Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

Abstract

The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.

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

“…The same type of model could apply in other contexts. In the case of b2AR (b2-adrenergic receptor), challenging results suggest that b-arrestin 2, as well as ARRDC3, are necessary for Nedd4-dependent ubiquitylation and degradation of the receptor after its activation by isoproterenol (Han et al, 2012;Nabhan et al, 2010;Shea et al, 2012;Shenoy et al, 2008;Shenoy et al, 2001). Therefore, and in accordance with our hypothesis, it is tempting to speculate that b-arrestin 2 and ARRDC3 (like b-arrestin 1 and ARRDC1 in our case) could heterodimerize to fulfil this scaffolding function.…”

Ubiquitin-Dependent Sorting in Endocytosis

“…For instance, Bul1/2 has a critical role in ubiquitinating and sorting the Gap1 transporter to the vacuole, whereas Art1 plays a critical role in trafficking the Mup1 transporter (Helliwell et al 2001;Soetens et al 2001;Lin et al 2008). In mammalian cells, ARRDC3 controls ubiquitination and down-regulation of b2 and b3 adrenergic receptors as well as the b4 integrin (Draheim et al 2010;Nabhan et al 2010;Patwari et al 2011;Shea et al 2012). Recent studies have shown that some a-arrestin proteins are regulated by phosphorylation, in particular kinases downstream from Tor1, thus allowing cell global regulation of plasma membrane protein levels in response to nutrient status (MacGurn et al 2011;Merhi and Andre 2012).…”

Structural and Biochemical Basis for Ubiquitin Ligase Recruitment by Arrestin-related Domain-containing Protein-3 (ARRDC3)

“…These arrestin-related proteins seem in many cases to target their substrates for degradation. In particular, ARRDC3 has been proposed to have a major role in the down-regulation of ␤2-adrenergic receptor in mice and in human cells (13,14,36), although contradictory observations have been reported (15). The human proteome includes nine members of the Nedd4 ubiquitin ligase family (16).…”