DEVELOPMENT OF POLYCLONAL ANTI-D2 DOPAMINE RECEPTOR ANTIBODIES USING SEQUENCE-SPECIFIC PEPTIDES

Multiple subtypes of dopamine receptors with similar properties have b een described. Ligands that have been shown to interact with a single subtype of receptor do not yet exist. The use of immunologic methods p rovides an alternative approach to distinguish receptors and receptor isoforms. Synthetic peptides corresponding to portions of the third in tracellular 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 bot h the D2L and the D2s isoforms. Rabbits were immunized monthly with pe ptide coupled to keyhole limpet hemocyanin. The immunogenicity of the peptides was established using a solid-phase radioimmunoassay. Both im munogens elicited antipeptide antibodies within 10 weeks of the primar y immunization, with titers of at least 1/10(4). An immunoprecipitatio n assay using receptors in digitonin-solubilized extracts of rat or ca nine caudate labeled with the high affinity D2 antagonist I-125-NCQ 29 8 showed that antipeptide antisera could recognize solubilized D2 rece ptors. At a dilution of 1/1000, antisera to peptide D2-284 quantitativ ely immunoprecipitated I-125-NCQ 298 binding sites from both rat and c anine striatal tissue, whereas antisera against peptide D2-244 immunop recipitated 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 t o D2-284, at a dilution of 1/1000, were able to quantitatively immunop recipitate receptor from both 293-D2L and 293-D2s cells. Antisera to D 2-244 were specific for the D2L isoform, immunoprecipitating I-125-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. Immun ohistochemical studies with anti-D2-284 demonstrated the presence of t he D2 receptor in several regions of rat brain. Immunostaining was mos t dense in the striatum, with a lateral to medial gradient and patches of lighter staining. Immunoreactivity was negligible with preimmune s erum or peptide-blocked immune serum. Immunoreactive processes were se en in the nucleus accumbens and ventral pallidum, as well as in the hy pothalamus. The high affinity binding of agonist to D2 dopamine recept ors was disrupted by anti-D2-284 but not anti-D2-244 antisera, implica ting the internal region of the third intracellular loop represented b y peptide D2-284 as a potential determinant of receptor-guanine nucleo tide-binding protein coupling.