Development of Polyclonal Anti-D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction

Authors

  • Virginia A. Boundy,

    1. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
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  • Robert R. Luedtke,

    1. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
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    • Dr. R. R. Luedtke is Department of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107, U.S.A.

  • Perry B. Molinoff

    Corresponding author
    1. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
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Address correspondence and reprint requests to Dr. P. B. Molinoff at M102 John Morgan Building, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104–6084, U.S.A.

Abstract

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [125I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [125I]-NCQ 298 bound reversibly and with high affinity (KD= 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [125I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins 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 caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.

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