Harden T K, Petch L A, Traynelis S F, Waldo G L
J Biol Chem. 1985 Oct 25;260(24):13060-6.
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.
将1321N1人星形细胞瘤细胞与卡巴胆碱一起温育，导致在4℃下完整细胞上测得的[3H]N-甲基东莨菪碱（[3H]NMS）与毒蕈碱型胆碱能受体的结合迅速丧失；而在37℃下用[3H]喹核醇基苯甲酸酯（[3H]QNB）测定的来自相同细胞的裂解物中毒蕈碱型受体的丧失速率较慢。当从培养基中去除激动剂后，完整细胞上测得的丧失的[3H]NMS结合位点以约20分钟的半衰期恢复，但仅恢复到[3H]QNB结合位点丧失的水平；在同一时期内，“丧失”的[3H]QNB结合位点没有恢复。基于这些数据以及Galper等人（Galper, J. B., Dziekan, L. C., O'Hara, D. S., and Smith, T. W. (1982) J. Biol. Chem. 257, 10344 - 10356）关于[3H]NMS与[3H]QNB相对亲水性的观点，有人提出卡巴胆碱诱导毒蕈碱型受体迅速隔离，随后这些受体从细胞中丧失。这些卡巴胆碱诱导的变化伴随着毒蕈碱型受体膜形式的改变。尽管来自对照细胞的基本上所有毒蕈碱型受体在蔗糖密度梯度上与质膜部分共纯化，但用100μM卡巴胆碱处理30分钟的细胞中20 - 35%的毒蕈碱型受体迁移到低得多的蔗糖密度。毒蕈碱型受体向“轻囊泡”形式的这种转变以约10分钟的半衰期发生，并以约20分钟的半衰期逆转。与该细胞系中先前关于β - 肾上腺素能受体的结果（Harden, T. K., Cotton, C. U., Waldo, G. L., Lutton, J. K., and Perkins, J. P. (1980) Science 210, 441 - 443）相反，从卡巴胆碱处理的细胞获得的轻囊泡部分中毒蕈碱型受体的激动剂结合仍受GTP调节。对这些数据的一种解释是，激动剂诱导毒蕈碱型受体内化，并保留其与鸟嘌呤核苷酸调节蛋白的功能相互作用。
