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

Virginia A. Boundy, Robert R. Luedtke, Perry B. Molinoff

Research output: Contribution to journalArticle

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2181-2191
Number of pages11
JournalJournal of Neurochemistry
Volume60
Issue number6
DOIs
StatePublished - Jun 1993

Fingerprint

Dopamine D2 Receptors
Dopamine Receptors
Rats
Immune Sera
Fusion reactions
Antibodies
Proteins
Chromatography
GTP-Binding Proteins
Immunoprecipitation
Escherichia coli
Radioimmunoassay
Canidae
Assays
Protein Isoforms
Complementary DNA
Rabbits
Peptides
NCQ 298

Keywords

  • Anti‐(D2 dopamine receptor) antibodies
  • Fusion proteins
  • G protein interaction. J. Neurochem.
  • Immunoprecipitation
  • Receptor‐G protein coupling. Boundy V. A. et al. Development of polyclonal anti‐D2 dopamine receptor antibodies to fusion proteins: Inhibition of D2 receptor
  • [I]NCQ 298

Cite this

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title = "Development of Polyclonal Anti‐D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction",
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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Development of Polyclonal Anti‐D2 Dopamine Receptor Antibodies to Fusion Proteins : Inhibition of D2 Receptor–G Protein Interaction. / Boundy, Virginia A.; Luedtke, Robert R.; Molinoff, Perry B.

In: Journal of Neurochemistry, Vol. 60, No. 6, 06.1993, p. 2181-2191.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Development of Polyclonal Anti‐D2 Dopamine Receptor Antibodies to Fusion Proteins

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AU - Boundy, Virginia A.

AU - Luedtke, Robert R.

AU - Molinoff, Perry B.

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AB - 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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