Antibodies against synthetic peptides predicted from the nucleotide sequence of D2 receptor recognize native dopamine receptor protein in rat striatum.
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)
Farooqui
S M
SM
Laboratory of Neurosciences, Pennington Biomedical Research Center, Baton Rouge, LA 70808.
Brock
J W
JW
Hamdi
A
A
Prasad
C
C
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
D000906:Antibodies; D001549:Benzamides; D010455:Peptides; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; C030265:nemonapride