Human m3 muscarinic acetylcholine receptor carboxyl-terminal threonine resides are required for agonist-induced receptor down-regulation.

The mechanisms involved in agonist-induced down-regulation of the human m3 muscarinic acetylcholine receptor were investigated by site-directed mutagenesis of the receptor cytoplasmic carboxyl terminus. Threonine residues (Thr550,553,554) were converted into alanines collectively and individually. The mutated and wild-type receptor cDNAs stably expressed in Chinese hamster ovary cells displayed similar antagonist- and agonist-binding properties. Furthermore, mutant receptors showed the same efficacy and potency for carbachol-induced activation of phosphoinositide hydrolysis as did the wild-type clone. In all cases the maximal increase in phosphoinositide hydrolysis was 8-9-fold. In contrast to normal intracellular signaling, however, the mutant receptor with all three threonines changed to alanines (Ala550,553,554) failed to undergo normal down-regulation in response to carbachol. After a 24-hr incubation in the presence of 1 mM carbachol, subsequent N-[3H]methylscopolamine binding was reduced by 66% for the wild-type clone but by only 12% for the mutant receptor. The Ala553,554 mutant also showed a profound reduction in receptor down-regulation. Subsequent studies showed that a small but significant blockage of receptor down-regulation also could be produced by converting a single threonine residue (Thr553) to alanine. The fact that these effects were not due to nonspecific conformational changes was suggested by the lack of effects on binding, signal transduction, and down-regulation of converting Thr550 to alanine or converting two cysteine residues (Cys561,563) to glycines in an adjacent region. A similar reduction in receptor number also was observed in binding studies using the membrane-permeant ligand [3H]scopolamine. These results show that threonine residues in the carboxyl-terminal domain of the human m3 muscarinic acetylcholine receptor are important in agonist-induced receptor down-regulation.