Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors

T. K. Harden, Leslie Anne Petch Lee, S. F. Traynelis, G. L. Waldo

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4°C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate ([3H]QNB) at 37°C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t( 1/2 ) of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of 'lost' [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper at a1. regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35% of the muscarinic receptors from cells treated for 30 min with 100 μM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a 'light vesicle' form occurred with a t( 1/2 ) (~) 10 min, and reversed with a t( 1/2 ) (~) 20 min. In contrast to previous results in this cell line regarding β-adrenergic receptors, agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.

Original languageEnglish (US)
Pages (from-to)13060-13066
Number of pages7
JournalJournal of Biological Chemistry
Volume260
Issue number24
StatePublished - Dec 1 1985
Externally publishedYes

Fingerprint

Cholinergic Receptors
Muscarinic Receptors
Cholinergic Agents
Carbachol
Membranes
Binding Sites
Sucrose
N-Methylscopolamine
Cells
Quinuclidinyl Benzilate
Adrenergic Agonists
Light
Hydrophilicity
Cell membranes
Guanosine Triphosphate
Astrocytoma
GTP-Binding Proteins
Hydrophobic and Hydrophilic Interactions
Cell Membrane
Recovery

ASJC Scopus subject areas

  • Biochemistry

Cite this

Harden, T. K., Petch Lee, L. A., Traynelis, S. F., & Waldo, G. L. (1985). Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. Journal of Biological Chemistry, 260(24), 13060-13066.

Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. / Harden, T. K.; Petch Lee, Leslie Anne; Traynelis, S. F.; Waldo, G. L.

In: Journal of Biological Chemistry, Vol. 260, No. 24, 01.12.1985, p. 13060-13066.

Research output: Contribution to journalArticle

Harden, TK, Petch Lee, LA, Traynelis, SF & Waldo, GL 1985, 'Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors', Journal of Biological Chemistry, vol. 260, no. 24, pp. 13060-13066.
Harden TK, Petch Lee LA, Traynelis SF, Waldo GL. Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. Journal of Biological Chemistry. 1985 Dec 1;260(24):13060-13066.
Harden, T. K. ; Petch Lee, Leslie Anne ; Traynelis, S. F. ; Waldo, G. L. / Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 24. pp. 13060-13066.
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abstract = "Incubation of 1321N1 human astrocytoma cells with carbachol resulted in a rapid loss of binding of [3H]N-methylscopolamine ([3H]NMS) to muscarinic cholinergic receptors measured at 4°C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate ([3H]QNB) at 37°C occurred at a slower rate. Upon removal of agonist from the medium, the lost [3H]NMS binding sites measured on intact cells recovered with a t( 1/2 ) of approximately 20 min, but only to the level to which [3H]QNB binding sites had been lost; no recovery of 'lost' [3H]QNB binding sites occurred over the same period. Based on these data and the arguments of Galper at a1. regarding the relative hydrophilicity of [3H]NMS versus [3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the cell. These carbachol-induced changes are accompanied by a change in the membrane form of the muscarinic receptor. Although essentially all of the muscarinic receptors from control cells co-purified with the plasma membrane fraction on sucrose density gradients, 20-35{\%} of the muscarinic receptors from cells treated for 30 min with 100 μM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a 'light vesicle' form occurred with a t( 1/2 ) (~) 10 min, and reversed with a t( 1/2 ) (~) 20 min. In contrast to previous results in this cell line regarding β-adrenergic receptors, agonist binding to muscarinic receptors in the light vesicle fraction obtained from carbachol-treated cells was still regulated by GTP. One interpretation of these data is that agonists induce an internalization of muscarinic receptors with the retention of their functional interaction with a guanine nucleotide regulatory protein.",
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