Human m3 muscarinic acetylcholine receptor carboxyl-terminal threonine resides are required for agonist-induced receptor down-regulation
Yang, J.; Logsdon, C.D.; Johansen, T.E.; Williams, J.A.
Molecular Pharmacology 44(6): 1158-1164
1993
ISSN/ISBN: 0026-895X PMID: 8264552 Document Number: 407737
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 (Thr-550,553,554) 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 (Ala-550,553,554) 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 Ala-553,554 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 (Thr-553) 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 Thr-550 to alanine or converting two cysteine residues (Cys-561,563) 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.