β-Arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2

Shenoy, Sudha K.; Modi, Aalok S.; Shukla, Arun K.; Xiao, Kunhong; Berthouze, Magali; Ahn, Seungkirl; Wilkinson, Keith D.; Miller, William E.; Lefkowitz, Robert J.

doi:10.1073/pnas.0901083106

Cited by 141 publications

(140 citation statements)

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“…Given experimental evidence provided within this study and the documented scaffolding function of ␤-arrestins for E3 ligases, ␤-arrestin-1 most likely recruits Nedd4-1 to the NHE1 C terminus for subsequent NHE1 ubiquitylation (14,15,44). However, other scenarios are theoretically also possible, we cannot exclude the possibility that ␤-arrestin-1 affects NHE1 ubiquitylation independently of Nedd4-1.…”

Section: Discussionmentioning

confidence: 84%

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

Simonin

Fuster

2010

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 84%

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

Simonin

Fuster

2010

Journal of Biological Chemistry

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“…␤arr2, USP20, and TRAF6 Associate with Each Other-In the course of regulating 7TMR trafficking and endocytosis, ␤arr2 associates with and is deubiquitinated by ubiquitin-specific protease 33 (USP33) (23). To determine whether ␤arr2 could be regulated by other USP family members, we asked whether ␤arr2 associates with USP20, which shares 59% identity with USP33 (25).…”

Section: Resultsmentioning

confidence: 99%

“…Purified rat ␤-arrestin2 was provided by Dr. Robert J. Lefkowitz (Duke University) (30). Using protocols we have reported previously (23,25), we purified HA-USP20 from COS-7 cells transfected with a pcDNA3-HA construct that contained the human USP20 cDNA insert (23,25).…”

Section: Methodsmentioning

confidence: 99%

“…Apparent paradoxes in ␤arr2-regulated inflammatory signaling may be reconciled by extrapolating from studies demonstrating reciprocal functions of ubiquitinated ␤arr2 and nonubiquitinated ␤arr2 in 7TMR signaling (22,23). Does reversible ubiquitination of ␤arr2 explain the proinflammatory versus anti-inflammatory dimensions of ␤arr2 activity?…”

mentioning

confidence: 99%

“…Does reversible ubiquitination of ␤arr2 explain the proinflammatory versus anti-inflammatory dimensions of ␤arr2 activity? Deubiquitination of ␤arr2 itself is mediated by USP33 (23), and the USP33 homolog USP20 has been shown to deubiquitinate TRAF6 in heterologous systems (24). By scaffolding TRAF6 and USP20, could ␤arr2 block canonical NFB activation?…”

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confidence: 99%

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Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Jean-Charles

Zhang

et al. 2016

Journal of Biological Chemistry

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Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ␤-arrestin2. Although ␤-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ␤-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti-and proinflammatory dimensions of ␤-arrestin2 activity could be dictated by ␤-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ␤-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20 in vivo and in vitro. Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFB activation and neointimal hyperplasia. We found that ␤-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ␤-arrestin2 was augmented when ␤-arrestin2 ubiquitination was prevented and reduced when ␤-arrestin2 ubiquitination was rendered constitutive. Constitutive ␤-arrestin2 ubiquitination also augmented NFB activation. We infer that pro-and anti-inflammatory activities of ␤-arrestin2 are determined by ␤-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ␤-arrestin2.␤-Arrestin2 (␤arr2) is an ϳ46-kDa multifunctional scaffolding protein that was discovered originally for its ability to desensitize G protein-mediated signaling evoked by seventransmembrane receptors (7TMRs) 4 (1, 2). However, ␤arr2 modulates the signaling and/or endocytosis of not only most 7TMRs but also of several receptor protein tyrosine kinases, cytokine receptors, ion channel receptors, and the LDL receptor (3, 4). Both the endocytic and signaling functions of ␤arr2 are intertwined with its ubiquitination, which in turn is stimulus-driven and regulated by specific E3 ubiquitin ligases or deubiquitinases (DUBs) (4). ␤arr2 not only undergoes dynamic ubiquitination/deubiquitination but also recruits E3 ubiquitin ligases to other substrates. Indeed, ␤arr2 is integral to the ubiquitination of cell surface receptors, channels, and non-receptor proteins (4, 5). However, thus far there is no clear demonstration that ␤arr2 can scaffold a DUB to specific substrates and affect signal transduction by mediating deubiquitination.A role in deubiquitination c...

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Glucagon's Metabolic Action in Health and Disease

Zeigerer

Sekar

Kleinert

et al. 2021

Comprehensive Physiology

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β-Arrestin-dependent signaling and trafficking of 7-transmembrane receptors is reciprocally regulated by the deubiquitinase USP33 and the E3 ligase Mdm2

Cited by 141 publications

References 31 publications

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

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

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Glucagon's Metabolic Action in Health and Disease

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