Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

Abstract

Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.

hTAC internalizes via both clathrin-dependent and clathrin-independent endocytosis in mammalian cells

“…Thus, we speculate that hTAC internalizes via both the CDE and CIE pathways. This complicated endocytosis mechanism involving internationalization via multiple pathways not only occurs in hTAC but also in other PM proteins, including muscarinic acetylcholine receptor 4 (Boucrot et al, 2015 ; Wan et al, 2015 ), platelet-derived growth factor receptor β (Jastrzebski et al, 2017 ) and EGFR (Boucrot et al, 2015 ; Caldieri et al, 2017 ). Altogether, these results and our findings expand our understanding of the endocytic characteristics of certain PM protein cargoes.…”

IC/BPS‐associated alterations of M2 and M3 muscarinic acetylcholine receptor trafficking in human detrusor

“…Internalization and recycling is essential for receptor resensitization after attenuation of the cellular response by increased/prolonged agonist exposure; thus enhanced abundance of those complexes is in accordance with increased M3 protein expression in IC/BPS disease. In contrast, the M2 is thought to be internalized via a clathrin‐independent endocytosis pathway and then targeted to lysosomes for degradation . Delaney et al suggested that once M2 mAChRs are initially internalized via clathrin‐independent endocytosis, they are quickly transferred to early endosomes of the clathrin endocytotic pathway in cultured HeLa cells.…”

“…M1, M3, and M4 all undergo internalization via clathrin‐mediated endocytosis in a dynamin‐dependent manner . In contrast, several observations indicate that M2 mAChRs internalize via a poorly characterized clathrin‐independent endocytotic pathway . Even it is evident that clathrin is not involved in M2 endocytosis, the involvement of caveolin (Cav) and the GTPase dynamin could not be finally clarified.…”

“…Dynamin was essential for clathrin‐independent M2 internalization in HEK‐293 cells and interactions of M2 and Cav were observed in cardiac myocytes . M2 mAChR internalization leads to degradation and receptor downregulation, while internalized M1, M3, and M4 recycle back to the cell surface …”

Endocytosis of Activated Muscarinic m2 Receptor (m2R) in Live Mouse Hippocampal Neurons Occurs via a Clathrin-Dependent Pathway

“…Some studies demonstrated that m2R internalization pathway involves CCP only ( Pals-Rylaarsdam et al, 1997 ; Jones et al, 2006 ; Yamanushi et al, 2007 ). Other claimed that m2R is internalized through a clathrin-independent process ( Vogler et al, 1999 ; Delaney et al, 2002 ; Wan et al, 2015 ). Ockenga and Tikkanen (2015) propose that m2R endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of CCP.…”