ARRDC3 tyrosine phosphorylation functions as a switch to control c-Src versus WWP2 interactions and distinct scaffolding functions.

Mammalian α-arrestins are members of the same arrestin family as the ubiquitously expressed and extensively studied β-arrestins. Arrestins share common structural elements, including the conserved N- and C-arrestin-fold domains, polar core, finger loop, and C-terminal tail, all of which mediate protein-protein interactions. In β-arrestins, these domains enable the control of G protein-coupled receptor (GPCR) signaling and scaffolding interactions with various signaling proteins including c-Src. By contrast, the repertoire of α-arrestin scaffolding partners and regulatory mechanisms that control their interactions are not well-understood. α-arrestins differ considerably from β-arrestins in the C-terminal region; β-arrestins contain clathrin adaptor β-adaptin-binding sites, whereas α-arrestins harbor PPxY motifs, demonstrated to interact with WW domains of E3 ubiquitin ligases such as WWP2. Here we report the identification of a novel phosphorylation site, tyrosine (Y) 394, embedded in the C-terminal PPxY motif of α-arrestin ARRDC3. The Y394 site functions as a phospho-regulatory switch to enable distinct ARRDC3 binding partners and scaffolding functions. We found that ARRDC3 Y394 phosphorylation promotes interaction with c-Src via its SH2 domain, whereas the non-phosphorylated form binds to WWP2. Our results further show that ARRDC3 Y394 phosphorylation and c-Src SH2 domain-dependent interaction enables regulation of c-Src activity, whereas ARRDC3 Y394 phosphorylation disrupts WWP2 interaction and perturbs ARRDC3-dependent lysosomal trafficking of the GPCR, protease-activated receptor-1. Together, these findings indicate that ARRDC3 Y394 functions as a phospho-regulatory switch to enable selective binding to different partners that impact distinct scaffolding functions.