NEDD4–2 (neural precursor cell expressed, developmentally downregulated 4-like) encoded by the Nedd4L gene, belongs to the NEDD4 family of ubiquitin protein ligases. NEDD4 family members are HECT-type ubiquitin ligases (E3) that act at the final step of the ubiquitin cascade to accept ubiquitin from a ubiquitin-conjugating enzyme (E2) and transfer it to their cognate substrates. Ubiquitination of a protein often targets it for degradation; however it may also affect protein localization, trafficking, and recognition by signaling or regulatory complexes. The other members of the NEDD4 family include NEDD4, ITCH, SMURF1, SMURF2, WWP1, WWP2, NEDL1, and NEDL2 (Rotin and Kumar 2009).
NEDD4–2 (originally submitted in the database as mouse KIAA0439 or human NEDD4L gene) is most closely related to the founding member, NEDD4, which was originally identified in the early embryonic central nervous system as a developmentally downregulated gene (Kumar et al. 1992). NEDD4 and NEDD4–2 share approximately 78% similarity, and while homologs of NEDD4 are found in all eukaryotes demonstrating high evolutionary conservation, NEDD4–2 likely arose much later by gene duplication as its homologs are only found in vertebrates (Harvey and Kumar 1999; Yang and Kumar 2010).
NEDD4–2 Structure and Expression
NEDD4–2 has distinctive modular domain architecture similar to other NEDD4 family members, comprising an amino terminal Ca2+ phospholipid binding (C2) domain, four WW (protein–protein interaction) domains, and a HECT domain at the carboxyl terminus (Fig. 1) (Harvey and Kumar 1999). The WW domains generally bind PY (PPxY) or similar motifs in substrates and regulatory proteins. The number and position of WW domains vary among NEDD4 family members (generally 2–4), which are thought to contribute to substrate specificity and involvement in distinct biological processes.
A number of proteins have been shown to be targets of NEDD4–2 (reviewed by Goel et al. (2015)); however, interactions with many of the putative targets have only been demonstrated in vitro and have not been validated using physiologically relevant animal models, such as in gene knockout mice. Here we focus on some key validated NEDD4–2 substrates.
Regulation of Sodium Homeostasis Through ENaC and NCC
The epithelial sodium channel (ENaC) plays an essential role in fluid and electrolyte homeostasis, and in kidney, it is necessary for Na+ homeostasis and maintenance of blood pressure. In the lung, ENaC is responsible for normal fluid clearance from alveolar spaces and subsequently normal exchange of gases.
In mixed genetic background, an apparently incomplete NEDD4–2 knockout in mice results in slightly elevated ENaC expression in the kidney and a mild salt-sensitive hypertension (Shi et al. 2008). Importantly the hypertension can be partially rescued by Amiloride, a specific inhibitor of ENaC. A null allele of NEDD4–2 in C57Bl6 background is perinatal lethal (Boase et al. 2011). Most animals die at time of birth due to inability to inflate their lungs, and pups that are born will die approximately 20 days after birth with severe lung inflammation. The NEDD4–2 deficiency results in increased ENaC expression and activity in the lung, presumably causing premature clearance of fetal lung fluid and subsequent drying of epithelia in surviving animals (Boase et al. 2011). A lung-specific knockout of NEDD4–2 also leads to perinatal lethality approximately 20 days after birth (Kimura et al. 2011); These mice present with a cystic fibrosis-like phenotype including airway mucus obstruction and inflammation, which was reversed by treatment with Amiloride (Kimura et al. 2011). These studies demonstrate the critical role of Nedd4–2 in regulating ENaC function.
The Na+-Cl− cotransporter (NCC) is expressed in the distal collecting duct of kidney and is important for regulation of Na+ balance and blood pressure. NEDD4–2 binds to and ubiquitinates NCC, however, as NCC does not have a classical PY motif, the mechanism of interaction remains unclear. Regulation of NCC by NEDD4–2 has been demonstrated to be critical for Na+ absorption leading to an increase in blood pressure in mice with a kidney specific deletion of NEDD4–2 (Ronzaud et al. 2013). These mice also show increased levels of βENaC and γENaC, and renal outer medullary K+ channel (ROMK) (Ronzaud et al. 2013). Treatment with thiazide was able to partially reverse the hypertension suggesting that NCC is also an important physiological target of NEDD4–2 (Ronzaud et al. 2013).
Regulation of Voltage-Gated Sodium Channels and Neuropathic Pain
Voltage-gated sodium channels (Navs) are expressed in many cell types, particularly in the nervous system. Navs mediate the influx of Na+ in response to local depolarizing stimuli, which generates action potentials in electrically excitable cells. There are nine members of the Nav family, seven of which contain PY motifs and can interact with the WW domains of NEDD4 and NEDD4–2 (Fotia et al. 2004); Several of these channels have been shown to be ubiquitinated and inhibited by NEDD4–2 (Fotia et al. 2004; van Bemmelen et al. 2004). In mouse cortical neurons, NEDD4–2 regulates Navs specifically in response to elevated intracellular Na+, but does not affect steady-state Nav activity (Ekberg et al. 2014). In animal models of neuropathic pain, a condition associated with hyperexcitability of neurons following nerve injury, NEDD4–2 is implicated in altered ubiquitination and regulation of Navs (Cachemaille et al. 2012; Laedermann et al. 2013; Ekberg et al. 2014).
Regulation of Mast Cell Function
Following antigen binding, crosslinking the IgE receptor on mast cells plays a critical role in the pathology of IgE-dependent allergic disorders. Recently NEDD4–2 and the adaptor NDFIP1 have been shown to limit the intensity and duration of such IgE-dependent signaling, through, in part, ubiquitination of phospho-Syk, a kinase required for downstream signaling (Yip et al. 2016). Thus NEDD4–2 is a negative regulator in IgE-dependent mast cell activity that plays a role in dampening the IgE-dependent allergic response.
Other Possible Targets and Functions
In vitro data implicate NEDD4–2 in the regulation of many other proteins, including several potassium and chloride ion channels, surfactant protein C, glutamate and dopamine transporters, EGFR, TGF-β receptor, WNT signaling, dopamine transporter, and divalent metal ion transporter (DMT1) (reviewed by Goel et al. (2015)). In addition, NEDD4–2 has also been show to play a role in nerve growth factor mediated functions through the regulation of TrkA (Arevalo et al. 2006; Yu et al. 2014; Yu et al. 2011). Despite a long list of potential substrates, most of the interacting proteins for NEDD4–2 have been discovered by in vitro methods, and more mechanistic and in vivo work is required before these functions of NEDD4–2 can be fully verified.
Regulation of NEDD4–2
The WW domains within NEDD4–2 can weakly bind to the LPxY motif in its HECT domain. This interaction is thought to stabilize NEDD4–2 and prevent its autoubiquitination, resulting in more NEDD4–2 available to bind to its substrates, including ENaC (Bruce et al. 2008). In addition, deubiquitination of NEDD4–2 by USP2–45 is also known to maintain NEDD4–2 protein stability (Krzystanek et al. 2012; Oberfeld et al. 2011; Pouly et al. 2013).
Proteins that don’t have a PY motif require adaptor proteins to bind to the WW domains of NEDD4 E3s, including NEDD4–2. One such family of adaptor proteins is NDFIPs (NDFIP1 and NDFIP2). Both proteins have PY motifs to bind to the WW domains of NEDD4 family E3s (Harvey et al. 2002; Mund and Pelham 2009; Shearwin-Whyatt et al. 2006), and through NDFIP1, NEDD4–2 has been shown to regulate divalent metal ion transporter, DMT1 (Foot et al. 2008; Howitt et al. 2009); the water channel, aquaporin 2 (AQP2) (de Groot et al. 2014); and may also modulate NEDD4–2s regulation of ENaC (Konstas et al. 2002).
In response to insulin and aldosterone signaling, NEDD4–2 is phosphorylated by kinases, SGK1 and AKT, which triggers its interaction with 14-3-3 proteins (Snyder et al. 2004; Bhalla et al. 2005; Lee et al. 2007). The binding of 14-3-3 to NEDD4–2 inhibits its function by preventing it from interacting with its substrates, such as the ENaC (Nagaki et al. 2006) (Fig. 2).
NEDD4–2/NEDD4L variants have been linked to salt-sensitive and essential hypertension in human subjects (Knight et al. 2006). In addition, mutations in β and γENaC, which disrupt or delete the PY motif in Liddle syndrome, abrogate the ability of NEDD4–2 to bind to and ubiquitinate ENaC. This results in accumulation of the functional ENaC on the cell membrane, triggering increased sodium reabsorption, which leads to increased reabsorption of water and consequentially hypertension (Foot et al. 2016). Based on mouse studies stated above, NEDD4–2 may also be important in IgE-dependent allergic disorders and controlling neuropathic pain.
NEDD4–2 is a member of the NEDD4 ubiquitin ligase family that comprises nine members of HECT type E3s. It is a close relative of NEDD4, which is highly conserved during evolution. Most of the known substrates of NEDD4–2 are membrane proteins, including ion channels and transporters. In particular, NEDD4–2 plays an essential role in sodium homeostasis in the lung and kidney through the regulation of ENaC, and the variants of NEDD4–2 gene are linked to human hypertension.
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