β-Arrestins and G Protein-Coupled Receptor Trafficking

Abstract

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 (β-arrestin1 and β-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 β-arrestins with clathrin, adaptor protein 2, and phosphoinositides such as PIP₂/PIP₃. 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 chapter, we provide an overview of the reagents and techniques used to study β-arrestin-mediated receptor trafficking.

Introduction

It has now been over 15 years since the discovery that β-arrestins stimulate agonist-promoted internalization of the β₂-adrenergic receptor (Ferguson et al., 1996; Goodman et al., 1996). Many subsequent studies have revealed that β-arrestins promote trafficking of many G protein-coupled receptors (GPCRs) as well as additional classes of receptors (Moore et al., 2007, Shenoy and Lefkowitz, 2011). However, there remain many receptors where this has not been investigated. Moreover, many new strategies have been developed over the past 5–10 years that have significantly advanced our ability to understand these processes. These include the use of RNA interference to knockdown β-arrestin expression, β-arrestin1/2 knockout (KO) mouse embryonic fibroblasts (MEFs) (Kohout, Lin, Perry, Conner, & Lefkowitz, 2001), and a better understanding of the mechanisms that mediate β-arrestin-promoted trafficking. This process involves the coordinated interaction of β-arrestins with the GPCR (Vishnivetskiy et al., 2011, Vishnivetskiy et al., 2004), clathrin (Kang et al., 2009, Krupnick et al., 1997, Krupnick et al., 1997), adaptor protein 2 (AP2) (Burtey et al., 2007, Kim and Benovic, 2002, Laporte et al., 1999, Schmid et al., 2006), and phosphoinositides (Gaidarov et al., 1999, Milano et al., 2006). Moreover, GPCR binding appears to promote β-arrestin interaction with the endocytic machinery thereby linking the binding and trafficking events (Kim and Benovic, 2002, Nobles et al., 2007, Xiao et al., 2004). The X-ray crystal structure of β-arrestin1 along with the regions involved in mediating GPCR binding and endocytic trafficking are shown in Fig. 5.1.

While previous publications have provided detailed methodology for evaluating arrestin-mediated trafficking of GPCRs (Gurevich et al., 2000, Mundell et al., 2002), here we will provide an overview of these methods highlighting the use of β-arrestin mutants, RNA interference, and β-arrestin KO MEFs as tools to study GPCR trafficking.