Nedd4-1 and β-Arrestin-1 Are Key Regulators of Na+/H+ Exchanger 1 Ubiquitylation, Endocytosis, and Function

Alexandre Simonin,Daniel Fuster

doi:10.1074/jbc.m110.115089

Alexandre Simonin, Daniel Fuster

Open Access

https://doi.org/10.1074/jbc.m110.115089

Copy DOI

Abstract

The ubiquitously expressed mammalian Na(+)/H(+) exchanger 1 (NHE1) controls cell volume and pH but is also critically involved in complex biological processes like cell adhesion, cell migration, cell proliferation, and mechanosensation. Pathways controlling NHE1 turnover at the plasma membrane, however, are currently unclear. Here, we demonstrate that NHE1 undergoes ubiquitylation at the plasma membrane by a process that is unprecedented for a mammalian ion transport protein. This process requires the adapter protein β-arrestin-1 that interacts with both the E3 ubiquitin ligase Nedd4-1 and the NHE1 C terminus. Truncation of NHE1 C terminus to amino acid 550 abolishes binding to β-arrestin-1 and NHE1 ubiquitylation. Overexpression of β-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knock-down of Nedd4-1 or β-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from β-arrestin-1 and Nedd4-1 knock-out mice show loss of NHE1 ubiquitylation, increased plasmalemmal NHE1 levels and greatly enhanced NHE1 transport compared with wild-type fibroblasts. These findings reveal Nedd4-1 and β-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for β-arrestins in the regulation of membrane ion transport proteins than currently known.

Highlights

Ized cells and at basolateral membranes in polarized epithelial cells, where it is required for assembly of stress fibers and focal adhesions
We find that Na؉/H؉ exchanger 1 (NHE1) turnover at the plasma membrane is regulated by ubiquitylation through a process that is unprecedented for mammalian ion transport proteins, requiring the concerted action of both ␤-arrestin-1 and Nedd4-1
Our data suggest that Nedd4-1 and ␤-arrestin-1 are key regulators of NHE1 ubiquitylation, endocytosis, and function

Summary

Introduction

Ized cells and at basolateral membranes in polarized epithelial cells, where it is required for assembly of stress fibers and focal adhesions. Up-regulation of NHE1 is associated with increased tumor growth and tumor cell invasion [9, 10] Regulation of these complex biological processes is thought to require both NHE transport directly as well as anchoring of cytoskeletal elements and scaffolding of signaling molecules by the large, intracellular C terminus of NHE1 [4]. Turnover of eukaryotic ion transport proteins often involves the posttranslational conjugation of one or several ubiquitin residues to target proteins by E3 ubiquitin ligases [11] This process, called ubiquitylation, typically targets plasmalemmal proteins for internalization by endocytosis, sorting into multivesicular bodies and delivery to lysosomes. We find that NHE1 turnover at the plasma membrane is regulated by ubiquitylation through a process that is unprecedented for mammalian ion transport proteins, requiring the concerted action of both ␤-arrestin-1 and Nedd. Our data suggest that Nedd and ␤-arrestin-1 are key regulators of NHE1 ubiquitylation, endocytosis, and function

Objectives

Conclusion