β-Arrestin1 and GRK2 play permissive roles in Src-mediated endocytosis of α4β2 nicotinic acetylcholine receptor

Background and purpose α4β2 nicotinic acetylcholine receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and implicated in several neurological disorders. The endocytosis of nAChR plays important roles in the pathogenesis of neurological diseases, but its molecular mechanisms remain poorly understood. Experimental approach Loss-of-function approaches and mutants of α4β2 nAChR that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signaling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of α4β2 nAChR was determined and crosschecked using an enzyme-linked immunosorbent assay and radioligand assay. Key results Endocytosis of α4β2 nAChR occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3η-dependent Src-mediated phosphorylation of the nAChR α4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of β-arrestin1 and G protein-coupled receptor (GPCR) kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homolog-mediated ubiquitination and subsequent downregulation of α4β2 nAChR. Conclusions and implications α4β2 nAChR, an ionophore receptor, employs the metabotropic signaling pathway required for endocytosis, which leads to ubiquitination and downregulation. Further, GRK2 and β-arrestin1, representative players of GPCRs, are involved in the endocytosis of α4β2 nAChR via different mechanisms. Considering the functional and pathological implications of nAChR endocytosis, results obtained in this study are crucial for the progression of basic research and clinical investigations.