Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation

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Abstract

G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse “effector” functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

Introduction

G-protein-coupled receptor kinases (GRKs) are a family of serine-threonine protein kinases composed of seven members (GRK1–GRK7) that share a global homology of 60–70% and are grouped in three subfamilies: visual GRKs, present in cones and rods, formed by GRK1 (or rhodopsin kinase) and GRK7; the β-adrenergic receptor (β-AR), subfamily, to which GRK2 and GRK3 belong; and a third subfamily, which includes GRK4, 5, and 6.1 Nonvisual kinases are ubiquitous, except GRK4, which is predominantly expressed in testes and, to a lower extent, in brain and kidney.

GRKs have a multidomain structure, with a preserved central catalytic domain of c. 500–520 residues. The N-terminal domain comprises 183–188 residues and, except in GRK1 and GRK7, incorporates an N-terminal RGS-homology (RH) domain, which in the case of GRK2 or GRK3, has been shown to allow their interaction with Gαq/11 proteins.2 The C-terminal domain of GRKs is more variable in extension and function and determines the localization of these proteins at the membrane. GRK2/3, although cytosolic at the basal state, incorporate a pleckstrin homology domain (PH) which is able to interact with phospholipids and G protein βγ (Gβγ) subunits at the membrane surface,3, 4 whereas GRK5 has predominantly basic sequences in both the N- and C-terminus through which it associates with membrane lipids. GRK1/4/6 and GRK7 are constitutively anchored to the membrane through short C-terminal prenylation sequences (GRK1 and 7) or through palmitoylation sites (GRK4 and GRK6).5, 6 Interestingly, and unlike many other kinases, activation of GRKs does not require a previous phosphorylation in their activation loop. Instead, docking to agonist-stimulated GPCRs directly triggers their catalytic activity.6

The first function of GRKs to be characterized was the ability to phosphorylate activated GPCRs and thus to be involved in their homologous desensitization.7 Such process is triggered by the phosphorylation of agonist-occupied GPCRs by GRKs, followed by the high-affinity association of cytosolic proteins termed arrestins to the receptor. These events result not only in the uncoupling of GPCR from G proteins, but also in the recruitment of the endocytic machinery, ultimately leading to receptor internalization, which in turn can promote either GPCR-recycling/resensitization or degradation. In addition, GRKs and arrestins are emerging as new signal transducer elements in GPCR signaling cascades as a result of functional or scaffolding interactions with a wide variety of substrates, and also arise as new players in non-GPCRs dependent pathways.8, 9

However, the dogma that GRK-mediated GPCR phosphorylation is strictly dependent on agonist occupancy and receptor activation has been recently called into question.10 Specifically, the GRK4 subfamily possesses the ability to phosphorylate inactive GPCRs that couple to different G-protein subfamilies and can trigger the recruitment of arrestins in the absence of agonist. Conversely, neither GRK2 nor GRK3 significantly phosphorylate inactive receptors, even in conditions that enforced their constitutive localization to the plasma membrane. The fact that membrane-tethered GRK2 mutants display very little basal kinase activity toward GPCRs raises the question of whether concurrent allosteric activation (by interaction with other receptor types or membrane proteins or via posttranslational modifications)6 could endow GRK2 with the ability to phosphorylate inactive GPCRs, in contrast to the intrinsic capacity of the GRK4 subfamily. If confirmed in other experimental systems, this surprising aspect of GRK4/5/6 function may have important repercussions in GPCR signaling and desensitization, since it would allow the formation of arrestin-driven signaling complexes into inactive receptors, while such predesensitized naive receptors could be refractory to trigger G-protein-dependent signaling.

The existence of only seven GRKs to regulate hundreds of GPCRs suggests the existence of some functional redundancy among GRKs and requires mechanisms to guarantee and drive their substrate specificity. However, several evidences show that, even when several GRKs can phosphorylate the same receptor, the functional consequences in receptor regulation are not equivalent. As an example, phosphorylation of the angiotensin receptor by GRK2 and GRK3 promotes β-arrestin mediated GPCR internalization, whereas phosphorylation by GRK5 or GRK6 only has an effect in the β-arrestin dependent capacity to signal to MAPK.11 Thus, different mechanisms modulating the action of distinct GRKs toward a same substrate, might exist depending on the requirements of the cell context.

The ability of GPCRs to divert signaling through different routes (G-protein- or β-arrestin-dependent pathways) according to the conformation stabilized by bound ligands (termed biased signaling) is related to GRK-specific phosphorylation patterns of the receptor conformers that determine which arrestin isoform is preferentially recruited, and which conformation arrestins adopted in the receptor complex in order to scaffold different signalosomes. In a given cell, the potential for β-arrestin/receptor biased signaling may be influenced by several factors, including the precise cellular dosage of each β-arrestin isoform; the biased features of ligands; the extent and duration of receptor stimulation promoting conformations differently engaged by GRKs leading to the assembly of diverse multiprotein complexes (signalosomes); or the competence of each GRK isoform to interact with active (or inactive) receptors.

In this regard, the abundance of a particular GRK in a given cellular context and/or subcellular site is an important variable that shapes receptor signaling and desensitization. Cellular levels of proteins ultimately result from the regulated integration of transcription, protein translation, and protein degradation processes. It is increasingly appreciated that nontranscriptional mechanisms are very actively involved in the regulation of the expression levels of GRKs, particularly of GRK2. Protein turnover relies on several intracellular protein degradation machineries that often converge in the same target, responding coordinately to external stimuli to balance protein levels. Such main degradation pathways include the lysosomal, caspase, calpain, and ubiquitin-dependent proteasome systems. In the last two decades many reports have shown that degradation of GRKs (most of evidence relating to GRK2), is mediated by calpains and the proteasome and coupled to receptor activation.

Altered protein levels of GRKs that are not paralleled by corresponding changes in mRNA levels have been noted in several pathological conditions including chronic inflammatory processes sustained by overdrive of diverse chemokines12; models of Gαq-mediated cardiac hypertrophy13; hypothyroidism14; different tumoral contexts such as in differentiated thyroid carcinoma,15 ovarian cancer,16 or breast cancer (Nogués L, Reglero C, Rivas V, Salcedo A, Lafarga V, Neves M, Ramos P, Mendiola M, Berjón A, Stamatakis K, Zhou XZ, Lu KP, Hardisson D, Mayor Jr F, Penela P. G protein-coupled receptor kinase 2 (GRK2) promotes breast tumorigenesis through a novel HDAC6-Pin1 axis. Submitted. 2016); or in the perinatal period (P. Penela and F. Mayor, unpublished observations), a situation characterized by acute stimulation of adrenergic receptors due to high levels of circulating catecholamines. In these settings protein turnover of GRKs emerges as a rapid and flexible mechanism that would allow for the occurrence of feedback and crosstalk mechanisms modulating GPCR desensitization and other cellular processes. In the next sections we will discuss different mechanisms of degradation of GRKs with a special focus on GRK2, which may help to understand how particularly relevant pathophysiological settings characterized by sustained stimulation of GPCRs or different types of stress (oxidative, genotoxic) can promote the downregulation of steady-state kinase levels, and their significance in GPCR responsiveness and cellular signaling.

Section snippets

GRK2 can be Degraded by the Proteasome System

GRK2 is a short-lived protein (half-life of c. 1 h) as determined by metabolic pulse–chase assays either in heterologous systems or in endogenous conditions in different cell lines (HEK293, Hela, mammary tumor cells lines, Jurkat, or C6 glioma cells).17, 18, 19, 20, 21 When GRK2 degradation rates are estimated by other less-accurate experimental approaches, such as, in conditions of protein synthesis shut-off caused by the presence of the protein translation inhibitor cycloheximide (CHX chase

Proteasome-Dependent Degradation of Other GRKs

Similar to GRK2, GRK3, GRK5, and GRK6 proteins exogenously expressed in HEK293 cells also display rapid turnover in metabolic pulse–chase assays although with slightly delayed kinetics, particularly for GRK6, whose half-life was above 3 h. The addition of the highly specific proteasome inhibitor lactacystin fully blocks degradation of GRK3 or GRK6, indicating that these proteins are basally degraded by the proteasome pathway (A. Elorza, F. Mayor, P. Penela, unpublished results). Moreover, Hsp90

Roles of GRK Ubiquitination Beyond Protein Degradation

Ubiquitination of GRK2 at the N-terminal lysine residues K19, K20, K30, and K31 marks the protein for its proteasome-dependent degradation. However, ubiquitination of these N-terminal residues might have per se additional, direct consequences on GPCR phosphorylation and kinase activity. Different studies of receptor and nonreceptor phosphorylation by GRKs using specific amino-terminus blocking antibodies, N-terminal deletion, or site-directed mutations of the kinase within this region as well

Phosphorylation of Ser670 at the Crossroads of GRK2 Regulation: When Does it Signal Protein Degradation?

Emerging evidence indicates that phosphorylation of S670 is of paramount importance in the regulation of GRK2. The sequence surrounding S670 is highly conserved in all mammalian GRK2 proteins, and different signaling inputs seems to converge in the modification of this residue through the action of several proline-directed kinases, which play critical roles in cell cycle, stress responses, and survival or metabolic control. Mitogen-activated protein kinases ERK1/2 and p38 have been reported to

Multifaceted Roles of the Hsp90 Chaperone in GRK Regulation: The Cardiac Connection

Hsp90 (heat shock protein, 90 kDa) is a very abundant protein (∼1–2% of total cellular protein) devoted to regulate the folding and functionality of a wide array of cellular proteins, referred as “clients.”112 Expression of Hsp90 increases as cells undergo different types of stress (oxidation, radiation) and enhanced levels of Hsp90 are associated with diverse pathological conditions (cancer, inflammatory conditions, Alzheimer disease, hypertension). The interaction of Hsp90 with its clients is

GRK Turnover in Ischemic Conditions: Proteasome and Calpains Coshape GPCR Responsiveness

Heart diseases resulting from myocardial ischemia, such as myocardial infarction or ischemic heart failure involve profound deregulation of cardiac GPCR responsiveness, which is mostly caused by altered patterns of GRK expression, particularly of the predominant cardiac isoforms GRK2 and GRK5.125 Deleterious effects of myocardial ischemia result from the initial interruption of blood supply that leads to the rapid depletion of intramyocardial ATP and contractile arrest, but also and more

Concluding Remarks

Active protein turnover seems to be a general regulatory feature of GRKs in diverse cellular systems and conditions. Although protein turnover is the balance between protein synthesis and protein degradation, this latter effect is more critically involved in the adaptation of GRK turnover to changing cellular conditions. Indeed, GRKs are short-lived proteins as a result of both intrinsic structural factors and extrinsically induced modifications by means of which GRK proteins engage

Acknowledgments

We thank Dr F. Mayor, Jr (CBMSO, Universidad Autónoma de Madrid, Spain) for critical reading of the manuscript and M. Sanz for technical assistance. Our laboratory is funded by grants from The Cardiovascular Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Instituto Carlos III (PI11/00859, PI14-00435), Fundación Eugenio Rodriguez Pascual and Fundación Ramón Areces to P.P.

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