Analysis of the trafficking of the M2 muscarinic acetylcholine receptor.

摘要

The M2 muscarinic acetylcholine receptor (mAChR) is a member of the G-protein coupled receptor (GPCR) superfamily that plays an important role in the regulation of various physiological processes, such as mediating the negative inotropic and chronotropic effects on heart rate. It has previously been demonstrated that the M2 mAChR is regulated differently than a number of GPCRs that have been studied. When the M2 mAChR was heterologously expressed in HEK293 cells, agonist-induced internalization was independent of the functions of arrestin and dynamin. However, the pathway operating for the internalization of the M2 mAChR in this cell type is unknown. In the studies presented in this report, we have demonstrated numerous novel aspects concerning the regulation of the trafficking of the M2 mAChR. We have demonstrated that the M2 mAChR traffics between the cell surface and the Golgi apparatus. We have also demonstrated that the M2 mAChR does not undergo down-regulation in response to prolonged treatment with agonist, and that the recovery of receptors to the cell surface following both agonist-dependent and -independent internalization is a very slow process.;Of particular interest, pretreatment with pertussis toxin, which results in the inactivation of G-proteins of the Gi/o family, revealed a previously unappreciated delivery of intracellular M2 mAChRs to the cell surface following agonist treatment. In addition, the recovery of receptors to the cell surface following agonist-mediated internalization was significantly enhanced following pertussis toxin pretreatment. We hypothesize that pertussis toxin neutralized an inhibitory signal that regulates the delivery of M2 mAChRS to the cell surface. We further hypothesize that this inhibitory signal is mediated by an intracellular G-protein of the G i/o family. In order to explore the possibility that the trafficking of other GPCRs is regulated in a similar manner, we tested the effects of pertussis toxin on the M3 and M4 mAChRs. Treatment with pertussis toxin did not affect the trafficking of the Gq-coupled M3 mAChR, but did reveal agonist-induced delivery of the G i/o-coupled M4 mAChR to the cell surface, suggesting that this mechanism of regulation may be applicable to other GPCRs.