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 M-1, M-3, and M-4 subtypes of muscarinic acetylcholine receptors internalize through the well-characterized mechanism of clathrin coated vesicles, the mechanism Of M-2 endocytosis is not well defined. Because the M-2 and M-4 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 M-2 and M-4 in JEG-3 human choriocarcinoma cells. Dominant-negative Rab5 as well as both wild-type and dominant-negative Rab11 inhibited M-4 but not M-2 endocytosis. In contrast, a dominant-negative Arf6 as well as wild-type Rab22 increased M-2 but not M-4 endocytosis. We used immunocytochemistry to show that in unstimulated cells, the M-2 and M-4 receptors co-localize on the cell surface, whereas after stimulation M-2 and M-4 are in distinct vesicular compartments. In this study, we demonstrate that agonist-induced internalization of the M-2 receptor utilizes an Arf6, Rab22 dependent pathway, while the M-4 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. (c) 2008 Elsevier Inc. All rights reserved.