Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling

Abstract

G protein-coupled receptors (GPCRs) belong to one of the largest family of signaling receptors in the mammalian genome [1]. GPCRs elicit cellular responses to multiple diverse stimuli and play essential roles in human health and disease. GPCRs have important clinical implications in various diseases and are the targets of approximately 25–50% of all marketed drugs [2], [3]. Understanding how GPCRs are regulated is essential to delineating their role in normal physiology and in the pathophysiology of several diseases. Given the vast number and diversity of GPCRs, it is likely that multiple mechanisms exist to regulate GPCR function. While GPCR signaling is typically regulated by desensitization and endocytosis mediated by phosphorylation and β-arrestins, it can also be modulated by ubiquitination. Ubiquitination is emerging an important regulatory process that may have unique roles in governing GPCR trafficking and signaling. Recent studies have revealed a mechanistic link between GPCR phosphorylation, β-arrestins and ubiquitination that may be applicable to some GPCRs but not others. While the function of ubiquitination is generally thought to promote receptor endocytosis and endosomal sorting, recent studies have revealed that ubiquitination also plays an important role in positive regulation of GPCR signaling. Here, we will review recent developments in our understanding of how ubiquitin regulates GPCR endocytic trafficking and how it contributes to signal transduction induced by GPCR activation.

Introduction

G protein-coupled receptors (GPCRs) belong to the largest family of signaling receptors [1]. GPCRs elicit cellular responses to multiple diverse stimuli and play essential roles in human health and have important clinical implications in various diseases [2], [3]. Upon binding to their cognate ligand, GPCRs typically signal via heterotrimeric GTP-binding proteins (G proteins) [1], [4], [5]. Heterotrimeric G proteins are comprised of an α-subunit (Gα) and a tightly associated β and γ-subunits (Gβγ). In the inactive state Gα is bound to GDP and once the GPCR is activated by its cognate ligand, conformational changes in the receptor induce the exchange of GDP for GTP on Gα leading to its activation and dissociation from the βγ subunits. The activated Gα (Gα-GTP) and dissociated βγ subunits activate downstream effector molecules contributing to GPCR signaling. One common effector molecule is adenylyl cyclase that catalyzes formation cyclic AMP (cAMP), which in turn activates the protein kinase A (PKA), a serine/threonine kinase that phosphorylates numerous substrates. Another effector molecule that is activated by predominantly Gαq is phospholipase C, which mediates the hydrolysis of phosphatidyl 4,5 bisphosphate to produce inositol 1,4,5-trisphosphate and diacyclglycerol, which in turn leads to calcium mobilization from intracellular stores and activation of protein kinase C (PKC), respectively. GPCRs may also signal independent of heterotrimeric G proteins, and this typically involves signaling by β-arrestins [6], [7]. β-Arrestins are best known to negatively regulate GPCR signaling via desensitization and endocytosis; however, β-arrestins also function as scaffolds that initiate new modes of GPCR signaling [8], [9], [10]. These properties of β-arrestins will be discussed below.

To ensure that signals are of the appropriate magnitude and duration, GPCR signaling is tightly regulated. Regulation of GPCR signaling involves multiple distinct temporal events that occur at the level of the receptor, G protein and downstream effector molecules [11], [12]. The latter steps include inactivation of the G protein and degradation of second messengers [13], [14]. Regulation at the level of the receptor involves a series of events, including receptor interactions with various cytosolic proteins and regulation by post-translational modifications such as phosphorylation and ubiquitination [11], [12]. Phosphorylation may occur by second-messenger-dependent protein kinases PKA and PKC, which promote GPCR signaling by phosphorylating effector molecules, but also function in a negative feedback loop by phosphorylating and desensitizing the GPCR to attenuate further signaling in a homologous or heterologous manner. Phosphorylation may also occur by another family of serine/threonine kinases known as G protein-coupled receptor kinases (GRKs). These kinases preferentially phosphorylate the activated or ligand bound form of the GPCR leading to homologous desensitization [11].

Phosphorylation by GRKs enhances GPCR binding to arrestins. Mammalian arrestins comprise a family of four proteins that can be sub-divided into two groups: visual (arrestin-1 and arrestin-4) and non-visual arrestins (β-arrestin-1 and β-arrestin-2, also known as arrestin-2 and arrestin-3, respectively) [15]. Expression of arrestin-1 and -4 is restricted to the visual system. Arrestin-1 is found in high abundance in rod cells whereas arrestin-4 is found in cone cells. In contrast, non-visual arrestins are ubiquitously expressed and regulate the signaling of most GPCRs. The classical function of arrestins is to mediate GPCR desensitization. Arrestins are typically recruited to the plasma membrane by activated GPCRs that are phosphorylated by GRKs [11]. Arrestin binding uncouples the receptor from G proteins via steric hindrance culminating in attenuated signaling [16]. Non-visual or β-arrestins promote GPCR internalization through clathrin-coated pits by binding directly to clathrin and β₂-adaptin, two important components of the internalization machinery [17], [18]. Arrestins may also contribute to signal termination by promoting degradation of certain second messengers [13], [14].

In addition to established roles of phosphorylation and arrestin binding, recent studies have shown a critical function for ubiquitination of GPCRs in signal regulation. Over the past 10 years, numerous studies have documented that GPCRs and associated proteins are post-translationally modified by ubiquitination and shown an important role for ubiquitination in regulation of various aspects of receptor signaling and trafficking. Here, we will discuss how certain GPCRs are modified by ubiquitination, the function of GPCR ubiquitination and recent developments of the role for ubiquitin and other ubiquitin-like post-translational modifications on GPCR trafficking and signaling.