The arrestin clan can now be broadly divided into three structurally similar subgroups: the originally identified arrestins (visual and β-arrestins), the α-arrestins and a group of Vps26-related proteins. The visual and β-arrestins selectively bind to agonist-occupied phosphorylated G protein-coupled receptors (GPCRs) and inhibit GPCR coupling to heterotrimeric G proteins while the β-arrestins also function as adaptor proteins to regulate GPCR trafficking and G protein-independent signaling. The α-arrestins have also recently been implicated in regulating GPCR trafficking while Vps26 regulates retrograde trafficking. In this review, we provide an overview of the α- and β-arrestins with a focus on our current understanding of how these adaptor proteins regulate GPCR trafficking.

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin ␤-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrincoated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I) 2 GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IB kinase promotes phosphorylation and degradation of NFB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.Pathogen-associated molecular patterns in microbes serve as ligands to activate a recently described class of pattern recognition receptors called Toll-like receptors (TLRs).3 13 TLRs have been identified to date, with TLR1, -2, -4, -5, -6, and -11 displayed on the cell surface and TLR3, -7, -8, and -9 localized intracellularly (1). Activation of TLR signaling constitutes one of the earliest responses of an organism to microbe invasion and understanding the signaling pathways stimulated by these TLRs is an area of intense interest. The TLRs as well as interleukin receptors have a conserved ϳ200-amino acid region in their cytoplasmic tails, known as the Toll/interleukin-1 receptor domain. Within the TIR domain are conserved regions that are crucial for signaling. After ligand binding, the TLRs dimerize and undergo conformational changes that are required for the recruitment of downstream signaling molecules (2). These include the adaptor molecule Myd88 (myeloid differentiation primary response protein 88), interleukin-1 receptor-associated kinases, TAK1 (transforming growth factor-␤-activated kinase), TAK1-binding protein (TAB1 and -2), and TRAF-6 (tumor necrosis factor (TNF) receptor-associated factor 6) (3-6).TLRs primarily activate two major signaling pathways, nuclear factor -B (NFB) and mitogen-activated protein kinase (ERK, JNK, and p38) pathways. The NFB pathway results in activation of transcription factors that are thought to act as a "master switch" for inf...

Non-visual arrestins (β-arrestin-1 and β-arrestin-2) are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. β-arrestins are ubiquitously expressed and function to inhibit GPCR/G protein coupling, a process called desensitization, and promote GPCR trafficking and arrestin-mediated signaling. β-arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of β-arrestins with clathrin, adaptor protein 2 (AP2) and phosphoinositides. These interactions are facilitated by a conformational change in β-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this review, we provide an overview of the key interactions involved in β-arrestin-mediated trafficking of GPCRs.

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