The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein

S. E. Senogles, J. L. Benovic, N. Amlaiky, C. Unson, G. Milligan, R. Vinitsky, Allen M. Spiegel, M. G. Caron

Research output: Contribution to journalArticle

108 Citations (Scopus)

Abstract

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca 2+ mobilization/phosphoinositide pathways. The D 2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) ([ 35S]GTPγS) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [ 32P]ADP-ribosylation of affinity-purified D 2 receptor preparations by pertussis toxin revealed the presence of a substrate of M(r) 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [ 32P]ADP-ribosylated endogenous N protein, transducin, and N(i) and N(o) from brain revealed similarities but not identity between the endogenous pituitary N protein and brain N(i) and N(o). Immunoblotting of the partially purified D 2 receptor preparations showed an M(r) 39,000-40,000 band with an N(i)-specific antiserum raised against a synthetic peptide, and with RV3, an N(o)-specific antiserum, but not with CW6, an antiserum strongly reactive with brain N(i). Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D 2 receptor. As measured by [ 35S]GTPγS binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D 2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that N(o) and/or a form of N(i) distinct from the M(r) 41,000 pertussis toxin substrate (N(i)) is the predominant N protein functionally coupled with the D 2-dopamine receptor of anterior pituitary.

Original languageEnglish (US)
Pages (from-to)4860-4867
Number of pages8
JournalJournal of Biological Chemistry
Volume262
Issue number10
StatePublished - 1987
Externally publishedYes

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Guanine Nucleotides
Pertussis Toxin
Dopamine Receptors
Carrier Proteins
Immune Sera
Brain
Proteins
GTP Phosphohydrolases
Adenosine Diphosphate
Transducin
Affinity chromatography
Peptides
Guanosine
Pancreatic Elastase
Substrates
Phosphatidylinositols
Electrophoresis
Affinity Chromatography
Immunoblotting
Adenylyl Cyclases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Senogles, S. E., Benovic, J. L., Amlaiky, N., Unson, C., Milligan, G., Vinitsky, R., ... Caron, M. G. (1987). The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein. Journal of Biological Chemistry, 262(10), 4860-4867.

The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein. / Senogles, S. E.; Benovic, J. L.; Amlaiky, N.; Unson, C.; Milligan, G.; Vinitsky, R.; Spiegel, Allen M.; Caron, M. G.

In: Journal of Biological Chemistry, Vol. 262, No. 10, 1987, p. 4860-4867.

Research output: Contribution to journalArticle

Senogles, SE, Benovic, JL, Amlaiky, N, Unson, C, Milligan, G, Vinitsky, R, Spiegel, AM & Caron, MG 1987, 'The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein', Journal of Biological Chemistry, vol. 262, no. 10, pp. 4860-4867.
Senogles, S. E. ; Benovic, J. L. ; Amlaiky, N. ; Unson, C. ; Milligan, G. ; Vinitsky, R. ; Spiegel, Allen M. ; Caron, M. G. / The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein. In: Journal of Biological Chemistry. 1987 ; Vol. 262, No. 10. pp. 4860-4867.
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abstract = "Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca 2+ mobilization/phosphoinositide pathways. The D 2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) ([ 35S]GTPγS) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [ 32P]ADP-ribosylation of affinity-purified D 2 receptor preparations by pertussis toxin revealed the presence of a substrate of M(r) 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [ 32P]ADP-ribosylated endogenous N protein, transducin, and N(i) and N(o) from brain revealed similarities but not identity between the endogenous pituitary N protein and brain N(i) and N(o). Immunoblotting of the partially purified D 2 receptor preparations showed an M(r) 39,000-40,000 band with an N(i)-specific antiserum raised against a synthetic peptide, and with RV3, an N(o)-specific antiserum, but not with CW6, an antiserum strongly reactive with brain N(i). Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D 2 receptor. As measured by [ 35S]GTPγS binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D 2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that N(o) and/or a form of N(i) distinct from the M(r) 41,000 pertussis toxin substrate (N(i)) is the predominant N protein functionally coupled with the D 2-dopamine receptor of anterior pituitary.",
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T1 - The D 2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein

AU - Senogles, S. E.

AU - Benovic, J. L.

AU - Amlaiky, N.

AU - Unson, C.

AU - Milligan, G.

AU - Vinitsky, R.

AU - Spiegel, Allen M.

AU - Caron, M. G.

PY - 1987

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N2 - Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca 2+ mobilization/phosphoinositide pathways. The D 2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) ([ 35S]GTPγS) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [ 32P]ADP-ribosylation of affinity-purified D 2 receptor preparations by pertussis toxin revealed the presence of a substrate of M(r) 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [ 32P]ADP-ribosylated endogenous N protein, transducin, and N(i) and N(o) from brain revealed similarities but not identity between the endogenous pituitary N protein and brain N(i) and N(o). Immunoblotting of the partially purified D 2 receptor preparations showed an M(r) 39,000-40,000 band with an N(i)-specific antiserum raised against a synthetic peptide, and with RV3, an N(o)-specific antiserum, but not with CW6, an antiserum strongly reactive with brain N(i). Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D 2 receptor. As measured by [ 35S]GTPγS binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D 2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that N(o) and/or a form of N(i) distinct from the M(r) 41,000 pertussis toxin substrate (N(i)) is the predominant N protein functionally coupled with the D 2-dopamine receptor of anterior pituitary.

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