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

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 ␤-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 ␤-arrestin-1 and NHE1 ubiquitylation. Overexpression of ␤-arrestin-1 or of wild type but not ligase-dead Nedd4-1 increases NHE1 ubiquitylation. siRNA-mediated knockdown of Nedd4-1 or ␤-arrestin-1 reduces NHE1 ubiquitylation and endocytosis leading to increased NHE1 surface levels. Fibroblasts derived from ␤-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 ␤-arrestin-1 as key regulators of NHE1 ubiquitylation, endocytosis, and function. Our data suggest a broader role for ␤-arrestins in the regulation of membrane ion transport proteins than currently known. Na ϩ /H ϩ exchangers (NHEs) 2 are ion transporters catalyzing the exchange of sodium with protons in prokaryotes and eukaryotes (1). The ubiquitous mammalian NHE isoform 1 (NHE1) controls cellular volume and pH and is therefore often referred to as the "housekeeping" NHE (2). In recent years, however, it has become clear that NHE1 is a highly dynamic protein at the plasma membrane with pivotal importance for mammalian biology that extends beyond ion translocation (3, 4). NHE1 regulates cell shape, adhesion, proliferation and migration (5). Moreover, NHE1 senses mechanical stress directly, thereby serving as a cellular mechanosensor (6). In resting cells, NHE1 is found at sites of focal adhesions in nonpolarized cells and at basolateral membranes in polarized epithelial cells, where it is required for assembly of stress fibers and focal adhesions. In migrating cells, NHE1 is endocytosed at the rear end of the cell and enriched at the leading edge of the cell, promoting cell migration by the development of pseudopodial protrusions and retraction at the rear end (7, 8). NHE1-deficient cells display reduced adhesion, loss of polarity and greatly diminished motility and chemotaxis (7). Conversely, 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). Clearly, the dynamics of NHE1 at the plasma membrane mandate rapid and tight regulation of NHE1 turnover. Th...

Unique Regulation of Human Na+/H+ Exchanger 3 (NHE3) by Nedd4-2 Ligase That Differs from Non-primate NHE3s

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“…Dilutions of the total and surface NHE3 were resolved by SDS-PAGE and immunoblotted with an anti-VSVG antibody. Sequential cell surface biotinylation and immunoprecipitation were performed using monomeric avidin-agarose beads (Pierce), which allow elution of biotinylated proteins by excess free biotin as described (29). Densitometric analysis was performed using ImageJ software (National Institutes of Health).…”

“…It was shown recently that NHE1 indirectly interacts with Nedd4-1 via ␤-arrestin (29). Hence, we examined the effect of silencing Nedd4-1 on NHE3 activity.…”

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“…NHE1 abundance at the plasma membrane was recently shown to be regulated by direct ubiquitylation of the exchanger [154].…”

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