A role for ubiquitin ligases of the Nedd4/Rsp5 family in the downregulation of channels, transporters and receptors

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In parallel to the discovery that Rsp5p is crucial for the downregulation of several yeast plasma membrane proteins [66, 69], the crucial role played by one isoform of Nedd4s, namely Nedd4-2 [24] in downregulation of the sodium channel ENaC was demonstrated [115-117]. ENaC plays an essential role in renal sodium management. This channel consists of three subnits (a, b, g), each of which contains a conserved PXY motif at its C-terminus. Deletion or mutation of the PXY motifs of the ß or g subnit of ENaC results in Liddle’s syndrome, a hereditary form of arterial hypertension in which ENaC activity is abnormally high. The PXY motifs of ENaC serve as binding sites for the WW domains of Nedd4, and mutations in these PY motifs abolishes the binding of this enzyme. Consistent with its ability to associate with Nedd4, ENaC is regulated by  ubiquitylation, which takes place primarily on a cluster of lysine residues in the g subnit. Mutation of these lysines leads to the impairment of channel ubiquitylation and an increase in channel density at the cell surface. Moreover, in a Xenopus oocyte system, it has been shown that the overproduction of wild-type Nedd4, but not of catalytically inactive Nedd4, leads to the inhibition of channel activity in a PXY-dependent manner, providing conclusive proof that Nedd4 is responsible for the ubiquitylation and downregulation of ENaC (reviewed in [21]). Two other channels also appear to display ubiquitin-dependent downregulation controlled by E3s of the Nedd4 family (Table II). The cardiac voltage-gated Na+ channel (rh1), which contains PXY motifs, is also negatively regulated by Nedd4 when produced in Xenopus oocytes [118]. The C-terminus of the chloride channel ClC5 has a PXY motif that is critical for its downregulation in response to interaction with an E3 of the Nedd4 family, possibly WWP2 [119].

A few receptors belonging to different families were also demonstrated to undergo downregulation mediated by ubiquitin ligases of the Nedd4 family (Table II). Insulin-like growth factor I receptor (IGF-IR), previously known to interact with the adapter Grb10, was demonstrated to undergo Nedd4-dependent ubiquitylation. Surprisingly, in this case, interaction of Nedd4 with its substrate was not mediated by Nedd4 WW domain, but by Grb10, acting as an adapter linked to Nedd4 C2 domain, as first evidenced by Nedd4/Grb10 two hybrid interaction [120]. AIP4/Itch was found to promote agonist-induced ubiquitylation of the chemokine receptor CXCR4, a representative of the superfamily of G protein-coupled receptors (GPCRs) [121], that plays a critical role in HIV infection [122]. CXCR4 is ubiquitylated (likely monoubiquitylated) at the plasma membrane, as inhibition of internalisation by expression of dominant negative form of dynamin leads to an accumulation of ubiquitylated CXCR4 [123]. In this case, ubiquitylation seems to be required for late steps of receptor endocytosis rather than internalization [123].

Ubiquitin ligases of the Nedd4 protein family are not always constitutively active on their plasma membrane substrates. It has long been known that ENaC activity is subject to complex regulation by a number of hormones, including aldosterone. This regulation seems to involve Sgk1 kinase (serum and glucocorticoid regulated kinase), a member of the Akt family of Ser/Thr kinases, which is induced by aldosterone, and stimulates ENaC. Based on the observation that Sgk1 has a PXY motif, and that Nedd4-2 includes two consensus sites for phosphorylation by Sgk1, Staub and coworkers showed that Sgk1 phosphorylates Nedd4-2 in a PXY-dependent manner in Xenopus oocytes, and that this phosphorylation reduces the interaction between Nedd4-2 and ENaC, leading to high levels of ENaC at the cell surface [124]. Extensive use of the Xenopus oocyte system injected with cRNA encoding active or inactive forms of Nedd4-2 and Sgk1 has revealed similar opposite role of these two enzymes in the downregulation of several transporters, the astrocyte aminoacid transporter SN1 [125], the glial glutamine transporter EAAT1 [126], and the intestinal phosphate transporter NaPi [127], supporting the idea that Sgk-dependent regulation of the interaction of Nedd4-2 with its substrates might be a general process.

A further level of complexity in the role of the ubiquitin system in the endocytic pathway was also illustrated in several cases with the demonstration that ubiquitylation of a given substrate sometimes involves several E3s. Ubiquitylation and downregulation of the epidermal growth factor receptor (EGFR) appear, in some cases, dependent on both the HECT ligase AIP4, and the RING finger protein Cbl3 that were described to interact [128]. Similarly, the receptor Notch, a protein involved in cell fate decision in many mammalian cell types, was described to interact with the mouse Nedd4-like Itch [129] and the RING cCbl [130], and to undergo ubiquitylation (polyubiquitylation ?) by both enzymes. The precise function of ubiquitylation in Notch signalling was recently documented, and is quite unsual. Binding of Notch ligands (such as Delta) was known to trigger successively a proteolytic cleavage in the extracellular domain of the receptor due to a protease of the ADAM family, TACE [131], followed by a cleavage in the transmembrane domain of the remaining protein by g-secretase. This leads to release of the intracellular (ICv) domain, which then translocates to the nucleus. It was recently demonstrated that g-secretase cleavage requires prior monoubiquitylation and subsequent endocytosis of Notch [132].The E3 responsible for this precise modification of Notch remains to be identified.

In this rapid overview of the involvement of proteins of the Nedd4 family in the endocytic pathway of various plasma membrane substrate, it seems that in most cases, ubiquitylation is a plasma membrane event leading to cargo internalization (channels, transporters, some receptors). In at least one case (CXCR4), ubiquitylation, although occuring at plasma membrane, appears required for later endocytic steps. In addition, it seems that Nedd4 might also display a dual regulation of some receptors at two trafficking steps, in scenarios similar to the downregulation of yeast transporters by Rsp5p-dependent ubiquitylation at two intracellular levels. Drosophila Nedd4 has been shown to control the Roundabout (Robo) receptor in axon guidance at the central nervous system (CNS) midline. It was previously defined by genetic studies that the transmembrane protein Comm is a negative regulator of Robo. Myat et al. showed that the internalization of Comm and of Comm/Robo from the cell surface is a DNedd4-mediated, ubiquitin-dependent event. They demonstrated that DNedd4 binds and ubiquitylates Comm in a PPxY-dependent way. These data suggested that DNedd4 activity is necessary for the internalization of Comm and that Comm acts as an adaptor-like protein that can cotarget Robo for internalization [133]. Data reported by Keleman et al. showed that transient production of Com prevents Robo from reaching the cell surface by binding to this receptor and targeting it directly to endosomes [134]. The two processes might also work together for optimal downregulationof Robo, by similarity to Rsp5p-dependent trafficking of yeast transporters [50].