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

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.