Beta-Arrestin1 And Gpcr Kinase2 Play Permissive Roles In Src-Mediated Endocytosis Of Alpha 4 Beta 2 Nicotinic Ach Receptors

Background and Purpose The alpha 4 beta 2 nicotinic ACh receptor (nAChR), a subtype of the ligand-gated ion channel, is abundantly expressed in the brain and is implicated in several neurological disorders. The endocytosis of nAChRs plays important roles in the pathogenesis of neurological diseases, but the underlying molecular mechanisms remain poorly understood.Experimental Approach Loss-of-function approaches and mutants of alpha 4 beta 2 nAChRs that display different endocytic properties were used to identify the cellular components and processes responsible for endocytosis. The signalling cascade that leads to endocytosis was deduced via protein interactions in predicted cellular components. The endocytosis of alpha 4 beta 2 nAChRs was determined and crosschecked using an ELISA and radioligand assay.Key Results Endocytosis of alpha 4 beta 2 nAChRs occurred through clathrin-mediated endocytosis in a dynamin-dependent manner. 14-3-3 eta-dependent Src-mediated phosphorylation of the nAChR alpha 4 subunit at Y575 was required for nAChR endocytosis, and this occurred with the assistance of beta-arrestin1 and GPCR kinase 2 (GRK2) without the need for kinase activity. Endocytosis triggered the mouse double minute 2 homologue-mediated ubiquitination and subsequent down-regulation of alpha 4 beta 2 nAChRs.Conclusions and Implications alpha 4 beta 2 nAChR, an ionophore receptor, employs the metabotropic signalling pathway required for endocytosis, which leads to ubiquitination and down-regulation. Further, GRK2 and beta-arrestin1, usually associated with GPCR signalling, are involved in the endocytosis of alpha 4 beta 2 nAChRs 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.