Cristina Murga

The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

GRK2 is a ubiquitous member of the G protein‐coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein‐coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist‐regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non‐GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context‐specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2.

Phosphorylation of p38 by GRK2 at the Docking Groove Unveils a Novel Mechanism for Inactivating p38MAPK

p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.

G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

OBJECTIVE Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.

Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts.

Abstract: The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein‐coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate‐treated rats as well as in opiate‐dependent rats after spontaneous or naloxone‐precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane‐associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose‐ and time‐dependent increases (8–22%) in total GRK2 concentrations [higher increases were observed for the membrane‐associated enzyme (46%)]. Spontaneous and naloxone‐precipitated withdrawal after chronic morphine or methadone induced a marked up‐regulation in the levels of total GRK2 in the rat frontal cortex (18–25%). These results suggest that GRK2 is involved in the short‐term regulation of μ‐opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.

High Affinity Binding of β-Adrenergic Receptor Kinase to Microsomal Membranes MODULATION OF THE ACTIVITY OF BOUND KINASE BY HETEROTRIMERIC G PROTEIN ACTIVATION

The β-adrenergic receptor kinase (βARK) modulates β-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that βARK also associates with intracellular microsomal membranes both “in vitro” and “in situ” (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and 5or, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of βARK. In this report, we used recombinant [35S]methionine-labeled βARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 ± 1 nM) present in salt-stripped rat liver microsomal membranes. Although βARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound βARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AlF4− or mastoparan/guanosine 5′-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated βARK. Interestingly, βARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of βARK1 (the proposed location of the βγ subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an ∼60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between βARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of βARK.

Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gβγ subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent K m values in the range of 40–100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gβγ subunits. Phosphorylation of phosducin by GRK2 markedly reduces its Gβγ binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

Interfering with MAP kinase docking interactions: implications and perspective for the p38 route.

Docking interactions are key to understand the dynamic assembly of signal transductioncomplexes in the cell. In particular, the docking domain (D domain)-dependentinteractions described so far for several MAPK routes are essential to specify theupstream regulators, downstream mediators and also inactivators that complex with thep38, JNK and ERK proteins. In addition to contributing to the maintenance of thelinearity and specificity of these pathways, novel data have revealed that dockingcontacts also regulate the activity, subcellular distribution and substrate selection ofeach MAPK. Moreover, phosphorylation inside or around a docking domain isemerging as a novel mechanism of regulation of MAPK association with cellularpartners, suggesting new potential strategies for the design of selective MAPKinhibitors. Here, we discuss these novel data and the biochemical and cellularimplications they may have with specific emphasis on the p38 route.

Increased Nitric Oxide Bioavailability in Adult GRK2 Hemizygous Mice Protects Against Angiotensin II–Induced Hypertension

G protein–coupled receptor kinase 2 (GRK2) is a ubiquitous serine/threonine protein kinase able to phosphorylate and desensitize the active form of several G protein–coupled receptors. Given the lack of selective inhibitors for GRK2, we investigated the effects elicited by GRK2 inhibition in vascular responses using global adult hemizygous mice (GRK2+/−). The vasodilator responses to acetylcholine or isoproterenol were increased in aortas and mesenteric resistance arteries from GRK2+/− mice compared with wild-type (WT) littermates. After angiotensin II (AngII) infusion, GRK2+/− mice were partially protected against hypertension, vascular remodeling, and mechanical alterations, even when resting basal blood pressures were not significantly different. AngII infusion also (1) increased GRK2 levels in WT but not in GRK2+/− vessels; (2) increased vasoconstrictor responses to phenylephrine in WT but not in GRK2+/− mice; and (3) decreased vasodilator responses to acetylcholine and vascular pAkt and eNOS levels more in WT than in GRK2+/− animals. Vascular NO production and the modulation of vasoconstrictor responses by endothelial-derived NO remained enhanced in GRK2+/− mice infused with AngII. Thus, GRK2+/− mice are resistant to the development of vascular remodeling and mechanical alterations, endothelial dysfunction, increased vasoconstrictor responses, and hypertension induced by AngII at least partially through the preservation of NO bioavailability. In conclusion, our results describe an important role for GRK2 in systemic hypertension and further establish that an inhibition of GRK2 could be a beneficial treatment for this condition.

The Origin of Behavioral Bursts in Decision-Making Circuitry

From ants to humans, the timing of many animal behaviors comes in bursts of activity separated by long periods of inactivity. Recently, mathematical modeling has shown that simple algorithms of priority-driven behavioral choice can result in bursty behavior. To experimentally test this link between decision-making circuitry and bursty dynamics, we have turned to Drosophila melanogaster. We have found that the statistics of intervals between activity periods in endogenous activity-rest switches of wild-type Drosophila are very well described by the Weibull distribution, a common distribution of bursty dynamics in complex systems. The bursty dynamics of wild-type Drosophila walking activity are shown to be determined by this inter-event distribution alone and not by memory effects, thus resembling human dynamics. Further, using mutant flies that disrupt dopaminergic signaling or the mushroom body, circuitry implicated in decision-making, we show that the degree of behavioral burstiness can be modified. These results are thus consistent with the proposed link between decision-making circuitry and bursty dynamics, and highlight the importance of using simple experimental systems to test general theoretical models of behavior. The findings further suggest that analysis of bursts could prove useful for the study and evaluation of decision-making circuitry.

GRK2 contribution to the regulation of energy expenditure and brown fat function

Obesity is a major health problem and an important risk factor for the development of multiple disorders. Previous studies in our laboratory have revealed that down‐regulation of GRK2 decreases age‐related adiposity, but the physiological and molecular mechanisms underlying this outcome remain unclear. We evaluate whether the lean phenotype results from a direct effect of GRK2 on energy homeostasis. The study of white adipose tissue (WAT) in wild‐type (WT) and GRK2+/− littermates showed a reduced expression of lipogenic enzymes and enhanced lipolytic rate in adult GRK2+/− mice. Moreover, hemizygous mice display higher energy expenditure and lower respiratory exchange ratio. Analysis of brown adipose tissue (BAT) from adult GRK2+/− mice showed a less deteriorated morphology associated with age compared to WT, which is correlated with a higher basal core temperature. BAT from young GRK2+/− mice showed an increase in gene expression of thermogenesis‐related genes. Accordingly, hemizygous mice displayed better thermogenic capacity and exhibited a more oxidative phenotype in both BAT and WAT than WT littermates. Overexpression of GRK2 in brown adipocytes corroborated the negative effect of this kinase in BAT function and differentiation. Collectively, our data point to GRK2 inhibition as a potential tool for the enhancement of brown fat activity, which may have important therapeutic implications for the treatment of obesity and associated metabolic disorders.—Vila‐Bedmar, R., Garcia‐Guerra, L., Nieto‐Vazquez, I., Mayor, F., Jr., Lorenzo, M., Murga, C., Fernández‐Veledo, S. GRK2 contribution to the regulation of energy expenditure and brown fat function. FASEB J. 26, 3503–3514 (2012). www.fasebj.org