Role of known and unknown E3s in ubiquitin-dependent downregulation of cytokine receptors

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The historical example of ubiquitin-dependent downregulation of GHR differs considerably from the ubiquitin-dependent endocytosis of ENaC, or of most other studied receptors. GHR is a member of the cytokine receptor superfamily. In reponse to growth hormone (GH), two receptor polypeptides dimerize, turning on a cascade of events leading to signal transduction and degradation of the receptor [135]. In 1987, it was observed that the growth hormone receptor (GHR) was ubiquitylated [135]. Nine years later, Strous and coworkers made the link between ubiquitin and endocytosis of the GHR: GHR was not degraded upon ligand binding at restrictive temperature in Chinese hamster ovary cells, which present a temperature-sensitive defect in ubiquitin conjugation [136]. Thus, the ubiquitin system is required for ligand-induced GHR endocytosis. The amino acid sequence DSWVEF₃₃₇IELD was shown to be required for the ubiquitin-dependent internalization of GHR, and was designated the UbE motif [137]. However mutation of lysines in the cytoplasmic tail of a truncated version of the GHR (GHR 1-399), the internalization of which depends on the UbE motif and the integrity of the ubiquitin conjugation system [138], did not impair internalization of the truncated GHR. Thus, internalization requires recruitment of the ubiquitin conjugation system to the GHR UbE motif, rather than the conjugation of ubiquitin to the GHR [137]. These data suggest that ancillary proteins of the endocytic machinery may be ubiquitylated, or that factors of the ubiquitin conjugation system itself may act as adaptors for the endocytosis machinery (reviewed in [139]). Strikingly, the UbE motif has been demonstrated to be required for recruitment of the GHR to clathrin-coated pits [140]. The use of a glutathione S-transferase (GST)-pulldown assay indeed allowed identification of a protein binding the UbE (small glutamine-rich tetratricopeptide repeat (TPR)-containing protein), but in an ubiquitin-independent way [141], suggesting that other UbE partners have yet to be indentified. Although GHR ubiquitylation does not appear to be required for endocytosis, GHR nonetheless undergoes ubiquitylation during endocytosis. The use of two different approaches to inhibit internalization, b-methyl cyclodextrin treatment, which inhibits endocytosis at the stage of coated vesicle formation, and overproduction of a dominant negative mutant form of dynamin, which prevents the detachment of clathrin-coated vesicles from plasma membrane, made it clear that GHR is ubiquitylated at the plasma membrane before endocytosis [142]. 

The b and g chains of the interleukin-2 receptor (IL2R) also belong to the cytokine receptor superfamily, whereas the IL2R  chain does not. The IL2R is internalized following the binding of interleukin-2 (IL2). After endocytosis, the three subunits are sorted differently: the a chain is recycled, whereas, the b and g chains are targeted to late endocytic compartments [143]. The b subunit of this receptor is monoubiquitylated. Neither this monoubiquitylation, nor an intact ubiquitin conjugation system, is required for the internalization step of endocytosis. However, ubiquitylation seems to be a signal involved in sorting from the early/recycling endosome to late endocytic compartments [144]. Hence, ubiquitylation events are associated with the endocytosis of several receptors of the cytokine receptor superfamily, some of which are internalized by clathrin-coated vesicles (GHR), and some of which are not (IL2R) [48]. It should be noted that, neither for IL2R nor for GHR has the E3 involved in ubiquitylation been identified.

            The type I interferon (IFN) receptor, another cytokine receptor consisting of IFNAR1 and IFNAR2 subunits, is still todate, to our knowledge, the unique example of a receptor ubiquitylated by a RING finger enzyme of the SCF (Skp1/Cullin/F-box) family of E3. In these multisubunits E3s, the substrates are recognized by specific F-box proteins. IFNAR1 was shown to interact with the Homolog of Slimb (HOS) F-box protein, an interaction promoted by interferon alpha (IFNa) that triggers IFNARI phosphorylation. SCF^HOS expression and activity is required for IFNalpha-stimulated ubiquitylation and downregulation of IFNAR1, probably associated with IFNAR2 [16]. In contrast to the case of IL2R, SCF^HOS- dependent ubiquitylation of IFNAR1 appears required for receptor internalization.