α-arrestin 1 (ARRDC1) and β-arrestins cooperate to mediate Notch degradation in mammals

Abstract

SummaryNotch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that b-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, b-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or a-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itchmediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in b-arrestin 2/2 or Itch 2/2 cells. Our data show for the first time that ARRDC1 and b-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

α-Arrestins – new players in Notch and GPCR signaling pathways in mammals

“…This heterodimerization of a-and b-arrestins has been addressed experimentally by Shea and colleagues; they demonstrated that ARRDC3 or ARRDC4 can heterodimerize with b-arrestins upon agonist binding by b2AR, and that this hetero-association is essential to recruit Nedd4 to the activated receptor (Shea et al, 2012). This scenario is very similar to the model that we propose for Notch degradation, in which ARRDC1-barrestin heterodimerization, observed in co-immunoprecipitation experiments and GST pulldown assays, is necessary to recruit Itch to unactivated Notch receptor (Puca et al, 2013) (Fig. 4).…”

“…Beyond observations in Drosophila, our recent study has addressed the functions of a-and b-arrestins in Notch signaling in mammals (Puca et al, 2013). In contrast to Drosophila, mammalian Notch receptors (with the exception of Notch3) have no PPXY motif and are not able to bind directly to the E3 ubiquitin ligase Itch (Chastagner et al, 2008).…”

“…ARRDC1, ARRDC3 and ARRDC4 show stronger affinity for E3 ubiquitin ligases than do ARRDC2 and TXNIP, which lack a detectable interaction with WWP2 and Itch (Masutani et al, 2011;Rauch and Martin-Serrano, 2011). By contrast, b-arrestins do not harbor PPXY motifs and are not able to interact directly with Itch (Puca et al, 2013), suggesting that the lack of PPXY motifs affects Itch binding. However, previous studies have described b-arrestins as scaffolding proteins for various E3 ubiquitin ligases, including those of the HECT family.…”

“…In summary, b-arrestins can interact with proteins of the HECT family; however, most of the experiments have been performed with lysates from transfected cells under conditions in which intermediary factors necessary for these interactions could be present (Bhandari et al, 2007;Shenoy et al, 2008). The recent discoveries that ARRDC3 and ARRDC1 can, respectively, recruit Nedd4 to the b2AR (Shea et al, 2012), and Itch to Notch (Puca et al, 2013), strongly argue in favor of the possibility that ARRDCs and b-arrestins could work together in recruiting E3 ubiquitin ligases to receptors. In any case, it is possible that a single PPXY E3-docking site is not sufficient to anchor the substrate to the E3 ubiquitin ligase, and several interactions (through scaffolding molecules) could increase the stability of the enzyme-substrate complex.…”

cDNA cloning, molecular characterization, and expression analyses of two novel porcine ARRDC genes—ARRDC1 and ARRDC5

“…Ubiquitin modification of ARRDC1 may be required for ARMM release (Nabhan et al, 2012). ARRDC1 can also act as a negative regulator of Notch signaling by heterodimerization with β-arrestin to promote the degradation of nonactivated Notch receptor (Puca et al, 2013).…”