Associate editor: K.A. Neve
The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

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Abstract

The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin–receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These “custom-designed” arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling.

Introduction

In the animal kingdom from Caenorhabditis elegans to humans, 3–4% of the genes encode various members of the largest and most diverse family of signaling proteins, G-protein-coupled receptors (GPCRs). The growing realization over the last 20 years of the amazing conservation of the core 7 transmembrane domain structure of these receptors, their signaling via heterotrimeric G proteins, and the regulation of their signaling and trafficking by G-protein-coupled receptor kinases (GRKs) and arrestins led to the formulation of the “classic” model of these processes (Fig. 1) (reviewed in Carman & Benovic, 1998, Claing et al., 2002, Marchese et al., 2003). In a nutshell, the model posits that the same active receptor conformation that preferentially interacts with G proteins is specifically phosphorylated by GRKs. Arrestin binds the active phosphoreceptor and shields its cytoplasmic surface, thereby precluding further G protein activation (desensitization). Receptor-bound arrestin also serves as an adaptor linking receptors to the internalization machinery of the coated pit, promoting receptor endocytosis. The internalized receptor can then be recycled back to the plasma membrane (resensitization) or directed to lysosomes and destroyed (down-regulation). Although it is not clear how the cell decides the fate of the internalized receptor, overall the model is beautiful and logical. The accumulating mechanistic evidence fit this model perfectly. The major activation-dependent conformational change in the receptor was demonstrated in a series of elegant experiments (reviewed in Hubbell et al., 2003). GRKs were found to specifically phosphorylate active GPCRs simply because the active receptor itself activates the kinase (Palczewski et al., 1991a). An ingenious mechanism involving activation and phosphorylation sensors in arrestin, and its transition into its active receptor-binding conformation when both sensors are engaged simultaneously, was found to ensure arrestin selectivity for the active phosphorylated receptor (Gurevich & Benovic, 1993). The arrestin sensor for receptor-attached phosphates was first identified by mutagenesis (Gurevich & Benovic, 1995) and then nicely confirmed by the crystal structure (Hirsch et al., 1999, Vishnivetskiy et al., 1999). A simple competition between G protein and arrestin was shown to underlie receptor desensitization (Krupnick et al., 1997b). The absence of arrestin (Xu et al., 1997a), receptor kinase (Chen et al., 1999), or receptor sites for GRK phosphorylation (Mendez et al., 2000) produced essentially the same expected phenotype: a severe deficit in receptor desensitization. Finally, direct interaction of receptor-bound arrestin with clathrin (Goodman et al., 1996) and clathrin adaptor complex AP-2 (Laporte et al., 1999) logically explained arrestin's role in receptor endocytosis, and receptor sequestration was found to be deficient in cells lacking non-visual arrestins (Kohout et al., 2001).

However, the great majority of these experiments were performed with just two model GPCRs, rhodopsin and the β2-adrenergic receptor (b2AR). Although most GPCRs studied are phosphorylated and interact with arrestins, with other receptors things do not seem so simple and straightforward. Arrestins were shown to bind a number of unphosphorylated receptors (Mukherjee et al., 1999a, Min & Ascoli, 2000, Min et al., 2002, Mukherjee et al., 2002, Galliera et al., 2004, Jala et al., 2005). Every imaginable mechanism of internalization of different GPCRs has been described: arrestin- and clathrin-dependent; arrestin- and clathrin-independent; arrestin-independent and clathrin-dependent; as well as the most puzzling arrestin-dependent dynamin- and clathrin-independent (reviewed in Marchese et al., 2003, Prossnitz, 2004). In some cases the same receptor apparently uses different internalization pathways under different circumstances (Pals-Rylaarsdam et al., 1997, Lee et al., 2000). Arrestin was found to be necessary for desensitization but not for the internalization of some receptors (Pals-Rylaarsdam et al., 1997), and even the active receptor conformation recognized by G proteins and GRKs/arrestins was reported to be different in some cases (Qian et al., 2001, Vilardaga et al., 2001, Whistler et al., 2002b, Kohout et al., 2004, Hunton et al., 2005, Ponimaskin et al., 2005). Thus, one is left wondering whether these data can be reconciled with the “central dogma” of GRK and arrestin function in GPCR regulation.

Here we attempt to understand the mechanistic basis of these disparate results and provide the conceptual framework for their interpretation based on the available structural and functional information about receptors and arrestins. Because more than 40 review articles covering various aspects of arrestin function have been published since 2000, to avoid unnecessary repetition we did not attempt to make this review comprehensive. We also focus on vertebrate arrestins due to the relative paucity of the structural data on invertebrate homologues.

Section snippets

The molecular mechanism of the arrestin–receptor interaction

The first (and undisputed) arrestin function in life is to stop (arrest) receptor signaling via G proteins. This is how the first member of the family, visual arrestin, was discovered (Kuhn, 1978, Kuhn et al., 1984, Pfister et al., 1985) and how it got its name. The fact that rhodopsin activation and phosphorylation both enhance the binding of arrestin (known at the time as 48 kDa protein) was established before the functional role of this phenomenon was understood (Kuhn, 1978, Kuhn et al., 1984

The arrestin–receptor complex: one size does not fit all

Structural diversity of the members of the GPCR superfamily defies imagination (Probst et al., 1992). Even though only their cytoplasmic surface matters from arrestins' point of view, at least 2 elements on the intracellular side of the receptor vary wildly. The 3rd loop (i3) ranges from a modest 16-residue-long connector to a large domain comparable in size with the arrestin itself. Similarly, receptor C-termini range from a dozen to several hundred residues. Arrestins, on the other hand, are

The functional consequences of complexity

It seems hard to believe now that less than 10 years ago the commonly accepted paradigm was that as far as signaling is concerned arrestin binding to the receptor was the end of the story. The first indication that arrestins do more than simply “arrest” G-protein-mediated signaling was reported in 1996 (Ferguson et al., 1996). Arrestin and GRK2 overexpression was able to rescue the internalization of a mutant b2AR that did not internalize otherwise, indicating that arrestin binding plays a role

Conclusions

In the 20 years since the discovery that arrestin binding to rhodopsin is enhanced by rhodopsin phosphorylation (Kuhn et al., 1984), our understanding of arrestin structure and function has improved enormously. In the first 10 years after this seminal discovery, it became increasingly clear that receptor phosphorylation followed by arrestin binding is a common mechanism regulating the signaling of the great majority of GPCRs. Subsequent identification of arrestin and receptor elements involved

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