Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary

Susan Senogles, N. Amlaiky, P. Falardeau, M. G. Caron

Research output: Contribution to journalArticle

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Abstract

The D2-dopamine receptor from bovine anterior pituitary has been purified ~33,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized carboxymethyleneoximinospiperone-Sepharose, Datura stramonium lectin-agarose, and hydroxylapatite chromatography. The purification yields a single polypeptide band of M(r) ~120,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed by labeling with radioiodinated Bolton-Hunter reagent, Coomassie Blue, or silver staining. The purified D2 receptor preparations display a specific activity of ~5.3 nmol of [3H]spiperone bound per mg of protein. In detergent solutions, the purified receptor has a K(D) for [3H]spiperone of 5-8 nM; however, after reinsertion of the purified protein into phospholipid vesicles, a K(D) of ~160 pM is obtained, similar to that found for the receptor in crude membrane preparations. Several lines of evidence document that this polypeptide contains the ligand binding site as well as the functional activity of the D2 receptor. The M(r) ~120,000 peptide can be covalently labeled by the affinity probe, 125I-bromoacetyl-N-(p-aminophenethyl)spiperone, with the pharmacological specificity expected of a D2-dopamine receptor. Agonist and antagonist ligands compete for [3H]spiperone binding to purified receptors in phospholipid vesicles with a rank order of potency and selectivity typical of a D2-dopamine receptor. Moreover, when reinserted into phospholipid vesicles with purified brain G(i)/G(o), the purified D2 receptors mediate the agonist stimulation of 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-proteins with a typical D2-dopaminergic order of potency. These data suggest that we have purified an intact functional D2-dopamine receptor.

Original languageEnglish (US)
Pages (from-to)18996-19002
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number35
StatePublished - Jan 1 1988

Fingerprint

Spiperone
Dopamine D2 Receptors
Purification
Phospholipids
Sepharose
Peptides
Brain
Ligands
Affinity chromatography
Agarose Chromatography
Silver Staining
Guanosine
Durapatite
Chromatography
Electrophoresis
Affinity Chromatography
Silver
GTP-Binding Proteins
Sodium Dodecyl Sulfate
Detergents

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary. / Senogles, Susan; Amlaiky, N.; Falardeau, P.; Caron, M. G.

In: Journal of Biological Chemistry, Vol. 263, No. 35, 01.01.1988, p. 18996-19002.

Research output: Contribution to journalArticle

Senogles, Susan ; Amlaiky, N. ; Falardeau, P. ; Caron, M. G. / Purification and characterization of the D2-dopamine receptor from bovine anterior pituitary. In: Journal of Biological Chemistry. 1988 ; Vol. 263, No. 35. pp. 18996-19002.
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abstract = "The D2-dopamine receptor from bovine anterior pituitary has been purified ~33,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized carboxymethyleneoximinospiperone-Sepharose, Datura stramonium lectin-agarose, and hydroxylapatite chromatography. The purification yields a single polypeptide band of M(r) ~120,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed by labeling with radioiodinated Bolton-Hunter reagent, Coomassie Blue, or silver staining. The purified D2 receptor preparations display a specific activity of ~5.3 nmol of [3H]spiperone bound per mg of protein. In detergent solutions, the purified receptor has a K(D) for [3H]spiperone of 5-8 nM; however, after reinsertion of the purified protein into phospholipid vesicles, a K(D) of ~160 pM is obtained, similar to that found for the receptor in crude membrane preparations. Several lines of evidence document that this polypeptide contains the ligand binding site as well as the functional activity of the D2 receptor. The M(r) ~120,000 peptide can be covalently labeled by the affinity probe, 125I-bromoacetyl-N-(p-aminophenethyl)spiperone, with the pharmacological specificity expected of a D2-dopamine receptor. Agonist and antagonist ligands compete for [3H]spiperone binding to purified receptors in phospholipid vesicles with a rank order of potency and selectivity typical of a D2-dopamine receptor. Moreover, when reinserted into phospholipid vesicles with purified brain G(i)/G(o), the purified D2 receptors mediate the agonist stimulation of 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-proteins with a typical D2-dopaminergic order of potency. These data suggest that we have purified an intact functional D2-dopamine receptor.",
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