Development of polyclonal anti-D2 dopamine receptor antibodies using sequence-specific peptides.

Multiple subtypes of dopamine receptors with similar properties have been described. Ligands that have been shown to interact with a single subtype of receptor do not yet exist. The use of immunologic methods provides an alternative approach to distinguish receptors and receptor isoforms. Synthetic peptides corresponding to portions of the third intracellular loops of the two isoforms of the rat D2 dopamine receptor were used to elicit polyclonal antipeptide antibodies. Peptide D2-244 is unique to the D2L isoform, whereas peptide D2-284 is present in both the D2L and the D2S isoforms. Rabbits were immunized monthly with peptide coupled to keyhole limpet hemocyanin. The immunogenicity of the peptides was established using a solid-phase radioimmunoassay. Both immunogens elicited antipeptide antibodies within 10 weeks of the primary immunization, with titers of at least 1/10(4). An immunoprecipitation assay using receptors in digitonin-solubilized extracts of rat or canine caudate labeled with the high affinity D2 antagonist 125I-NCQ 298 showed that antipeptide antisera could recognize solubilized D2 receptors. At a dilution of 1/1000, antisera to peptide D2-284 quantitatively immunoprecipitated 125I-NCQ 298 binding sites from both rat and canine striatal tissue, whereas antisera against peptide D2-244 immunoprecipitated 40% of the D2 receptors solubilized from rat caudate. The selectivity of the antisera was determined using 293 cells transfected with cDNA encoding the D2L or the D2S isoform of receptor. Antisera to D2-284, at a dilution of 1/1000, were able to quantitatively immunoprecipitate receptor from both 293-D2L and 293-D2S cells. Antisera to D2-244 were specific for the D2L isoform, immunoprecipitating 125I-NCQ 298 binding sites from 293-D2L cells but not from 293-D2S cells. Anti-D2-284 specifically recognized multiple bands of 100 kDa, 68 kDa, and 50 kDa in immunoblots of denatured preparations of rat caudate. Immunohistochemical studies with anti-D2-284 demonstrated the presence of the D2 receptor in several regions of rat brain. Immunostaining was most dense in the striatum, with a lateral to medial gradient and patches of lighter staining. Immunoreactivity was negligible with preimmune serum or peptide-blocked immune serum. Immunoreactive processes were seen in the nucleus accumbens and ventral pallidum, as well as in the hypothalamus. The high affinity binding of agonist to D2 dopamine receptors was disrupted by anti-D2-284 but not anti-D2-244 antisera, implicating the internal region of the third intracellular loop represented by peptide D2-284 as a potential determinant of receptor-guanine nucleotide-binding protein coupling.