Functional reconstitution of β-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase

S. E. Pedersen, E. M. Ross

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Abstract

A procedure for the functional reconstitution of β-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. β-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of β-adrenergic agonists. This stimulation of the activation of G/F was specific for β-adrenergic agonists and could be specifically blocked by β-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active β-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F receptor was reconstituted.

Original languageEnglish (US)
Pages (from-to)7228-7232
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume79
Issue number23 I
StatePublished - 1982

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GTP-Binding Proteins
Adenylyl Cyclases
Adrenergic Receptors
Adrenergic Agonists
Phospholipids
Dihydroalprenolol
Adrenergic Antagonists
Guanine Nucleotides
Guanosine
Erythrocyte Membrane
Phosphatidylcholines
Detergents
Gel Chromatography
Carrier Proteins
Cell Membrane
Rabbits
Liver

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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abstract = "A procedure for the functional reconstitution of β-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. β-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of β-adrenergic agonists. This stimulation of the activation of G/F was specific for β-adrenergic agonists and could be specifically blocked by β-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active β-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F receptor was reconstituted.",
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T1 - Functional reconstitution of β-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase

AU - Pedersen, S. E.

AU - Ross, E. M.

PY - 1982

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N2 - A procedure for the functional reconstitution of β-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. β-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of β-adrenergic agonists. This stimulation of the activation of G/F was specific for β-adrenergic agonists and could be specifically blocked by β-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active β-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F receptor was reconstituted.

AB - A procedure for the functional reconstitution of β-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. β-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of β-adrenergic agonists. This stimulation of the activation of G/F was specific for β-adrenergic agonists and could be specifically blocked by β-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active β-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F receptor was reconstituted.

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