Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.

Aosa Kamezaki,Kazuhiro Aoki,Fumio Matsuzaki,Koichi Kawakami,Atsuko Sehara-Fujisawa,Fuminori Sato,Kazuhide Asakawa

doi:10.1038/srep28873

Abstract

Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as ‘ectodomain shedding’, is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.

Highlights

Many membrane proteins are subjected to limited proteolysis, which sheds the ectodomain, a process termed “ectodomain shedding”
The involvement of ADAM family proteases including ADAM10, ADAM17, and ADAM19 in Neuregulin 1 (NRG1) ectodomain shedding has been shown in cultured cells, little is known about their roles in NRG1 processing in vivo
We developed the fluorescent N-CISSOR probe to monitor NRG1 ectodomain shedding in living cells and zebrafish embryos

Summary

Introduction

Many membrane proteins are subjected to limited proteolysis, which sheds the ectodomain, a process termed “ectodomain shedding”. Such post-translational modifications of membrane proteins are critical for proper cell-cell interactions in various biological processes It is not well known when and where ectodomain shedding takes place in vivo and how such shedding regulates intercellular signalling or adhesion. The β-secretase 1 (BACE1)-dependent processing of NRG1 is well characterized and required for the proper myelination of Schwann cells, as well as the development and maintenance of muscle spindles, indicating that NRG1 ectodomain shedding plays critical roles in its in vivo functions[5,6,7,8]. Our findings reveal that N-CISSOR provides a novel tool for investigating the spatiotemporal regulation of NRG1-ErbB signalling by visualizing NRG1 ectodomain shedding in single cells at subcellular resolution, both in vitro and in vivo

Methods

Results

Conclusion