Agonist-dependent delivery of M₂ muscarinic acetylcholine receptors to the cell surface after pertussis toxin treatment

The internalization of the M₂ muscarinic cholinergic receptor (mAChR) proceeds through an atypical pathway that is independent of arrestin and clathrin function and shows a unique sensitivity to dynamin when the receptor is expressed in human embryonic kidney 293 cells. In this report we demonstrate that the internalization of the M₂ mAChR was modulated by activation of heterotrimeric G proteins, because treatment with pertussis toxin, which ADP-ribosylates G proteins of the G_i/o family, caused a significant delay in the onset of internalization of the M₂ mAChR. The effects of pertussis toxin could not be explained by alteration of the agonist-dependent phosphorylation of the M₂ mAChR. The modulation of internalization by pertussis toxin was revealed to be due to recruitment of intracellular receptors to the cell surface upon agonist treatment. Pretreatment with pertussis toxin also greatly increased both the rate and extent of recovery of M₂ mAChRs to the cell surface after agonist-mediated internalization. These results demonstrate a novel aspect involved in the regulation of GPCRs. As with the tightly controlled internalization of GPCRs, the delivery of GPCRs to the cell surface is also highly regulated.