Journal of Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striaturn *Shakeel M. Farooqui, *Jeffery W. Brock, *Anwar Hamdi, and *tChandan Prasad *Laboratory of Neurosciences, Pennington Biomedical Research Center, Baton Rouge, and ?Section of Endocrinology, Department of Medicine, Louisiana State University Medical Center, New Orleans, Louisiana, U.S.A.

Abstract: Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEGKADRPHYC) and peptide I1 (NNTDQNECIIY), corresponding to 24-34 and 176- 185 from the NHz terminus, respectively, were conjugated to keyhole limpet hemocyanin and injected into rabbits. Peptide 1 showed a greater immunogenic response than did peptide 11. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2receptor by its high affinity for the D2selective photoaffinity probe '251-N'-azidospiperone (I2*I-NAPS).Photoaffinity labeling of the 90-kDa protein by I2%NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no Dz ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2,a dopamine Dz

receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of t3H]YM-0915 1-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in B,,, (203.5 k 11.0 and 164.6 t 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in K D . These results suggest that polyclonal antisera raised against peptide I exhibited specific antibodies for the dopamine D2receptor protein. The primary epitope for this antibody is at or near the ligand binding site which can be recognized in both denatured and native receptor protein in stnatal membranes. Key Words: Dz-dopamine receptors-Dz receptor antibodie~-[~H]YM-0915I 2 binding-'251-N'-azidospiperone affinity labeling. Farooqui S. M. et al. Antibodies against synthetic peptides predicted from the nucleotide sequence of D2receptor recognize native dopamine receptor protein in rat striatum. J. Neurochem. 57, 1363-1369 (1991).

Differences in biochemical, physiological, and pharmacological properties have led to the classification of dopamine (DA) receptors into three subtypes-D, ,DZ, and D3.For example, activation of Dl or Dz results in stimulation or inhibition of cyclic AMP (CAMP)production, respectively, in the same neuron (Seeman, 1980; Creese et al., 1983; Cooper et al., 1985; Sokoloff

et al., 1990). The DA receptors, particularly those of D2subtype, have been suggested to play an important role in a number of neurological and psychiatric disorders, including tardive dyskinesia (Cross et al., 1983), schizophrenia, and Alzheimer's and Parkinson's diseases (Seeman, 1985; Seeman et al., 1987; Farde et al., 1988). Therefore, a better understanding of factors reg-

Received February 28, 1991; accepted March 19, 1991. Address correspondence and reprint requests to Dr. C. Prasad at Laboratory of Neurosciences, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808, U.S.A. Abbreviations used: BSA, bovine serum albumin; CAMP, cyclic AMP; 4-CN, 4-chloronaphthol; DA, dopamine; D2 receptor, dopamine receptor subtype DZ; ELISA, enzyme-linked immunosorbent assay; Gi, inhibitory type GTP-binding protein; IgG, immunoglobulin

G; KLH, keyhole limpet hemocyanin; '"1-NAPS, '2SI-N'-azidospiperone; PBS, phosphate-buffered saline; SDS-PAGE, sodium dcdecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TMS, transmembrane spanning; ['H]YM-09 151-2, (+)-cis&( 1 -ben~yL2-[~H]methylpyrrolidin - 3 - yl) - 5 - chloro - 2 methoxy - 4 - methylamino benzamide.

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ulating D2 receptor synthesis and its tissue concentration will greatly facilitate the management and understanding of the above disorders. At present, the tissue concentration of D2 receptors is measured by calculating the maximum number of ligand binding sites (Bmax) using D2-selective radioactive or fluorescent ligand (Seeman, 1980; Severson and Wilcox, 1988; Ariano et al., 1989). An indirect determination of D2 receptors by measurement of the mRNA levels has recently been made possible by the availability of cDNA and RNA probes (Mengod et al., 1989; Meador-Woodruff et al., 1989; Mansour et al., 1990). Although there is generally a good correlation between the distribution of D2 receptor mRNA and D2-selective ligand binding, some areas showed a lack of such correlation (Mansour et al., 1990). Measurement of D2 receptor protein or its immunocytochemical localization in different regions of the brain was not possible due to lack of a specific D2 receptor antibody. The availability of such an antibody will allow us to achieve the goal of demonstrating the expression of D2 receptor protein in various regions of the brain. The D2 receptor from rat brain has been cloned and the primary structure was deduced from the cDNA sequence (Bunzow et al., 1989). Two oligopeptides corresponding to 24-34 and 176-1 85 amino acid from the amino terminus of the D2receptor, exhibiting little or no sequence homology with other inhibitory type GTP-binding protein (Gi)-coupled receptors such as P2-adrenergic receptor, a2-adrenergic receptor, muscarinic receptor, and substance K receptor (Bunzow et al., 1989), were chosen to raise antibodies to the DA D2 receptor in rabbits. The data presented here show the location of one antigenic site between amino acids 24-34 within the D2 receptor protein. The antibody raised against this epitope recognizes both native and denatured DA receptor protein and also inhibits D2 antagonist binding to receptors.

MATERIALS AND METHODS (+)-cis-N-(1-Benzyl-2-[3H]methylpyrrolidin-3-yl-5-chloro2-methoxy-4-methylaminobenzamide ([3H]YM-09I 5 1-2) (sp act 7 1 Ci/mmol) and '251-N'-azidospiperone('251-NAPS)(sp act 2,200 Ci/mmol) were purchased from New England Nuclear (Boston, MA, U.S.A.). Goat anti-rabbit (H & L) immunoglobulin G (IgG) conjugated with horseradish peroxidase was purchased from Bio-Rad (Richmond, CA, U.S.A.). RIBI emulsion adjuvant system was purchased from RIBI immunochemical Research (Hamilton, MT, U.S.A.). Iodobeads, BCA, and glutaraldehyde were purchased from Pierce Chemical (Rockford, IL, U.S.A.).

Peptide synthesis Two peptides were synthesized by LSU Medical Center Core Laboratories on a Pepsynthesizer 11, using 9-fluoroenylmethoxycarbonyl-protected pentafluorophenyl-activated ester amino acids with pepsyn KA solid support. Peptide I (CGSEGKADARPHYC)corresponds to amino acid 24-34

J Neurochi,m , Vol. 57, No 4, 1991

plus two additional cysteines at both termini, whereas peptide I1 (NTDQNECIIY) corresponds to amino acid 176-1 85 plus an additional tyrosine at the carboxy terminus. In peptide I, the amino and carboxyl terminal cysteines allowed reductive cyclization of the peptide, and thereby increased the probability of generating a "midportion" directed antiserum. A similar approach has been employed earlier to raise antibody against a thyrotropin-releasing hormone precursor polypeptide (Lechan et al., 1986). The purity of the peptides was assessed by HPLC on a CI8column (pBondpak, Millipore, Bedford, MA, U.S.A.) using a 5-100% (vol/vol) acetonitrile gradient in 0.1% (vol/vol) trifluoroaceticacid (TFA) in water. Peptides were detected at 240 nm, using on-line spectrophotometry.

Antigen preparation and immunization Peptides I and I1 were coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde as described by Duggan and Stephenson (1 989). In brief, peptides I and I1 (5 mg) and corresponding iodinated peptide (sp act, 145,000- 150,000 cpm/pg) and KLH (5 mg) were dissolved in 5 ml of 0.1 M NaHC03. Glutaraldehyde was added to a final concentration of 0.05% (vol/vol). The samples were incubated for 5 h at room temperature. The reaction was stopped by the addition ofglycine ethylester to a final concentration of 100 mM. The tubes were left at room temperature for an additional 30 min. The peptide-KLH conjugates were separated from the free peptide on a 5-ml Sephadex G-25 desalting column equilibrated with phosphate-buffered saline (PBS). The coupling efficiency of the peptide to KLH was measured by protein-bound radioactivity in trichloroacetic acid (TCA) precipitate and found to be 95-98% of the total peptide. The peptide-KLH conjugateswere resuspended in PBS and stored at -20°C. The peptide-KLH conjugates were emulsified in RIBI adjuvant system according to the manufacturer's instructions. One milliliter of emulsion (containing approximately 0.2 pmol of peptide) was injected into each rabbit as follows: 300 pI intradermally (50-pl aliquots at six sites), 400 p1 intramuscularly (200 pl in each hind leg), 100 pl subcutaneously in the neck region, and 200 pl intrapentoneally. All rabbits were boosted with 0.1 pmol of peptide conjugate every I5 days after a 15-mlblood sample was drawn from each rabbit for antibody titer determination.

Antibody screening and enzyme-linked immunosorbent assay (ELISA) All rabbits were checked for the immune response against the corresponding peptides on microtiter plates as described by Jaffe et al. (1987). In brief, the peptides ( I pg/ml), dissolved in 0.05 M sodium carbonate buffer, pH 9.6, were added to each well. Plates were rocked for 20 min and then kept at 4°C for 14-16 h. The wells were then washed twice with PBS, filled with 4% (wt/vol) fat-free milk in PBS, and incubated at 25°C for 2 h. Finally, the peptide-coated wells were washed three times with PBS, then filled with PBS and stored at 4°C. Fifty microliters of serially diluted antisera ( I X 10'-1 X lo5) in PBS were transferred to coated microtiter plates and incubated for 12-16 h at 4'C. The plates were washed twice with PBS, twice with 0.05% Tween-20 in PBS, and finally twice with PBS. The development of immunogenic response was measured by the addition of goat anti-rabbit antibodies conjugated with horseradish peroxidase [ 1:2,000

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ANTIBODIES TO DOPAMINE RECEPTORS dilution in PBS containing 0.05% bovine serum albumin (BSA)] to each well with an additional incubation for 1 h at 37°C. After washing as before, the bound antibodies were detected by color development in the presence of diaminobenzidine in 0.2 M citrate/phosphate buffer, pH 4.3, containing 0.05% (vol/vol) HzOz. After incubation in the dark at 25"C, the reaction was terminated by the addition of 25 pl of 3 M H2S04and the absorbance was measured at 492 nm in a Bio-Rad microplate reader (Model 3550). The antibody titer was defined as the serum dilution that gave a 50% absorbance at 495 nm compared to 1: 10 diluted serum.

Preparation of striatal membranes and ligand binding assays The striata from adult male Sprague-Dawley rats were quickly removed after decapitation and homogenized (Polytron setting 60 for 20 s) in 20 volumes of ice-cold buffer A (50 mM Tris-HC1, pH 7.4; 8 mM MgClz; 5 mM EDTA, containing protease inhibitors, 10 pg/ml of leupeptin, 5 pg/ ml of pepstatin, 5 pg/ml of aprotonin, and I mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 18,000 g for 20 rnin at 4OC. The pellet was resuspended in buffer B (50 mM Tris-HC1, pH 7.4; 120 mMNaC1; 5 mM KCI; 5 mM MgClz; 1.5 mM CaC12; 1 mM EDTA; 10 pM pargyline hydrochloride; and 0.1%ascorbic acid) at a protein concentration of 2 mg/ml. Protein was measured using BCA reagent according to the manufacturer's instructions. The [3H]YM-09I5 1-2 binding was camed out as described by Terai et al. ( 1989). In brief, membranes (200 pg of protein) were incubated for 60 rnin at 25°C under reduced light with increasing concentrations of [3H]YM-0915 1-2 (5-200 pM) in a final volume of 2 ml of buffer B. The binding was terminated by rapid filtration of the reaction mixture through a glass fiber filter (GF/B), followed by four washes with 4 ml of ice-cold buffer B. The filters were then dried and counted for radioactivity in 7 ml of liquid scintillation fluid. Nonspecific binding, defined in the presence of 100 p M spiperone, constituted