Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

Abstract

β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-βarr conformations can carry out distinct functions.O ver the past decade, significant efforts have been made to understand the molecular properties and regulatory mechanisms that control the function of β-arrestin (βarr) interactions with G protein-coupled receptors (GPCRs) (1, 2). Once activated, GPCRs initiate a highly conserved signaling and regulatory cascade marked by interactions with: (i) heterotrimeric G proteins, which mediate their actions largely by promoting second-messenger generation (3); (ii) GPCR kinases (GRKs), which phosphorylate activated conformations of receptors (4); and (iii) βarrs, which bind to the phosphorylated receptors to mediate desensitization of G protein signaling and receptor internalization (5, 6). In addition to their canonical function of desensitization and internalization, βarrs have been appreciated as independent signaling units by virtue of their crucial role as both adaptors and scaffolds for an increasing number of signaling pathways (7-11).There are two driving forces that mediate βarr interactions with an activated GPCR: phosphorylation of the C-terminal tail of the receptor by GRKs and/or binding to the transmembrane core of the receptor. How each of these interactions contributes to βarr functionality remains unclear. Moreover, GPCRs tend to either interact with βarr transiently, termed "class A" GPCRs [e.g., β 2 -adrenergic receptor (β 2 AR)], or tightly, known as "class B" GPCRs [e.g., vasopressin type 2 receptor (V 2 R)]. For the current study, we use a previously described chimeric β 2 V 2 R construct, which comprises the β 2 AR with its C-terminal tail exchanged with the V 2 R C-terminal tail (12-14). The β 2 V 2 R construct provides an ideal system for studying a GPCR-βarr complex in vitro, because it maintains identical pharmacological properties to the WT β 2 AR and has a robustly increased class B affinity for βarr1, which allows stable β 2 V 2 R-βarr complexes to be formed and purified.Structural insights have shed some light onto the complexity of the interaction between GPCRs and βarrs. A recent struc...

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

“…Active conformation of the receptor and subsequent GRK-mediated phosphorylation are generally thought to be prerequisite for β-arrestin binding (16), suggesting that the two main interactions occur simultaneously. Recent studies have shown that the phosphate and the core interactions can be separated in vitro or by using receptor chimeras and mutants (51)(52)(53). However, the question remained whether the distinct interactions could be independent in physiologically relevant situations.…”

Catalytic activation of β-arrestin by GPCRs

“…We next investigated how GPCR core engagement affects the activation of β-arrestin trafficking by focusing on a polar region in β-arrestin that is located proximal to the conserved finger loop and is thought to directly contact the ligand-activated GPCR core 12,14 . We identified three charged residues in this ‘finger loop proximal’ region that produce constitutive β-arrestin-2 accumulation in CCSs when mutated to alanine (R77A, K78A, D79A; Figure 3a, b).…”

“…A long-standing view is that all of these functions occur from a stable and stoichiometric GPCR/β-arrestin complex whose formation requires binding of the phosphorylated GPCR tail 7,8 . Emerging evidence suggests that GPCR/β-arrestin complexes can vary in structure but, nevertheless, all present concepts of cellular β-arrestin function require formation of a GPCR/β-arrestin complex driven by the phosphorylated GPCR tail 10,14–17 .…”

GPCR signaling: conformational activation of arrestins

“…7,8 It has been suggested that these are in turn mediated by two distinct sites on arrestins -a 'phosphorylation sensor' and an 'activation sensor'. 3 In the past, studies have largely focused on activation of arrestins by the R P -tail and its functional outcomes, 9 while the role of the receptor transmembrane core, if any, has remained obscure. However, the two new papers illuminate the distinct roles of both the R P -tail and the 7 TM core in mediating arrestin interaction with and activation by GPCRs.…”