The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the ß2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/ß-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the ß2-adrenergic receptor (ß2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the ß2AR in a ligand-independent manner. Although ARRDC3 has no effect on ß2AR endocytosis or degradation, it negatively regulates ß2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in ß2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the ß2AR.

ß-arrestins and G protein-coupled receptor trafficking.

Nonvisual arrestins (ß-arrestin-1 and ß-arrestin-2) are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. ß-arrestins are ubiquitously expressed and function to inhibit GPCR/G protein coupling, a process called desensitization, and promote GPCR trafficking and arrestin-mediated signaling. ß-arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of ß-arrestins with clathrin, adaptor protein 2 (AP2), and phosphoinositides. These interactions are facilitated by a conformational change in ß-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 ß-arrestin-mediated trafficking of GPCRs.

Detection and characterization of SUMO protease activity using a sensitive enzyme-based reporter assay.

In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His(6)-tagged small ubiquitin-like modifier (SUMO) to the amino terminus of the reporter enzyme phospholipase A(2) (PLA(2)). Following cleavage by a member of the sentrin specific proteases (SENPs), free PLA(2) is able to turn over its substrate, resulting in the release of a fluorescent product which is readily quantifiable using a fluorimeter or a fluorescence plate reader. The utility of this SUMO-CHOP-Reporter assay platform is demonstrated by its ability to determine K(m) values and to characterize inhibitors of SUMO proteases.

Role of ß-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking.

The arrestin clan can now be broadly divided into three structurally similar subgroups: the originally identified arrestins (visual and ß-arrestins), the α-arrestins and a group of Vps26-related proteins. The visual and ß-arrestins selectively bind to agonist-occupied phosphorylated G protein-coupled receptors (GPCRs) and inhibit GPCR coupling to heterotrimeric G proteins while the ß-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-arrestins and ß-arrestins with a focus on our current understanding of how these adaptor proteins regulate GPCR trafficking.

ß-Arrestins and G protein-coupled receptor trafficking.

Arrestins are adaptor proteins that function to regulate G protein-coupled receptor (GPCR) signaling and trafficking. There are four mammalian members of the arrestin family, two visual and two nonvisual. The visual arrestins (arrestin-1 and arrestin-4) are localized in rod and cone cells, respectively, and function to quench phototransduction by inhibiting receptor/G protein coupling. The nonvisual arrestins (ß-arrestin1 and ß-arrestin2, a.k.a. arrestin-2 and arrestin-3) are ubiquitously expressed and function to inhibit GPCR/G protein coupling and promote GPCR trafficking and arrestin-mediated signaling. Arrestin-mediated endocytosis of GPCRs requires the coordinated interaction of ß-arrestins with clathrin, adaptor protein 2, and phosphoinositides such as PIP(2)/PIP(3). These interactions are facilitated by a conformational change in ß-arrestin that is thought to occur upon binding to a phosphorylated activated GPCR. In this chapter, we provide an overview of the reagents and techniques used to study ß-arrestin-mediated receptor trafficking.

Convergence of G protein-coupled receptor and S-nitrosylation signaling determines the outcome to cardiac ischemic injury.

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving G protein-coupled receptors (GPCRs) or nitric oxide (NO). These regimens are thought to target distinct molecular pathways. We showed that these pathways were interdependent and converged on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function. Ischemic injury coupled to GPCR activation, including GPCR desensitization and myocyte loss, required GRK2 activation, and we found that cardioprotection mediated by inhibition of GRK2 depended on endothelial nitric oxide synthase (eNOS) and was associated with S-nitrosylation of GRK2. Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other, the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into the mechanism of action of classic drugs used to treat heart failure and new therapeutic approaches to ischemic heart disease.

Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format.

The reversible conjugation of ubiquitin and ubiquitin-like (UbL) proteins to protein substrates plays a critical role in the regulation of many cellular pathways. The removal of ubiquitin from target proteins is performed by ubiquitin proteases also known as deubiquitylases (DUBs). Owing to their substrate specificity and the central role ubiquitylation plays in cell signaling pathways, DUB are attractive targets for therapeutic development. The development of DUB inhibitors requires assays that are amenable to high-throughput screening and provide rapid assessment of inhibitor selectivity. Determination of inhibitor selectivity at an early stage of drug discovery will reduce drug failure in the clinic as well as reduce overall drug development costs. We have developed two novel assays, UbL-Enterokinase light chain and UbL-Granzyme B, for quantifying ubiquitin and UbL protease activity. In our quest to discover and characterize novel chemical entities, we have combined these assays with a previously developed assay in a multiplex format. This multiplex format allows for the detection of three distinct protease activities simultaneously, in a single well. We have demonstrated that the multiplex format is able to distinguish between selective and nonselective protease inhibitors. Specifically, we have used this assay format to characterize P022077, a selective ubiquitin-specific protease 7 inhibitor discovered at Progenra.

Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.