Interplay of β-arrestins and Nedd4 E3 ligase regulates ubiquitination and trafficking of mGluR7

Abstract

Metabotropic glutamate receptor 7A belongs to group III of metabotropic glutamate receptors as a G protein-coupled receptor. GPCRs undergo a number of post-translational modifications, which regulate receptor trafficking and function. These modifications of GPCR have been known as important role for mental disorders such as neurodegenerative diseases. β-arrestins are well known as adaptor protein of seven-transmembrane receptors. And β-arrestins mediate trafficking of many cell-surface receptors. In many previous reports, it have been revealed that β-arrestins-dependent ubiquitinylation plays a role in protein turn-over and receptor trafficking. Although mGluR7A can be an attractive drug target due to the GPCR properties, whether mGluR7A is a target of ubiquitin conjugation has not been identified yet. For the first time I investigated whether mGluR7A is ubiquitinylated in HEK293T cells and Neuronal cultured cells by immunoprecipitation. These experiments show that mGluR7A is ubiquitinylated by agonist. And also ubiquitinylation of mGluR7A is happened at C-terminus and Loop2 regions of mGluR7A. In this study I found that β-arrestin1 binds strongly to mGluR7A but not beta-arrestin2 in HEK293T cells using immunoprecipitation method. And this binding is regulated with metabotropic glutamate receptor group III specific agonist (L-AP4) dependent manner. In addition GST-pull down assay results show that β-arrestin1 binds to C-terminus and Loop2 of mGluR7A. Furthermore E3 ligase, Nedd4 also binds to mGluR7A and betaii arrestin1. I identified that this triplet-complex-form regulates mGluR7A ubiquitinylation in HEK293T cells and neuronal cultured cells. Also my confocal microscopy imaging results represent that surface stability of mGluR7A is regulated with the forming complex of mGluR7A, beta-arrestin1 and Nedd4 in neuronal cell. So it is reasonable to assume that beta-arrestin1 and Nedd4 interact with mGluR7A and make a form of multi-complex to regulate mGluR7A ubiquitinylation and cell surface stability.