Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies

Abstract

A baculovirus expression system provided an enriched source of biologically and immunologically active D3 dopamine receptors. Receptors expressed in Spodoptera frugiperda insect (Sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, N-propylnorapomorphine. The binding of agonists was not sensitive to GTP. Antisera raised against synthetic peptides in the third intracellular loop of the D3 dopamine receptor immunoprecipitated binding sites for (S)-3-[125I]-iodo-2-hydroxy-5,6-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)- methyl]-benzamide from solubilized extracts of infected Sf9 cells and detergent extracts of rat caudate. These antisera specifically recognized a single band on immunoblots of Sf9 cells infected with recombinant D3 baculovirus. Both the immunoprecipitation and immunoblot reactions were blocked by preincubation of the antisera with the immunization peptide. These results suggest that the D3 receptor protein is expressed in rat brain.