Transfer of M2 Muscarinic Acetylcholine Receptors to Clathrin-derived Early Endosomes following Clathrin-independent Endocytosis

Abstract

Upon agonist stimulation, many G protein-coupled receptors such as ␤ 2 -adrenergic receptors are internalized via ␤-arrestin-and clathrin-dependent mechanisms, whereas others, like M 2 muscarinic acetylcholine receptors (mAChRs), are internalized by clathrin-and arrestin-independent mechanisms. To gain further insight into the mechanisms that regulate M 2 mAChR endocytosis, we investigated the post-endocytic trafficking of M 2 mAChRs in HeLa cells and the role of the ADP-ribosylation factor 6 (Arf6) GTPase in regulating M 2 mAChR internalization. Here, we report that M 2 mAChRs are rapidly internalized by a clathrin-independent pathway that is inhibited up to 50% by expression of either GTPase-defective Arf6 Q67L or an upstream Arf6 activator, G␣ q Q209L. In contrast, M 2 mAChR internalization was not affected by expression of dominant-negative dynamin 2 K44A, which is a known inhibitor of clathrindependent endocytosis. Nevertheless, M 2 mAChRs, which are initially internalized in structures that lack clathrin-dependent endosomal markers, quickly localize to endosomes that contain the clathrin-dependent, early endosomal markers early endosome autoantigen-1, transferrin receptor, and GTPase-defective Rab5 Q79L, which is known to swell early endosomal compartments. These results suggest that M 2 mAChRs initially internalize via an Arf6-associated, clathrin-independent pathway but then quickly merge with the clathrin endocytic pathway at the level of early endosomes.

G Protein-coupled Receptor Endocytosis in ADP-ribosylation Factor 6-depleted Cells

“…Similarly to most other receptors studied, we observed that reduced levels of ARF6 did inhibit the agonistpromoted internalization of the M2MR. This result, together with other published observation where mutant forms of ARF6 were overexpressed (25), strongly hints at a preponderant role for ARF6 in this lesser known endocytic pathway. Using a similar RNA interference approach in HeLa cells, it was recently demonstrated that ARF6 was also involved in the clathrin-independent endocytosis of Tac, the interleukin-2 receptor ␣-subunit (37).…”

“…Its internalization cannot be blocked by dominant negatives of dynamin and ␤-arrestin or by overexpression of the ARF GAP GIT1 (19). However, one report has shown that expression of an ARF6 mutant can block the internalization of this receptor (25). Here, 27 Ϯ 7% of the M2MRs were found to internalize following acetylcholine stimulation (Fig.…”

“…Internalization via this pathway is independent of ␤-arrestin, GIT1, and dynamin (19,23,24) but was proposed to involve ARF6 (24). Of all the G protein-coupled receptors, only the acetylcholine muscarinic M2 receptor has been shown to be internalized by this endocytic route (25).…”

Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

“…NGFdifferentiated PC12 cells synthesize and release acetylcholine (Greene and Rein, 1977), which could lead to elevated levels of acetylcholine. In response to sustained agonist stimulation, m 2 and m 4 muscarinic receptors undergo desensitization, which involves dynamin-dependent pathways and either clathrin-independent or clathrin-dependent processes, respectively (Volpicelli et al, 2001;Delaney et al, 2002). Although the two pathways are separate initially, they likely merge in sorting endosomes (Delaney et al, 2002).…”

GEP 100 /BRAG2: Activator of ADP-ribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via E-cadherin and α-catenin

“…Fc␥ receptor-mediated phagocytosis by macrophages also was shown to be regulated by ARF6 (10,11). ARF6 functions in the internalization of both agonist-stimulated G protein-coupled receptors, such as ␤2-adrenergic receptors via ␤-arrestin-and clathrin-dependent mechanisms (5,6,23) and M2-muscarinic acetylcholine receptors via ␤-arrestin-and clathrin-independent mechanisms (4). In addition, ARF6 regulation of recycling to the plasma membrane of integrins (24,25), which mediate cell-cell adhesion in most solid tissues (12,15,26) and are crucial for cell migration, indicates that it can inf luence cell shape, migration, and scattering.…”