DEVELOPMENT OF POLYCLONAL ANTI-D2 DOPAMINE RECEPTOR ANTIBODIES TO FUSION PROTEINS - INHIBITION OF D2 RECEPTOR-G PROTEIN-INTERACTION

Portions of the cDNA encoding the third intracellular loop (i3 loop) o f the long and short isoforms of the rat D2 dopamine receptor were sub cloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to im munize rabbits for the production of polyclonal anti-receptor antisera . The anti-fusion protein antisera recognized synthetic peptides corre sponding to segments of the i3 loops of D2 dopamine receptors in a sol id-phase radioimmunoassay. Antisera were tested in an immunoprecipitat ion assay using the reversible D2 antagonist [I-125]NCQ 298 and digito nin-solubilized extracts of canine and rat caudate. [I-125]NCQ 298 bou nd reversibly and with high affinity (K(D) = 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. Th e anti-UBI-D2i3L and anti-UBI-D2i3S antisera were able to immunoprecip itate quantitatively D2 dopamine receptors labeled with [I-125]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ab ility to affect the coupling of D2 dopamine receptors to GTP-binding p roteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3L and anti-UBI-D2i3S antisera were able to inhibit the high-affinity binding of the agonist N-propylnorapomorphine to digitonin-solubilized rat ca udate. These findings indicate that the i3 loop of the D2 dopamine rec eptor is an important determinant for coupling of the G protein.