Development and characterization of polyclonal antibodies for the D2 and D3 dopamine receptors

The D2 and D3 dopamine receptors, members of the G protein-coupled receptor family, exhibit a similar pharmacologic profile. Antibodies to the D2 receptor isoforms (D2$\sb{\rm L},$ D2$\sb{\rm S})$ and the D3 receptor were developed to enable study of these receptor proteins. The D2 isoforms differ by the presence of an additional 29 amino acids in the third intracellular loop (i$\sb3$) of the D2$\sb{\rm L}$ receptor. A segment of the i$\sb3$ loop of each D2 receptor isoform was subcloned into the expression vector pNMHUBpoly. The expressed ubiquitin-D2i$\sb3$ loop fusion proteins were used as immunogens to generate anti-D2 receptor antibodies. Synthetic peptides corresponding to portions of the i$\sb3$ loops of the D2 and D3 receptors were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Isoform-selective anti-D2$\sb{\rm L}$ antibodies specifically recognize the D2$\sb{\rm L}$ receptor expressed in 293 cells. The non-isoform-selective anti-D2 receptor antibodies quantitatively immunoprecipitate D2$\sb{\rm L}$ and D2$\sb{\rm S}$ receptors from solubilized canine and rat caudate and transfected cell lines, and recognize the receptors in Western blots of these tissues. Examination of the coupling of G proteins to the D2 receptor using these antibodies implicates the internal portion of the i$\sb3$ loop as a potential determinant of this interaction. The anti-D2 antibodies raised to the i$\sb3$ loop fusion proteins or to a peptide present in both isoforms inhibit the high-affinity binding of agonist. In contrast, the isoform-selective anti-D2$\sb{\rm L}$ antibodies do not modulate agonist binding. A baculovirus expression system was used to generate large amounts of receptor for pharmacologic studies and antibody screening. Insect cells infected with recombinant D3 virus express 15-20 pmol receptor/mg protein, whereas those infected with recombinant D2$\sb{\rm L}$ or D2$\sb{\rm S}$ virus express 8-10 pmol receptor/mg protein. These receptors are pharmacologically similar to those expressed in mammalian systems, although no high-affinity, GTP-sensitive binding was seen. The anti-D3 receptor antibodies selectively immunoprecipitate the D3 receptor from rat caudate and specifically recognize the D3 receptor in Western blots of rat caudate, demonstrating expression of the receptor protein in mammalian brain