HUMAN M3 MUSCARINIC ACETYLCHOLINE-RECEPTOR CARBOXYL-TERMINAL THREONINE RESIDUES ARE REQUIRED FOR AGONIST-INDUCED RECEPTOR DOWN-REGULATION

The mechanisms involved in agonist-induced down-regulation of the huma n m3 muscarinic acetylcholine receptor were investigated by site-direc ted mutagenesis of the receptor cytoplasmic carboxyl terminus. Threoni ne residues (Thr550,553,554) were converted into alanines collectively and individually. The mutated and wild-type receptor cDNAs stably exp ressed in Chinese hamster ovary cells displayed similar antagonist- an d agonist-binding properties. Furthermore, mutant receptors showed the same efficacy and potency for carbachol-induced activation of phospho inositide hydrolysis as did the wild-type clone. In all cases the maxi mal increase in phosphoinositide hydrolysis was 8-9-fold. In contrast to normal intracellular signaling, however, the mutant receptor with a ll three threonines changed to alanines (Ala550,553,554) failed to und ergo normal down-regulation in response to carbachol. After a 24-hr in cubation in the presence of 1 mm carbachol, subsequent N-[H-3]methylsc opolamine binding was reduced by 66% for the wild-type clone but by on ly 12% for the mutant receptor. The Ala553,554 mutant also showed a pr ofound reduction in receptor down-regulation. Subsequent studies showe d that a small but significant blockage of receptor down-regulation al so could be produced by converting a single threonine residue (Thr553) to alanine. The fact that these effects were not due to nonspecific c onformational changes was suggested by the lack of effects on binding, signal transduction, and down-regulation of converting Thr550 to alan ine or converting two cysteine residues (Cys561,563) to glycines in an adjacent region. A similar reduction in receptor number also was obse rved in binding studies using the membrane-permeant ligand [H-3]scopol amine. These results show that threonine residues in the carboxyl-term inal domain of the human m3 muscarinic acetylcholine receptor are impo rtant in agonist-induced receptor down-regulation.