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PRODUCTIONANDCHARACTERIZATIONOFAMONOCLONALANTIBODYTODOPAMINED~ RECEPTOR: COMPARISONWITHAPOLYCLONALANTIBODYTOADIFFERENTEPITOPE Shakeel M. Farooqui’, Chandan Prasad**, and Massarat Ali Laboratory of Neurosciences and Gene Expression Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808 ** Department of Medicine, Section of Endocrinology, Received
March
17,
LSUMC, New Orleans, LA 70112
1992
Summary: A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 pM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein. 0 1992Academic PIcz55,Inc.
Molecular cloning of dopamine receptor has led to the identification of at least five different receptor subtypes, including Dl, D2 (long and short form), D3, D4, and D5 (l-3). Of these various subtypes, D2 receptor (DAR) has not only been implicated in the regulation of a variety of physiologic processes such as motor coordination, affective behavior and sexual performance, but also in hyper-dopaminergic disorders such as certain subtypes of schizophrenia and tardive dyskinesia (4-7). Therefore, in order to understand the etiology of hyperdopaminergic disorders as well as the control of normal dopaminergic functions, it will be essential to examine the status of DAR at the level of gene transcription, receptor protein synthesis and its maturation to 1 To whom correspondence
and reprint requests should be addressed. Abbreviations: BSA, bovine serum albumin; 4-CN, 4-chloronaphthol; DAR, dopamine D2 receptor; ELISA, enzyme linked immunosorbent assay; KLH, keyhole limpet hemocyanin; Mab, monoclonal antibody; NAPS-biotinyl; N(D-biotinylaminophenethyl)spiperone; PBS, phosphate buffered saline; PEG, polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; [3H]YM09151-2, (+)-cis-N-(l-benzyl-2[3H]-methylpyrrolidin-3-yl)-5-chloro2-methoxy-4-methylamino-benzamide.
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a functional entity. The studies on the structure/function and regulation of the DAR protein synthesis has been hampered by the unavailability of site specific antibodies to the receptor protein. To this end, we have recently raised and characterized a polyclonal antibody to a decapeptide representing amino-terminal (residue 24-34) sequence of DAR protein (8). Since polyclonal antibodies represent a mixture of different immunoglobulins molecules directed towards different epitopes on the same protein, the specificity of the antibody with regards to its ability to recognize endogenous protein can never be precisely determined. On the contrary, monoclonal antibodies (Mab) are one class of immunoglobulins directed against a single epitope and therefore, they posses a high degree of specificity. These considerations have led us to raise monclonal antibodies to a synthetic peptide predicted from DAR protein sequence (9), and compare its properties to a polyclonal antibody against a peptide from a different region of the dopamine receptor protein.
MATERIALS
AND
METHODS
Antigen preparation and immuniztztion:
Peptide I (24-34; CGSEGKADRPHYC) and peptide II (176- 185; NNTDQNECIIY) were synthesized on a Milligen Model # 9050 peptide synthesizer and purified on a Cl8 (p bondpack, Millipore) HPLC coulmn using a 5100% (v/v) acetonitrile gradient in 0.1% trifluoroacetic acid in water. The peptides were coupled individually to keyhole limpet hemocyanin (peptide-KLH) by the glutaraldehyde method (10). Balb/c mice were immunized every two weeks (4 i.p. injections; 100 pg peptide-KLH protein/injection) with peptide-KLH conjugate in phosphate buffered saline (PBS), emulsified in adjuvant (RIBI, Immunochemical Research). Titers of antisera were determined by ELISA using free peptide as antigen. The 96-well microtiter plate was coated with 50 rig/well peptide in 0.1 M sodium bicarbonate buffer, pH 9.6. After blocking with 5% bovine serum albumin (BSA, fraction V; Sigma), 50 pl of the serially diluted antibody solution was added. After incubation and washing, secondary antibody coupled to horseradish peroxidase (1: 1000 dilution; Biorad) was added. The color was developed with o-phenylenediamine and H202. The production of polyclonal antibody to peptide II in rabbits has been described elsewhere (8). Three days before fusion, the mice were boosted with 100 pg peptide-KLH conjugate. Fusions were performed using azaguanine-resistant NS/O myeloma cells. The fusion was achieved using polyethylene glycol (PEG) Mr. 1500 (Boehringer Mannheim), and the hybridoma selection was accomplished in HAT medium. Positive clones were selected by screening hybridoma supernatants in 96-well ELISA plates coated with either free peptide, peptide-KLH or KLH (50 rig/well). The hybridomas that reacted with peptide and peptide-KLH were single-cell cloned, expanded and isotyped. These clones were injected (107 cells/mouse) intraperitoneally to two mice to obtained ascites. The antibody isotypes were determined using a mouse monoclonal sub-isotyping kit (Hyclone). The clone SF4 described here secreted IgM subtype antibody.
Preparation and screening of hybridomas:
Preparation and solubilization of striatul membranes and ligand binding assays: Rat striatal membrane was prepared as described earlier (11). Briefly, the tissues were homogenized in 20 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.4; 8 mM MgC12; 5 mM EDTA), containing a mixture of protease inhibitors (10 pg/ml leupeptin, 5 pg/ml pepstatin, 5 pg/ml aprotanin and 1 mM phenylmethyl sulfonylflouride). The homogenate was centrifuged at 49,000 x g for 20 min at 4oC, the intermediate pellet was resuspended in the same buffer and the process was repeated once. The final pellet was suspended in buffer B (50 mM Tris-HCI, pH 7.4; 120 mM NaCl; 5 mM 662
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KCl; 5 mM MgC12; 1.5 mM CaC12; 1mM EDTA; 10 PM pargyline-HCl and 0.1% ascorbic acid) at a protein concentration of 2 mg/ml and stored at -800C. Liver membranes were prepared as described elsewhere (12). The striatal membranes were solubilized in CHAPS/NaCl buffer (50 mM Tris, pH 7.4; 10 mM CHAPS; 720 mM NaCl) containing protease inhibitors as described elsewhere (13). The [3H]YM-09151-2 (specific activity 71 mCi/mmol; New England Nuclear) binding to striatal membranes (8) and soluble receptor (13) was carried out as described earlier. Briefly, the soluble receptor binding assay was performed in 500 l.rl buffer B containing 60 pM radioligand and 100 lrg protein from solubilized membranes using incubation period of lhr at 370C. Following incubation, 500 l.tl of 3% (w/w) g globulin and 1 ml of 30% PEG (Mr. 78009000) in 50 mM Tris, pH 7.4 were added. The content of each tube was vortexed, kept on ice for 15 min, filtered under vacuum through GF/B filters, and washed three times with 5 ml of 10% PEG in ice cold 50 mM Tris pH, 7.4. The radioactivity on the filters was counted. Non-specific binding, defined in the presence of 2mM (-)sulpride, constituted less than 18% of the total bound radioactivity. The equilirium binding parameters were calculated after obtaining the best-fit curves for specific binding using computer program “Graph pad”. SDS-PAGE, Western blotting and ligand blotting of membranes in the presence of NAPSbiotinyl: Membrane proteins were subjected to 4-15% continuous sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose (Biorad) and were stained with Ponceau S (Sigma) to verify the uniform transfer of proteins. Nitrocellulose membranes were blocked in buffer C (10 mM Tris-HCl, pH 7.4; 154 mM NaCl; 0.02% thimerosal; 5% (w/v) non-fat dry milk) for 2 hr at room temperature and incubated with ascites or polyclonal rabbit antisera in tris buffered saline (25 mM Tris-Cl, pH 7.4; 154 mM NaCl). The membranes were washed three times in buffer D (10 mM Tris-HCl, pH 7.4; 154 mM NaCl; 0.05% Tween-20) followed by incubation at room temperature in buffer C containing 1: 1000 dilution of horseradish peroxidase conjugated secondary antibody. The blots were washed three times in Buffer D, and developed with 4-chloronaphthol(4-CN). For ligand blotting, the nitrocellulose membranes, treated overnight in buffer C containing protease inhibitors, were incubated with 200 pM NAPS-biotinyl and 50 nM ketanserin (Research Biochemical Inc.) in buffer B, washed in buffer D and were incubated with avidin-horseradish peroxidase conjugate (Biorad) in buffer C. The biotin bound proteins were detected in the presence of 4-CN. Membrane protein was determined by BCA reagent using BSA as standard (Pierce Chemicals).
RESULTS AND DISCUSSION
We have used DAR peptides 24-34 (peptide I) and 174-184 (peptide II) to raise monoclonal and polyclonal antibodies respectively. The characteristic of the polyclonal antibody against peptide II has been described in detail earlier (8). Briefly, the polyclonal antibody was specific for peptide II, and it showed no cross reactivity to peptides representing other regions of the DAR or Dl receptor (8 and unpublished observations). The low antibody titer reported earlier for peptide II antibody was increased significantly after subsequent boosters. The Mab clone SF4 hybridoma supernatant and ascites were tested for their reactivity against the peptide I in solid phase ELISA; the antibody detected free as well as peptide-KLH conjugate at relatively low antibody concentrations (l:lOO,OOO dilution). The addition of the free peptide suppressed the ELISA reactivity in a concentration-dependent manner; addition of 50 l.rg/ml peptide I resulted in to a 90% decrease in the Mab immune complex formation. On the contrary, the addition of peptide II at concentrations up to lmg/ml did not inhibit the immune complex formation (Data not 663
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12345
205
66.2-
45
31-
I
panel
A 1
L--
Panel
B-
Figure 1. Western blot detection of rat DAR using Mab (Panel A) and polyclonal antibody to peptide II (Panel B). Increasing concentration of rat striatal membrane protein (lane l-5; 10, 20, 40, 60, 80 pg protein respectively) was electrophoresed on a 4-15% SDS-PAGE. Antibodies (Mab, 1500; polyclonal antibody, I:250 ) were diluted in TBS and incubated with blots for 17 hr at 4°C. For compitition assay (Lane 6, Panel A), the Mab had previously been mixed with free pcptidc I (200 l.tg/lOO 1.11in TBS) and incubated overnight at 4oC. The relative molecular weights in kDa are indicated on the left.
shown). Thus, both the polyclonal and Mab appeared to be highly specific for their homologous peptides. The specificity of Mab and polyclonal antibodies were further tested on immunoblots using rat striatal membranes. As shown in Fig. 1 (Panel A) the Mab at 1500 dilution reacted with five major polypeptides with apparent molecular wights 220, 145, 95, 66 and 47 kDa. Pre-absorption of the Mab with an excess of free peptide I (2 mg/ml) blocked the recognition of all the immunoreactive proteins bands (Panel A, lane 6). The polyclonal antibody against peptide II recognized only three out of five Mab immunoreactive proteins with an apparent Mr. 220, 145 and 47 kDa (Fig. 1, Panel B). The tissue specificity of Mab was further characterized by Western blot analysis of rat striatal, cerebellar and liver membranes. Using 20 pg striatal, cerebellar and liver membranes and Mab dilution l:lOOO, only the 220 and 47 kDa proteins were clearly visible in the striatal membrane; however, under similar conditions the intensity of the 220 kDa protein band was reduced by 70-75% in cerebellum and was absent in liver (Fig. 2). The 47 kDa protein was the only Mab reactive entity present in the liver membranes and its concentration was about 50% 664
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8
20%
116.297.4-
116.297.4-
66.266.2-
i.< ‘. 45bil 31-
02 Figure 2.
Western blot analysisof DAR. Twenty pg protein of striatal (lane I), cercbellar (lane 2) and liver (lane 3) membranes were subjected to 4-15% SDS-PAGEand hlottcd on nitrocellulose as describedin Materials and methods. The blots were incubated with I: 1000 dilution of Mab and the immunoreactivc proteins were detected in 4-CN. Molecular weights in kDa arc indicated on the left of the figure. Figure 3.
Ligand blotting of striatal membrane with NAPS-biotinyl. increasing concentration of striatal membrane protein (Lane l-5; 10, 20.40, 60, 80 pg protein) or 80 pg ccrebellar membranes (lane 6) were clectrophoresed on a 4-1541 SDS-PAGE. The nitrocellulosc strips wcrc incuhatcd with (lane 1-7) or without (lane 8) 200 pM NAPS-biotinyl. The competition assaywas performed on 80 pg striatal membranes in the presenceof 2 pM (-)sulpridc (lane 7). The biotin labeled proteins were detected by avidin-HRP conjugate as described in Materials and methods. Molecular weights in kDa are indicated on the left of the figure.
lower compared to striatum (Fig. 2). The detection of 220, 145 and 47 kDa proteins in known dopaminergic rich regions by two different antibodies and their reduced levels in the cerebellum
and concipicous absence of 220 kDa protein in the liver suggest that these antibodies may recognize the intact DAR protein. Based on the nucleotide sequence of the DAR, the “receptor core protein” should have a molecular weight around 47 kDa (9). The presence of 47 kDa, but not 220 kDa protein (capable of binding D2 antagonist) and a Lotal absence of D2 selective ligand specific-binding by liver membranes suggests that liver may lack the mechanism(s) needed to process the receptor core protein to a ligand binding receptor form. Alternatively, it is conceivable that the Mab is recognizing a liver protein having similar epitope but unrelated to dopamine receptor. We have investigated the ability of Mab to recognize soluble DAR protein; this may facilitate affinity purification of both the ligand binding and non-ligand binding domains of the 665
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DAR. To this end, we solubilized the DAR from striatal membranes in CHAPS/NaCl buffer, followed by centrifugation at 200,000 x g for 30 min; the supernatant was evaluated for specific [3H]YM09151-2 binding in the absence and the presence of 2 PM (-)sulpride. The specific binding of [3H]YM09151-2 to straital membranes and soluble receptor at 60 pM ligand concentration was 120.6 + 12.5 and 32.9 + 1.8 fmoles/mg protein respectively. The total binding of [3H]YM09151-2 to both the intact membranes and the soluble DAR was decreased by (-)sulpride, an active antagonist, but not by (+)sulpride, an inactive isomer (data not shown), suggesting the presence of high degree of stereospecificty for the soluble DAR (14). While there was no difference between the levels of Mab reactive protein in the intact membranes and solubilized DAR preparations (Data not shown), only 27% of the total binding capacity was recovered in the soluble fraction. The reason(s) for this apparent loss of [3H]YM09151-2 binding sites following the membrane solubilization is not clear; such a decrease in the binding sites could result from many factors including receptor denaturation, proteolysis or change in the affinity of the soluble receptor. To determine the size of the ligand binding form of the DAR, ligand blot analyses were performed on striatal membranes. Incubation of the nitrocellulose membranes containing increasing concentration of striatal membrane proteins (lo-80 p.g> with DAR antagonist NAPSbiotinyl revealed three proteins of apparent Mr. 220, 120 and 80 kDa (Fig. 3). The control strips incubated in the absence of NAPS-biotinyl (Fig. 3, lane 8) or in the presence of 2 yM (-)sulpride (Fig. 3, lane 7) detected only two bands of Mr 120 and 80 kDa. In addition, the ligand blotting of cerebellar membrane (80 ug protein) failed to detect the 220 kDa protein band (Fig. 3, lane 6). These results suggest that the apparent size of DAR ligand binding subunit is a 220 kDa polypeptide. The 110 and 80 kDa proteins labeled with avidin are dopamine receptor unrelated proteins that are either endogenously biotinylated or have high affinity for avidin. In several earlier studies the size of the ligand binding form of DAR, determined after photoaffinity labeling, has been reported to be 95-l 10 kDa (8,15,16). The reason(s) for this apparent discrepancy can only be speculated at this time. These include, the degradation of ligand bound DAR protein during photoactivation to achieve cross-linking or an irreversible denaturation of 95-110 kDa DAR protein by SDS treatment prior to electrophoresis, making it incapable of recognizing NAPS-biotinyl on ligand blots. In order to study if the DAR antibodies are directed against epitopes which may be involved in the ligand binding, the effect of the two antibodies on [3H]YM09151-2 binding was performed. The [3H]YM-09151-2 binding to striatal membrane was studied at various ligand concentrations and the equilibrium binding parameters were calculated. Both Mab and polyclonal antibodies failed to significantly alter the Bmax (264.2 + 16.8, 287.7 + 7.3, 267.1 + 20.7 fmoles/mg protein for control, Mab and peptide II antibody respectively) or Kd (11.9 + 2.8; 13.8 + 2.3 and 15.7 + 2.1 pM or control, Mab and peptide II antibody respectively) for the [3H]YM09151-2 binding to the striatal membranes. The inability of Mab raised against peptide I to inhibit ligand binding is contrary to our earlier observation reporting a significant inhibition in the [3H]YM09151-2 binding to striatal membranes by the polyclonal antibody against the same 666
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antigen (8). These data suggests that Mab recognizes an epitope which may not be involved in the ligand binding. It is also conceivable that the observed inhibition of [3H]YM09151-2 binding by polyclonal antibody in our previous report (8) may be due to steric hindrance caused by polyclonal nature of the antibody. We are in the process of screening other clones for antibodies directed against the ligand binding motif of the DAR protein and their usefulness in the immunoprecipitaiton protocols. These site specific antibodies will serve as useful reagents to study the structure and the regulation of the dopamine D2 receptor protein.
ACKNOWLEDGMENTS
We thank Shiru Q. Farooqui for her help in typing and preparation of the manuscript. These studies were supported in part by US Army Research and Development Command (grant # DAMD-17-88-Z-8023). Opinions and interpretations are those of the author and are not necessarily endorsed by the US Army. In conducting research using animals, the investigators adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the Committee on Care and Use of Laboratory Animals of the Institute Laboratory Animal Resources, National Research Council (NIH), Publication # 86-23, revised 1985.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Seeman, P. (1980). Parmacol. Rev. 32,229-313. Sunahara, R. K., Guan, H. C., 0-Dowd, B. F., Seeman, P., Laurier, L. J., Ng, G., George, S. R., Torchia, J., Van Toll, H. H. M. and Niznik, H. B. (1991). Nature 350, 614-619. Van Tol, H. H. M., Bunzow, J. R., Guan, H. C., Sunhara, R. K., Seeman, P., Niznik, H. B. and Civelli, 0. (1991). Nature 350, 610-614. Stenback, A. (1979). Akt. Gerontol. 9, 277-282. Martin, C. (1977). In: Hand book of Sexology (eds: Money, J. and Musaph H.) pp 813824. Came, D. B. and Langston, J. W. (1983). Lancet 38, 1457-1459. Cross, A. J., Crow, T. J., Ferrier, I. N., Johnstone, E. C., McCreadie, R. M., Owen, F., Owens, D. G. C., and Poulter, M. (1983). J. Neural transm. (suppl) 18, 265-272. Farooqui, S. M., Brock, J., Hamdi, A. and Prasad, C. (1991). J. Neurochem. 57, 13631369. Bunzow, J. R., Van Toll, H. H. M., Grandy, D. K., Albert, P., Salon, J., Chritie, M., Machida, C. A., Neve, K. A. and Civelli, 0. (1988). Nature 783-787. Duggan, M. J. and Stephenson, F. A. (1989). J. Neurochem. 53, 132-139. Sakata, M. , Farooqui, S. M. and Prasad, C. (1992). Brain Res. (In Press). Prpi’c, V., Green K. C., Blackmore, P. F. and Exton, J. H. (1984). J. Biol. Chem. 259, 1382-1385. Kuno T., Saijoh K. and Tanaka C. (1983). J. Neurochem. 41, 8 41-847. Dewar, K. M., Montreuil, B., Grondin, L. and Reader, T. A. (1988). J. Pharmacol. Exp. Ther. 250,696-706. Amlaiky, N. and Caron, M. G. (1985). J. Biol. Chem. 260, 1983-1986. Senogles, S. E., Amlaiky, N., Falardeau, P., Caron, M. G. (1988). J. Biol. Chem. 263, 18996- 19002.
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PRODUCTIONANDCHARACTERIZATIONOFAMONOCLONALANTIBODYTODOPAMINED~ RECEPTOR: COMPARISONWITHAPOLYCLONALANTIBODYTOADIFFERENTEPITOPE Shakeel M. Farooqui’, Chandan Prasad**, and Massarat Ali Laboratory of Neurosciences and Gene Expression Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808 ** Department of Medicine, Section of Endocrinology, Received
March
17,
LSUMC, New Orleans, LA 70112
1992
Summary: A monoclonal antibody (Mab) that recognizes the rat dopamine D2 receptor (DAR) has been generated using DAR specific peptide. The Mab, IgM isotype recognizes five proteins (Mr 220, 145, 95, 66 and 47 kDa) in striatal membrane on Western blot. Preincubation of Mab with free peptide blocked the labeling of all five bands. A polyclonal antibody against peptide from a different region of the DAR, reacted with three out of five proteins (220, 66, and 47 kDa) in these membranes. The DAR antagonist NAPS-biotinyl binds to a 220 kDa protein in striatal membrane on ligand blotts; the labeling can be blocked by the addition of 2 pM sulpride. The 220 kDa Mab reactive protein was less in cerebellum and was absent in the liver. Neither the Mab nor polyclonal antibody inhibited binding of a DAR antagonist, [3H]YM09151-2, to the striatal membranes. These antibodies will enable us to study the structure/function and regulation of the synthesis of DAR protein. 0 1992Academic PIcz55,Inc.
Molecular cloning of dopamine receptor has led to the identification of at least five different receptor subtypes, including Dl, D2 (long and short form), D3, D4, and D5 (l-3). Of these various subtypes, D2 receptor (DAR) has not only been implicated in the regulation of a variety of physiologic processes such as motor coordination, affective behavior and sexual performance, but also in hyper-dopaminergic disorders such as certain subtypes of schizophrenia and tardive dyskinesia (4-7). Therefore, in order to understand the etiology of hyperdopaminergic disorders as well as the control of normal dopaminergic functions, it will be essential to examine the status of DAR at the level of gene transcription, receptor protein synthesis and its maturation to 1 To whom correspondence
and reprint requests should be addressed. Abbreviations: BSA, bovine serum albumin; 4-CN, 4-chloronaphthol; DAR, dopamine D2 receptor; ELISA, enzyme linked immunosorbent assay; KLH, keyhole limpet hemocyanin; Mab, monoclonal antibody; NAPS-biotinyl; N(D-biotinylaminophenethyl)spiperone; PBS, phosphate buffered saline; PEG, polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; [3H]YM09151-2, (+)-cis-N-(l-benzyl-2[3H]-methylpyrrolidin-3-yl)-5-chloro2-methoxy-4-methylamino-benzamide.
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a functional entity. The studies on the structure/function and regulation of the DAR protein synthesis has been hampered by the unavailability of site specific antibodies to the receptor protein. To this end, we have recently raised and characterized a polyclonal antibody to a decapeptide representing amino-terminal (residue 24-34) sequence of DAR protein (8). Since polyclonal antibodies represent a mixture of different immunoglobulins molecules directed towards different epitopes on the same protein, the specificity of the antibody with regards to its ability to recognize endogenous protein can never be precisely determined. On the contrary, monoclonal antibodies (Mab) are one class of immunoglobulins directed against a single epitope and therefore, they posses a high degree of specificity. These considerations have led us to raise monclonal antibodies to a synthetic peptide predicted from DAR protein sequence (9), and compare its properties to a polyclonal antibody against a peptide from a different region of the dopamine receptor protein.
MATERIALS
AND
METHODS
Antigen preparation and immuniztztion:
Peptide I (24-34; CGSEGKADRPHYC) and peptide II (176- 185; NNTDQNECIIY) were synthesized on a Milligen Model # 9050 peptide synthesizer and purified on a Cl8 (p bondpack, Millipore) HPLC coulmn using a 5100% (v/v) acetonitrile gradient in 0.1% trifluoroacetic acid in water. The peptides were coupled individually to keyhole limpet hemocyanin (peptide-KLH) by the glutaraldehyde method (10). Balb/c mice were immunized every two weeks (4 i.p. injections; 100 pg peptide-KLH protein/injection) with peptide-KLH conjugate in phosphate buffered saline (PBS), emulsified in adjuvant (RIBI, Immunochemical Research). Titers of antisera were determined by ELISA using free peptide as antigen. The 96-well microtiter plate was coated with 50 rig/well peptide in 0.1 M sodium bicarbonate buffer, pH 9.6. After blocking with 5% bovine serum albumin (BSA, fraction V; Sigma), 50 pl of the serially diluted antibody solution was added. After incubation and washing, secondary antibody coupled to horseradish peroxidase (1: 1000 dilution; Biorad) was added. The color was developed with o-phenylenediamine and H202. The production of polyclonal antibody to peptide II in rabbits has been described elsewhere (8). Three days before fusion, the mice were boosted with 100 pg peptide-KLH conjugate. Fusions were performed using azaguanine-resistant NS/O myeloma cells. The fusion was achieved using polyethylene glycol (PEG) Mr. 1500 (Boehringer Mannheim), and the hybridoma selection was accomplished in HAT medium. Positive clones were selected by screening hybridoma supernatants in 96-well ELISA plates coated with either free peptide, peptide-KLH or KLH (50 rig/well). The hybridomas that reacted with peptide and peptide-KLH were single-cell cloned, expanded and isotyped. These clones were injected (107 cells/mouse) intraperitoneally to two mice to obtained ascites. The antibody isotypes were determined using a mouse monoclonal sub-isotyping kit (Hyclone). The clone SF4 described here secreted IgM subtype antibody.
Preparation and screening of hybridomas:
Preparation and solubilization of striatul membranes and ligand binding assays: Rat striatal membrane was prepared as described earlier (11). Briefly, the tissues were homogenized in 20 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.4; 8 mM MgC12; 5 mM EDTA), containing a mixture of protease inhibitors (10 pg/ml leupeptin, 5 pg/ml pepstatin, 5 pg/ml aprotanin and 1 mM phenylmethyl sulfonylflouride). The homogenate was centrifuged at 49,000 x g for 20 min at 4oC, the intermediate pellet was resuspended in the same buffer and the process was repeated once. The final pellet was suspended in buffer B (50 mM Tris-HCI, pH 7.4; 120 mM NaCl; 5 mM 662
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KCl; 5 mM MgC12; 1.5 mM CaC12; 1mM EDTA; 10 PM pargyline-HCl and 0.1% ascorbic acid) at a protein concentration of 2 mg/ml and stored at -800C. Liver membranes were prepared as described elsewhere (12). The striatal membranes were solubilized in CHAPS/NaCl buffer (50 mM Tris, pH 7.4; 10 mM CHAPS; 720 mM NaCl) containing protease inhibitors as described elsewhere (13). The [3H]YM-09151-2 (specific activity 71 mCi/mmol; New England Nuclear) binding to striatal membranes (8) and soluble receptor (13) was carried out as described earlier. Briefly, the soluble receptor binding assay was performed in 500 l.rl buffer B containing 60 pM radioligand and 100 lrg protein from solubilized membranes using incubation period of lhr at 370C. Following incubation, 500 l.tl of 3% (w/w) g globulin and 1 ml of 30% PEG (Mr. 78009000) in 50 mM Tris, pH 7.4 were added. The content of each tube was vortexed, kept on ice for 15 min, filtered under vacuum through GF/B filters, and washed three times with 5 ml of 10% PEG in ice cold 50 mM Tris pH, 7.4. The radioactivity on the filters was counted. Non-specific binding, defined in the presence of 2mM (-)sulpride, constituted less than 18% of the total bound radioactivity. The equilirium binding parameters were calculated after obtaining the best-fit curves for specific binding using computer program “Graph pad”. SDS-PAGE, Western blotting and ligand blotting of membranes in the presence of NAPSbiotinyl: Membrane proteins were subjected to 4-15% continuous sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose (Biorad) and were stained with Ponceau S (Sigma) to verify the uniform transfer of proteins. Nitrocellulose membranes were blocked in buffer C (10 mM Tris-HCl, pH 7.4; 154 mM NaCl; 0.02% thimerosal; 5% (w/v) non-fat dry milk) for 2 hr at room temperature and incubated with ascites or polyclonal rabbit antisera in tris buffered saline (25 mM Tris-Cl, pH 7.4; 154 mM NaCl). The membranes were washed three times in buffer D (10 mM Tris-HCl, pH 7.4; 154 mM NaCl; 0.05% Tween-20) followed by incubation at room temperature in buffer C containing 1: 1000 dilution of horseradish peroxidase conjugated secondary antibody. The blots were washed three times in Buffer D, and developed with 4-chloronaphthol(4-CN). For ligand blotting, the nitrocellulose membranes, treated overnight in buffer C containing protease inhibitors, were incubated with 200 pM NAPS-biotinyl and 50 nM ketanserin (Research Biochemical Inc.) in buffer B, washed in buffer D and were incubated with avidin-horseradish peroxidase conjugate (Biorad) in buffer C. The biotin bound proteins were detected in the presence of 4-CN. Membrane protein was determined by BCA reagent using BSA as standard (Pierce Chemicals).
RESULTS AND DISCUSSION
We have used DAR peptides 24-34 (peptide I) and 174-184 (peptide II) to raise monoclonal and polyclonal antibodies respectively. The characteristic of the polyclonal antibody against peptide II has been described in detail earlier (8). Briefly, the polyclonal antibody was specific for peptide II, and it showed no cross reactivity to peptides representing other regions of the DAR or Dl receptor (8 and unpublished observations). The low antibody titer reported earlier for peptide II antibody was increased significantly after subsequent boosters. The Mab clone SF4 hybridoma supernatant and ascites were tested for their reactivity against the peptide I in solid phase ELISA; the antibody detected free as well as peptide-KLH conjugate at relatively low antibody concentrations (l:lOO,OOO dilution). The addition of the free peptide suppressed the ELISA reactivity in a concentration-dependent manner; addition of 50 l.rg/ml peptide I resulted in to a 90% decrease in the Mab immune complex formation. On the contrary, the addition of peptide II at concentrations up to lmg/ml did not inhibit the immune complex formation (Data not 663
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Figure 1. Western blot detection of rat DAR using Mab (Panel A) and polyclonal antibody to peptide II (Panel B). Increasing concentration of rat striatal membrane protein (lane l-5; 10, 20, 40, 60, 80 pg protein respectively) was electrophoresed on a 4-15% SDS-PAGE. Antibodies (Mab, 1500; polyclonal antibody, I:250 ) were diluted in TBS and incubated with blots for 17 hr at 4°C. For compitition assay (Lane 6, Panel A), the Mab had previously been mixed with free pcptidc I (200 l.tg/lOO 1.11in TBS) and incubated overnight at 4oC. The relative molecular weights in kDa are indicated on the left.
shown). Thus, both the polyclonal and Mab appeared to be highly specific for their homologous peptides. The specificity of Mab and polyclonal antibodies were further tested on immunoblots using rat striatal membranes. As shown in Fig. 1 (Panel A) the Mab at 1500 dilution reacted with five major polypeptides with apparent molecular wights 220, 145, 95, 66 and 47 kDa. Pre-absorption of the Mab with an excess of free peptide I (2 mg/ml) blocked the recognition of all the immunoreactive proteins bands (Panel A, lane 6). The polyclonal antibody against peptide II recognized only three out of five Mab immunoreactive proteins with an apparent Mr. 220, 145 and 47 kDa (Fig. 1, Panel B). The tissue specificity of Mab was further characterized by Western blot analysis of rat striatal, cerebellar and liver membranes. Using 20 pg striatal, cerebellar and liver membranes and Mab dilution l:lOOO, only the 220 and 47 kDa proteins were clearly visible in the striatal membrane; however, under similar conditions the intensity of the 220 kDa protein band was reduced by 70-75% in cerebellum and was absent in liver (Fig. 2). The 47 kDa protein was the only Mab reactive entity present in the liver membranes and its concentration was about 50% 664
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116.297.4-
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Western blot analysisof DAR. Twenty pg protein of striatal (lane I), cercbellar (lane 2) and liver (lane 3) membranes were subjected to 4-15% SDS-PAGEand hlottcd on nitrocellulose as describedin Materials and methods. The blots were incubated with I: 1000 dilution of Mab and the immunoreactivc proteins were detected in 4-CN. Molecular weights in kDa arc indicated on the left of the figure. Figure 3.
Ligand blotting of striatal membrane with NAPS-biotinyl. increasing concentration of striatal membrane protein (Lane l-5; 10, 20.40, 60, 80 pg protein) or 80 pg ccrebellar membranes (lane 6) were clectrophoresed on a 4-1541 SDS-PAGE. The nitrocellulosc strips wcrc incuhatcd with (lane 1-7) or without (lane 8) 200 pM NAPS-biotinyl. The competition assaywas performed on 80 pg striatal membranes in the presenceof 2 pM (-)sulpridc (lane 7). The biotin labeled proteins were detected by avidin-HRP conjugate as described in Materials and methods. Molecular weights in kDa are indicated on the left of the figure.
lower compared to striatum (Fig. 2). The detection of 220, 145 and 47 kDa proteins in known dopaminergic rich regions by two different antibodies and their reduced levels in the cerebellum
and concipicous absence of 220 kDa protein in the liver suggest that these antibodies may recognize the intact DAR protein. Based on the nucleotide sequence of the DAR, the “receptor core protein” should have a molecular weight around 47 kDa (9). The presence of 47 kDa, but not 220 kDa protein (capable of binding D2 antagonist) and a Lotal absence of D2 selective ligand specific-binding by liver membranes suggests that liver may lack the mechanism(s) needed to process the receptor core protein to a ligand binding receptor form. Alternatively, it is conceivable that the Mab is recognizing a liver protein having similar epitope but unrelated to dopamine receptor. We have investigated the ability of Mab to recognize soluble DAR protein; this may facilitate affinity purification of both the ligand binding and non-ligand binding domains of the 665
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DAR. To this end, we solubilized the DAR from striatal membranes in CHAPS/NaCl buffer, followed by centrifugation at 200,000 x g for 30 min; the supernatant was evaluated for specific [3H]YM09151-2 binding in the absence and the presence of 2 PM (-)sulpride. The specific binding of [3H]YM09151-2 to straital membranes and soluble receptor at 60 pM ligand concentration was 120.6 + 12.5 and 32.9 + 1.8 fmoles/mg protein respectively. The total binding of [3H]YM09151-2 to both the intact membranes and the soluble DAR was decreased by (-)sulpride, an active antagonist, but not by (+)sulpride, an inactive isomer (data not shown), suggesting the presence of high degree of stereospecificty for the soluble DAR (14). While there was no difference between the levels of Mab reactive protein in the intact membranes and solubilized DAR preparations (Data not shown), only 27% of the total binding capacity was recovered in the soluble fraction. The reason(s) for this apparent loss of [3H]YM09151-2 binding sites following the membrane solubilization is not clear; such a decrease in the binding sites could result from many factors including receptor denaturation, proteolysis or change in the affinity of the soluble receptor. To determine the size of the ligand binding form of the DAR, ligand blot analyses were performed on striatal membranes. Incubation of the nitrocellulose membranes containing increasing concentration of striatal membrane proteins (lo-80 p.g> with DAR antagonist NAPSbiotinyl revealed three proteins of apparent Mr. 220, 120 and 80 kDa (Fig. 3). The control strips incubated in the absence of NAPS-biotinyl (Fig. 3, lane 8) or in the presence of 2 yM (-)sulpride (Fig. 3, lane 7) detected only two bands of Mr 120 and 80 kDa. In addition, the ligand blotting of cerebellar membrane (80 ug protein) failed to detect the 220 kDa protein band (Fig. 3, lane 6). These results suggest that the apparent size of DAR ligand binding subunit is a 220 kDa polypeptide. The 110 and 80 kDa proteins labeled with avidin are dopamine receptor unrelated proteins that are either endogenously biotinylated or have high affinity for avidin. In several earlier studies the size of the ligand binding form of DAR, determined after photoaffinity labeling, has been reported to be 95-l 10 kDa (8,15,16). The reason(s) for this apparent discrepancy can only be speculated at this time. These include, the degradation of ligand bound DAR protein during photoactivation to achieve cross-linking or an irreversible denaturation of 95-110 kDa DAR protein by SDS treatment prior to electrophoresis, making it incapable of recognizing NAPS-biotinyl on ligand blots. In order to study if the DAR antibodies are directed against epitopes which may be involved in the ligand binding, the effect of the two antibodies on [3H]YM09151-2 binding was performed. The [3H]YM-09151-2 binding to striatal membrane was studied at various ligand concentrations and the equilibrium binding parameters were calculated. Both Mab and polyclonal antibodies failed to significantly alter the Bmax (264.2 + 16.8, 287.7 + 7.3, 267.1 + 20.7 fmoles/mg protein for control, Mab and peptide II antibody respectively) or Kd (11.9 + 2.8; 13.8 + 2.3 and 15.7 + 2.1 pM or control, Mab and peptide II antibody respectively) for the [3H]YM09151-2 binding to the striatal membranes. The inability of Mab raised against peptide I to inhibit ligand binding is contrary to our earlier observation reporting a significant inhibition in the [3H]YM09151-2 binding to striatal membranes by the polyclonal antibody against the same 666
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antigen (8). These data suggests that Mab recognizes an epitope which may not be involved in the ligand binding. It is also conceivable that the observed inhibition of [3H]YM09151-2 binding by polyclonal antibody in our previous report (8) may be due to steric hindrance caused by polyclonal nature of the antibody. We are in the process of screening other clones for antibodies directed against the ligand binding motif of the DAR protein and their usefulness in the immunoprecipitaiton protocols. These site specific antibodies will serve as useful reagents to study the structure and the regulation of the dopamine D2 receptor protein.
ACKNOWLEDGMENTS
We thank Shiru Q. Farooqui for her help in typing and preparation of the manuscript. These studies were supported in part by US Army Research and Development Command (grant # DAMD-17-88-Z-8023). Opinions and interpretations are those of the author and are not necessarily endorsed by the US Army. In conducting research using animals, the investigators adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the Committee on Care and Use of Laboratory Animals of the Institute Laboratory Animal Resources, National Research Council (NIH), Publication # 86-23, revised 1985.
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