The internalization of the M2 and M4 muscarinic acetylcholine receptors involves distinct subsets of small G-proteins

Multiple mechanisms exist for the endocytosis of receptors from the cell surface. While the M1, M3, and M4 subtypes of muscarinic acetylcholine receptors internalize through the well-characterized mechanism of clathrin coated vesicles, the mechanism of M2 endocytosis is not well defined. Because the M2 and M4 receptors transduce their signals through the same second messengers but internalize though different pathways, we tested the ability of several small G-proteins to regulate the agonist-induced endocytosis of M2 and M4 in JEG-3 human choriocarcinoma cells. Dominant-negative Rab5 as well as both wild-type and dominant-negative Rab11 inhibited M4 but not M2 endocytosis. In contrast, a dominant-negative Arf6 as well as wild-type Rab22 increased M2 but not M4 endocytosis. We used immunocytochemistry to show that in unstimulated cells, the M2 and M4 receptors co-localize on the cell surface, whereas after stimulation M2 and M4 are in distinct vesicular compartments. In this study, we demonstrate that agonist-induced internalization of the M2 receptor utilizes an Arf6, Rab22 dependent pathway, while the M4 receptor undergoes agonist-induced internalization through a Rab5, Rab11 dependent pathway. Additionally, we show that Rab15 and RhoA are not involved in either pathway in JEG-3 cells.