS and incubated in 1:250 dilution of preimmune or immune serum for 30 minutes; the cells were rinsed once in PBS and incubated with 300 pM PPHT-fluorescein in the binding buffer for an additional 30 minutes at 25oc. After washing the cells in PBS, the PPHT-fluorescence was measured in FACS-440; FACS generated histograms are untransfected cells (A-C) and transfected cells (D-F); A and D, Forward scatter; B and E, Fluorescence intensity; C and F, 900 scatter. Shaded area under the curve represents the cells incubated with immune sera.
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Mean Fluorescence intensity Fig. 3. Computer processed data for the forward scatter and fluorescence intensity of the large and small cells. Sub-confluent cultures (3x10 6 cells/flask) were incubated with PPHTfluorescein as described under the legend of figure 2. The ceils were washed in PBS and suspended in DMEM at 1 X 106 cells/ml. The cell population exhibiting PPHTfluorescein florescence were analyzed; Panel A. Forward scatter, Panel B. florescence intensity. Shaded area under the curve shows the florescence of the small cells.
19, 1992
Vol. 51, No. 19, 1992
Antibody to Dopamine D2 Receptor
1515
As shown in Figure 2 (panel D and F) the preincubation of cells with antiserum or preimmune serum had no effect on the forward scatter or the 90 ° scatter. However, the MFC value of the PPHT-fluorescien fluorescence was decreased significantly in cells incubated with immune serum compared with preimmune serum (preimmune : 88.7 + 7.8; immune: 69.2 + 4.8; p
51, pp.
1509-1516
Pergamon
Press
AN ANTIBODYTO DOPAMINED2 RECEPTORINHIBITSDOPAMINEANTAGONISTAND AGONIST BINDINGTO D O P A M I D2 RECEPTORc D N A TRANSFECTEDMOUSEFIBROBLASTCELLS. Shakeel M. Farooqui* and Chandan Prasad**.
Laboratory ofNeurosciences Pennington Biomedical Research Center. Baton Rouge, LA 70808 and **Department o f Medicine, LSUMC, New Orleans, L A 70112 (Received
in final
form September
4, 1992)
SUMMARY A polyclonal antibody to dopamine D2 receptor (D2-rece~otor) has been used to examine the immuno-inhibition in the binding of a D2 antagonist, [aH]YM09151-2 and an agonist, PPHT-fluorescein to dopamine receptor DNA transfected mouse fibroblast cells. The specific activity of the [3H]YM09151-2 binding to transfected (Ltk-RGB) cells is 4-5 fold higher than untransfected (Ltk-) cells. The antibody is able to inhibit the [3H]YM091512 binding to the cell membranes from Ltk-RGB cells (Bmax 110.56 + 5.26 and 76.20 + 5.18 fmoles/mg protein in the presence of preimmune and immune sera, respectively, with no change in the Kd). The flow cytometric analysis of the PPHT-fluorescein labeled Ltkand Ltk-RGB cells indicated that ligand specific fluorescence is associated only with small Ltk-RGB cells (second peak) and autofluorescence with large cells (first peak). Preincubation of the Ltk-RGB ceils with antibody, reduced the fluorescence intensity of the PPHT-fluorescein by 20-25% without changing the auto-fluorescence. These results suggest that peptide antibody recognize D2-receptor in both membranes and in intact cells and interact at or near the ligand binding site of the receptor
The nucleotide sequence for the dopamine D2 receptor (D2-receptor) gene suggests that molecular weight of the core D2-receptor protein should be around 46 kDa (1, 2). However, past studies devoted to purification of the functional D2-receptor (the receptor capable of binding antagonists or agonists) suggest an apparent molecular weight of 71 kDa to 220 kDa (3, 4, 5, 6, 7, 8). To facilitate the examination of the chemical basis for the apparent differences in the molecular weights of "core" and "functional" D2receptors, we have raised and characterized both monoclonal and polyclonal antibodies to D2 receptors (7, 8). The polyclonal antibody to an amino terminal (amino acid 23-34) D2-receptor peptide was found to selectively inhibit the binding of [3H]YM-09151-2 or 125I-azido-NAPS to striatai membrane (7); furthermore both 125I-azido NAPS and one of the polyclonal antibody label a protein of the same molecular weight (7). Although the above data suggest a specificity of the antibody to D2-receptor, it is * Correspondance and request for reprints to: Shakeel M. Farooqui, Laboratory of Neurosciences, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1510
Antibody to Dopamine D2 Receptor
Vol. 51, No. 19, 1992
conceivable that the antibody may also label some non-D2-receptor protein(s). For example, both [3H]YM09151-2 and 125I-azido-NAPS are known to label 5-HT as well as other dopamine receptor subtypes (9, 10, 11); in addition, both monoclonal and polyclonal antibodies recognize multiple striatal proteins ranging in molecular weights between 46kDa to 220 kDa (7,8). Therefore, it was of interest to examine the specificity of D2-receptor antibody using a system devoid of these interfering receptors. To this end, we examined the inhibition of the binding of a D2receptor antagonist, (+)-cis-N-(1-benzyi-2-[3H] -methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4methylamino-benzamide, [3H]YM09151-2 (12) and an agonist, (+)-2-(N-phenylethyl-N-porpyl)amino-5hydroxytetralin fluorescien, PPHT-fluorescein (13) to Ltk- cells (normally devoid of dopaminergic and serotonergic receptors) transfected with dopamine receptor DNA. The results of this study show that D2receptor antibody inhibits both the antagonist and agonist binding to the D2-receptor expressing cells and therefore, we conclude that the polyclonal peptide antibody (amino acid 23-34) is highly specific for D2receptor. Since PPHT-fluorescein binds only to the transfected cells, a relatively pure population of cells expressing D2-receptor can be obtained by sorting the cells for PPHT-fluorescence in a fluorescence activated cell sorter (FACS).
Materials and Methods Polyclonal antibody: The properties and characterization of the polyclonal D2-receptor antibody (pep I) has been described elsewhere (7, 8, 14). The pep I refers to the peptide containing amino acid 23 to 34 in rat D2-receptor sequence. The antibody was reactive to homologous peptide antigen (titer, 1:105 on ELISA) but failed to show cross reactivity to peptides from other regions of the D2-receptor and D1receptor (7, and unpublished observations). The amino acid sequence of 23-34 of the rat D2 receptor, and the oligopeptide antigen used for the production of peptide I antibody, share little sequence homology with other dopamine receptors subtypes or with other kno~aa receptor sequences: human D1 9%; rat D1 9%; human D2 27%; rat D3 18%; human D4 27%; and human D5 9% (14). This will give an inherent selectivity for the D2 receptor protein over other G-protein coupled receptors. Cell culture and receptor binding: The mouse fibroblasts (Ltk- cells) were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat denatured fetal calf serum under humidified 90% 02 and 10% CO2 atmosphere at 37°C and were fed every 3-4 days. The Ltk- cells were transfected with dopanfine receptor cDNA plasimd pZem3 and neomyacin resistant plasmid pRSVneo by standard CaPO 4 precipitation technique (1). The transfectants were isolated in 350 ~tg/ml of G418. The D2-receptor expressing Ltk- cells were screened by receptor binding in intact cells using a specific D2-receptor antagonist, [3H]YM-09151-2. Briefly, the cells [(1.2-1.4) X 106 cells/well] were washed 3 times in 10 mM phosphate buffer, pH 7.4 and 150 mM NaCI (PBS) and incubated in 2 ml of binding buffer (25 mM Hepes, pH 7.4; 0.2% bovine serum albumin in Hanks balanced salt solution) containing 300 pM [3H]YM-09151-2 (NEN, specific activity 87 Ci/mmoles) at 37°C for 30 minutes. The cells were washed three times in 2 ml PBS on ice, lysed in 10% KOH, and the radioactivity was measured. The non-specific binding was defined in the presence of 2 p.M (-)sulpride and it was 23-35% of the total binding. The specific binding of the [3H]YM09151-2 to Ltk-RGB cell membranes was carried out as described earlier for rat striatal membranes (7,15). Briefly, the cells were washed in PBS and homogenized in buffer A (50 mM Tris, pH 7.4; 8 mM MgC12; 5 mM EDTA) containing protease inhibitors (10 p.g/ml leupeptin; 5 gg/ml pepstatin; 5 p.g/ml aprotinin; 1 mM PMSF) and centrifuged at 65,000 xg for 30 minutes at 4°C. The membrane pellet was reconstituted in buffer B (50 mM Tris, pH 7.4; 120 mM NaCI; 5 mM KC1; 1.5 mM CaCI2; 1 mM EDTA and 0.1% ascorbic acid) containing protease inhibitors. The partially purified membranes were incubated with increasing concentration (10200 pM) of [3H]YM0915 I-2 in the binding buffer B under reduced light for 60 minutes at 25°C in a final volume of 500 /al. The binding was terminated by rapid filtration on a GF/B filters followed by three washes with ice cold buffer A and the radioactivity on the filters was counted. The non-specific binding was determined in the presence of 2 ~tM (-)sulpride and constituted less than 18% of the total bound
VOI. 51, NO. 19, 1992
Antibody to Dopamine D2 Receptor
1511
radioactivity. The saturation kinetics of the [3H]YM09151-2 binding was analyzed by non linear regression analyses. The equilibrium binding parameters were calculated after obtaining the best fit curves for single site analysis of the specific binding and transfroming the data for Scatchard plot analyses using the computer program "Graph Pad" (ISI, Philadelphia, PA). Flow cvtomet~ and data collection: For flow cytometric analysis, cells (2.5-3.2 X 106) were washed with PBS at 37oc and incubated with either preimmune or immune sera at 1:250 dilutions in DMEM for 30 minutes at 37°C. After briefly washing the cells with PBS at 25°C, 5 ml of binding buffer containing 300 pM PPHT-fluorescein was added and the cells were further incubated for an additional 30 minutes at 25°C. The cells were washed once with ice cold PBS and suspended in DMEM at 1-2 X 106 cells/ml on ice. PPHT-fluorescein fluorescence was analyzed in a FACS-440 (Becton-Dickinson, San Jose, CA) utilizing the 488 nm line from a 2-W argon laser (Coherent, Inc., Palo Alto, CA) operating at 200 mW. Data collection through the FACS-440 was triggered using only forward light scatter but list mode data were collected for forward light scatter, 90 ° light scatter and green fluorescence emission parameters (530/30 band pass filters). The data were analyzed using a DEC micro-VAX II workstation (Digital Equipment corporation, Westminster, MA) and the Consort-40 flow cytometry program package (BectonDickinson); histograms were composed of 104 cells per sample.
Results and Discussion The expression of D2-receptor in the transfected and untransfeeted mouse fibroblast cells was examined by measuring the specific binding of [3H]YM-09151-2 in these cells. The specific binding of [3H]YM-0915 I-2 was 4-5 fold higher in the Ltk-RGB cells compared with the recipient Ltk- cells (mean + SEM; transfected, 435 + 37, untransfected Ltk- cells 58 + 18 fmoles/mg protein, p< 0.05). The specific binding of [3H]YM-09151-2 was of 2-3 orders of magnitude higher compared to the spiperone binding in these cell membranes [1]. This could probably be due to marked differences in the affinity and specificity of [3H]YM09151-2 for D2 receptors compared with spiperone (13). In order to ascertain that dopamine receptor antibody interacts with the cloned D2-receptor similar to striatal D2 receptors, the equilibrium kinetics of the [3H]YM09151-2 binding to the membranes from the Ltk-RGB cells was studied at increasing ligand (10-200 pM) concentrations in the absence and presence of immune or preimmune sera. The specific binding of [3H]YM09151-2 was significantly reduced in the presence of D2-receptor antibody at all concentrations of ligand studied (Figure 1). The Scatchard analysis of the specific binding data indicated a single class of [3H]YM09151-2 binding sites in these membranes. The presence of immune serum led to a 30-35% decrease in the total number of [3H]YM09151-2 binding sites with no apparent change in the receptor affinity (Kd). In contrast, the addition of preimmune serum did not alter either the Bmax or the Kd of the D2-receptor (Table I). These results are in agreement with the antibody dependent inhibition of the [3H]YM09151-2 binding to rat striatal membranes (7), suggesting a common putative antibody binding site at or close to the ligand interaction motif on the cloned or striatal D2-receptors. Although the exact molecular nature of the D2receptor observed in these cells is unknown, the transfection of dopamine D2 receptor DNA in Chinese hamster ovary cells suggested DR to be a 71 kDa protein (3). In contrast, the DR in the brain is a 95-220 kDa protein (4, 5, 6, 7, 8). These observations suggest possible differences in the post-translational processing of the receptor core protein in cultured cells. In spite of the differences in the molecular weights of D2-receptor, the antibody recognized both the cloned and striatal receptor forms, suggesting that the epitope may reside on the receptor core protein. Next we studied the antibody dependent inhibition in the binding of D2-receptor agonist PPHTfluorescein to Ltk-RGB cells in a fluorescence activated cell sorter (FACS). Figure 2 shows the forward scatter, mean fluorescence channel (MFC) values and the 90° scatter of Ltk- and Ltk-RGB cells preincubated with preimmune or immune sera followed by PPHT-fluorescein. The PPHT-fluorescein labeled Ltk- cells exhibited a single peak for forward scatter, 90° scatter and a MFC value of 55 + 9 (Figure 2; Panel A, B and C). Similar values for the forward scatter, 90 ° scatter and MFC value were
1512
Antibody to Dopamine D2 Receptor
Vol. 51, No. 19, 1992
J
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(PM) Fig. 1. The saturation curves of the [3H]YM09151-2 binding to dopamine receptor DNA transfected Ltk-RGB ccll membranes. The cell membranes were incubated with increasing concentration of [3H]YM09151-2 in the binding buffer B at 25°C for 60 minutes in the absence of serum ( A ) or presence of preimmune ( O ) or immune ((~)) sera. The Scatchard plot of the of the specific binding data from preimmune and immune sera treated samples are shown in the inset.
T A B L E I. Equilibrium Kinetic Parameters o f Antibody Dependent Inhibition o f [3H]YM09151-2 Binding to the Ltk-RGB cells.
Additions
Bmax (fmol/mg protein)
Kd (pM)
None (control)
120.26 + 5.26
27.49 + 3.95
Preimmune serum
110.56 + 9.30
28.39 + 7.75
Immune serum
76.20 + 5.18"
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The binding of [3H]YM09151-2 to Ltk-RGB cell membranes in the absence and presence of preimmune or immune sera (2 ~tl serum/assay tube). The results are expressed as mean + S.E.M. * p < 0.05 as compared to preimmune group.
VOI. 51, No. 19, 1992
Antibody to Dopamine D2 Receptor
1513
obtained when untreated Ltk- cells were used in the FACS assay (data not shown). The forward and 90 ° scattering of light depends on the cell size and shape; the larger cells will scatter more light than the smaller ones. Thus these results suggest that untransfeeted Ltk- cells constitute a homogeneous population of cells exhibiting intense auto-fluorescence. When PPHT-fluorescein treated Ltk-RGB cells were analyzed in the FACS assay (figure not shown), the forward scatter (peak 1 = 45; peak 2 = 105) and MFC (MFC 1 = 48. + 6 and MFC 2 = 79 + 8) analyses yielded two peaks each. However, washed, but PPHT-fluorescein untreated Ltk-RGB cells exhibited similar forward scatter and 90 ° scatter values, but showed only one MFC peak (MFC value 51 + 3; data not shown). In sum, these results suggest that LtkRGB cells constitute large (55%) and small (45%) cells and the MFC2 peak is associated with the PPHTfluorecein fluorescence. In a separate set of experiments, transfeeted cells were incubated with preimmune or immune serum before labeling with the PPHT-fluorescein and were used for FACS analysis.
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Mean Fluorescence intensity Fig. 2. Flow cytometric analysis of PPHT-Fluorescein binding to Ltk- and Ltk-RGB cells. Subce.,tfluent cultures (3x106 ceils/flask) were washed with PBS and incubated in 1:250 dilution of preimmune or immune serum for 30 minutes; the cells were rinsed once in PBS and incubated with 300 pM PPHT-fluorescein in the binding buffer for an additional 30 minutes at 25oc. After washing the cells in PBS, the PPHT-fluorescence was measured in FACS-440; FACS generated histograms are untransfected cells (A-C) and transfected cells (D-F); A and D, Forward scatter; B and E, Fluorescence intensity; C and F, 900 scatter. Shaded area under the curve represents the cells incubated with immune sera.
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Mean Fluorescence intensity Fig. 3. Computer processed data for the forward scatter and fluorescence intensity of the large and small cells. Sub-confluent cultures (3x10 6 cells/flask) were incubated with PPHTfluorescein as described under the legend of figure 2. The ceils were washed in PBS and suspended in DMEM at 1 X 106 cells/ml. The cell population exhibiting PPHTfluorescein florescence were analyzed; Panel A. Forward scatter, Panel B. florescence intensity. Shaded area under the curve shows the florescence of the small cells.
19, 1992
Vol. 51, No. 19, 1992
Antibody to Dopamine D2 Receptor
1515
As shown in Figure 2 (panel D and F) the preincubation of cells with antiserum or preimmune serum had no effect on the forward scatter or the 90 ° scatter. However, the MFC value of the PPHT-fluorescien fluorescence was decreased significantly in cells incubated with immune serum compared with preimmune serum (preimmune : 88.7 + 7.8; immune: 69.2 + 4.8; p
