title: Antibodies against synthetic peptides predicted from the nucleotide sequence of D2 receptor recognize native dopamine receptor protein in rat striatum.
abstract: Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. 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 D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-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 D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 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 [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Neurosciences, Pennington Biomedical Research Center, Baton Rouge, LA 70808.
authors.forename: S M
authors.initials: SM
authors.lastname: Farooqui
authors.forename: J W
authors.initials: JW
authors.lastname: Brock
authors.forename: A
authors.initials: A
authors.lastname: Hamdi
authors.forename: C
authors.initials: C
authors.lastname: Prasad
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D001483:Base Sequence; D001549:Benzamides / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D005544:Forecasting; D015151:Immunoblotting; D008297:Male; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000235:genetics / Q000276:immunology*; D017448:Receptors, Dopamine D2
chemical_list: D000906:Antibodies; D001549:Benzamides; D010455:Peptides; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; C030265:nemonapride