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Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D 2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum
Author(s) -
Farooqui Shakeel M.,
Brock Jeffery W.,
Hamdi Anwar,
Prasad Chandan
Publication year - 1991
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1991.tb08302.x
Subject(s) - peptide , keyhole limpet hemocyanin , microbiology and biotechnology , peptide sequence , receptor , biochemistry , biology , chemistry , ligand (biochemistry) , antibody , gene , immunology
Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D 2 receptor were synthesized. Peptide I (CGSEG‐KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24–34 and 176–185 from the NH 2 terminus, respectively, were conjugated to keyhole 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 D 2 receptor by its high affinity for the D 2 selective photoaffinity probe 125 I‐AP‐azidospiperone ( 125 I‐NAPS). Photoaffinity labeling of the 90‐kDa protein by 125 I‐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 D 2 ligand binding sites. The antibody to peptide I inhibited the binding of [ 3 H] YM‐09151‐2, a dopamine D 2 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 p M ligand concentration. The data on the equilibrium inhibition kinetics of [ 3 H] YM‐09151‐2 binding to striatal membranes were examined in the presence of antibody and showed a 25–30% decrease in B max (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 K D . These results suggest that polyclonal antisera raised against peptide I exhibited specific antibodies for the dopamine D 2 receptor 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 striatal membranes.