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 degrees C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinyl benzilate [( 3H]QNB) at 37 degrees 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 t1/2 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 et al. (Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344-10356) 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 microM carbachol migrated to a much lower sucrose density. This conversion of muscarinic receptors to a "light vesicle" form occurred with a t1/2 approximately 10 min, and reversed with a t1/2 approximately 20 min. In contrast to previous results in this cell line regarding beta-adrenergic receptors (Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441-443), 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.
Highlights
Methylscopolamine ([3H]NMS) to muscarinic cholinergic receptorsmeasured at 4 O C on intact cells; loss of muscarinic receptors in lysates from the same cells measured with [3H]quinuclidinylbenzilate (r3H]QNB)
NMS uersus r3H]QNB, it is proposed that carbachol induces a rapid sequestration of muscarinic receptors that is followed by a loss of these receptors from the density lipoprotein (4) receptors, receptor-mediated endocytosis is thought to be involved in the transport of ligand to intracellular sites
Current technology has not permitted a morphological analysis of this process, the occurrence of 8-adrenergic receptors in a membrane form that is not associated with the plasma membrane (7-9) and that is relatively inaccessible to hydrophilic ligands (10, 11)is consistent with this idea, As with insulin and epidermal growth factor receptors, internalization of,&adrenergicreceptors may cell
Summary
Heart Association and United States Public Health Service Grant 150-mm plastic culture dishes or six-well plates in Dulbecco’s modi-. For assays using [3H]NMS at 4 "C and [3H]QNB binding assays with most experiments, cells were incubated with vehicle or a muscarinic lysates from the same cells a t 37 "C. receptor agonist for 30 min a t 37 "C. [3H]NMS bound to muscarinic r3HJ replaced with 4 "C Hepes-Eagle's containing 0.25 mg/ml of concana- receptors on intact 1321N1cells at 4 "Cwith a K d of 181& 41 pM (n valin A (19, 20), and the dishes were placed on ice for 20 min. Light vesicle fractions and plasma membrane fractions were identified by [3H]QNB binding assays (seebelow), pooled, diluted with 10 mM Hepes, pH 7.5, 10 mM EDTA, and centrifuged for 30 min at 170,000. X g.The supernatantwas discarded, and thepellet was resuspended in 10 mM Hepes, 10 mM EDTA using a hand-operated Teflon and glass homogenizer and centrifuged a second time a t 170,000 X g for
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