Showing papers by "Robert J. Lefkowitz published in 2004"

Desensitization of g protein–coupled receptors and neuronal functions

Abstract: ▪ Abstract G protein–coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein–coupled receptor kinases (GRKs) Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and βarrestins in major brain functions In particular, screening of genetically modified mice lacking individual GRKs or βarrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and μ-opioid receptor re

Activity-Dependent Internalization of Smoothened Mediated by ß-Arrestin 2 and GRK2

Abstract: Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo These two processes promote endocytosis of Smo in clathrin-coated pits Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo

β-Arrestin inhibits NF-κB activity by means of its interaction with the NF-κB inhibitor IκBα

Abstract: In addition to their roles in desensitization and signaling of seven-membrane-spanning receptors, β-arrestins have been more recently implicated in regulating non-seven-membrane-spanning receptor pathways. By using a yeast two-hybrid screen, we identified the inhibitor of NF-κB, IκBα, as a binding partner of β-arrestin 1. Both β-arrestin 1 and 2 interact with IκBα in transfected cells as assessed by immunoprecipitation experiments. Additionally, upstream kinases known to regulate the function of IκBα, such as IκB kinase α and β and NF-κB-inducing kinase, were also shown to interact with β-arrestin. Overexpression of either β-arrestin 1 or β-arrestin 2 led to marked inhibition of NF-κB activity, as measured by reporter gene activity. Inhibition of NF-κB activity was independent of the type of stimulus used for NF-κB activation. Conversely, suppression of β-arrestin 1, but not β-arrestin 2, expression by using RNA interference led to a 3-fold increase in tumor necrosis factor-stimulated NF-κB activity as measured by NF-κB mobility-shift analysis. These data uncover a role of β-arrestins in the regulation of NF-κB-mediated gene regulation.

Relative opioid efficacy is determined by the complements of the G protein-coupled receptor desensitization machinery.

Abstract: G protein-coupled receptor regulation by G protein-coupled receptor kinases and β-arrestins can lead to desensitization and subsequent internalization of the receptor. In in vitro and cellular systems, β-arrestins do not seem to play a major role in regulating μ opioid receptor (μOR) responsiveness. Removal of the βarrestin2 (βarr2) gene in mice leads paradoxically to enhanced and prolonged μOR-mediated antinociception. The βarr2 knockout (βarr2-KO) mice also fail to develop morphine antinociceptive tolerance in the hot-plate test, further indicating that the βarr2 protein plays an essential role in μOR regulation in vivo. In this study, the contribution of βarr2 to the regulation of the μOR was examined in both human embryonic kidney 293 cells and in βarr2-KO mice after treatment with several opiate agonists. A green fluorescent protein tagged βarr2 was used to assess receptor-βarr2 interactions in living cells. Opiate agonists that induced robust βarr2-green fluorescent protein translocation produced similar analgesia profiles in wild-type and βarr2-KO mice, whereas those that do not promote robust βarr2 recruitment, such as morphine and heroin, produce enhanced analgesia in vivo. In this report, we present a rationale to explain the seemingly paradoxical relationship between β-arrestins and μOR regulation wherein morphine-like agonists fail to promote efficient internalization and resensitization of the receptor.

Neuropathic pain activates the endogenous κ opioid system in mouse spinal cord and induces opioid receptor tolerance

Abstract: Release of endogenous dynorphin opioids within the spinal cord after partial sciatic nerve ligation (pSNL) is known to contribute to the neuropathic pain processes. Using a phosphoselective antibody [κ opioid receptor (KOR-P)] able to detect the serine 369 phosphorylated form of the KOR, we determined possible sites of dynorphin action within the spinal cord after pSNL. KOR-P immunoreactivity (IR) was markedly increased in the L4-L5 spinal dorsal horn of wild-type C57BL/6 mice (7-21 d) after lesion, but not in mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). In addition, knock-out mice lacking prodynorphin, KOR, or G-protein receptor kinase 3 (GRK3) did not show significant increases in KOR-P IR after pSNL. KOR-P IR was colocalized in both GABAergic neurons and GFAP-positive astrocytes in both ipsilateral and contralateral spinal dorsal horn. Consistent with sustained opioid release, KOR knock-out mice developed significantly increased tactile allodynia and thermal hyperalgesia in both the early (first week) and late (third week) interval after lesion. Similarly, mice pretreated with norBNI showed enhanced hyperalgesia and allodynia during the 3 weeks after pSNL. Because sustained activation of opioid receptors might induce tolerance, we measured the antinociceptive effect of the κ agonist U50,488 using radiant heat applied to the ipsilateral hindpaw, and we found that agonist potency was significantly decreased 7 d after pSNL. In contrast, neither prodynorphin nor GRK3 knock-out mice showed U50,488 tolerance after pSNL. These findings suggest that pSNL induced a sustained release of endogenous prodynorphin-derived opioid peptides that activated an anti-nociceptive KOR system in mouse spinal cord. Thus, endogenous dynorphin had both pronociceptive and antinociceptive actions after nerve injury and induced GRK3-mediated opioid tolerance.

Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.

Abstract: Arrestin regulates almost all G protein–coupled receptor (GPCR)–mediated signaling and trafficking. We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gβγ complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of α2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.

Anti–β1-adrenergic receptor antibodies and heart failure: causation, not just correlation

Abstract: Do anti–β-adrenergic receptor (anti–β-AR) antibodies play a role in the pathogenesis of chronic systolic heart failure (CHF)? This question emerged almost 30 years ago (1), when antibodies with β-adrenergic stimulating (agonist) activity were discovered in the serum of patients with Chagas disease, one of the most common causes of CHF worldwide (2). Since that time, IgGs with agonist activity for the β1-adrenergic receptor (β1-AR) have been found in sera not only from patients with Chagas disease, but also from patients with idiopathic dilated cardiomyopathy (3) as well as ischemic (4) cardiomyopathy. Whether these antibodies merely correlate with myocardial inflammation that leads to CHF, result from myocardial inflammation, or actually contribute to the pathogenesis of CHF could not be ascertained — until now. In this issue of the JCI, Jahns et al. employed isogenic injections of anti–β1-AR antiserum in inbred rats to produce a cardiomyopathy that appears to be non-inflammatory (5). In so doing, these authors conclusively demonstrated that agonistic, anti–β1-AR IgG — by itself — is sufficient to engender the sort of myocardial dysfunction characteristic of CHF. This finding fundamentally advances our understanding of CHF. However, it should not really surprise us, because it represents a logical extension of diverse but congruent investigations conducted over several decades. To provide historical and mechanistic perspectives for the elegant work of Jahns et al., we address several questions that relate their work to contemporary concepts of β1-AR pathophysiology: How might IgG activate the β1-AR, and how could chronic β1-AR activation result in cardiomyocyte toxicity? What molecular mechanisms regulate the β1-AR when it is chronically stimulated by IgG or other agonists, and how might these mechanisms affect the pathogenesis of CHF? Lastly, how can these perspectives elucidate the therapeutic efficacy of β-AR antagonists, or “beta blockers,” in CHF?

G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal

Abstract: Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G-protein receptor kinase mediated receptor phosphorylation and subsequent β-arrestin binding. To define the role of this regulatory mechanism in the control of the electrophysiological and behavioral responses to opioids, we used mice having a targeted disruption of the G-protein receptor kinase 3 (GRK3) gene. Mice lacking GRK3 did not differ from wild-type littermates neither in their response latencies to noxious stimuli on the hot-plate test nor in their acute antinociceptive responses to fentanyl or morphine. Tolerance to the electrophysiological response to the opioid fentanyl, measured in vitro in the hippocampus, was blocked by GRK3 deletion. In addition, tolerance to the antinociceptive effects of fentanyl was significantly reduced in GRK3 knockouts compared to wild-type littermate controls. Tolerance to the antinociceptive effects of morphine was not affected by GRK3 deletion although morphine tolerance in hippocampal slices from GRK3 knockout mice was significantly inhibited. Tolerance developed more slowly in vitro to morphine than fentanyl supporting previous work in in vitro systems showing a correlation between agonist efficacy and GRK3-mediated desensitization. The results of these studies suggest that GRK3-mediated mechanisms are important components of both electrophysiologic and behavioral opioid tolerance. Fentanyl, a high efficacy opioid, more effectively produced GRK3-dependent effects than morphine, a low efficacy agonist. British Journal of Pharmacology (2004) 141, 55–64. doi:10.1038/sj.bjp.0705595

The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels.

Abstract: Epithelial Na+ channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel β subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na+ channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

β-Arrestin-1 Competitively Inhibits Insulin-Induced Ubiquitination and Degradation of Insulin Receptor Substrate 1

Abstract: β-Arrestins are versatile adaptor proteins that form complexes with most seven-transmembrane receptors (7TMR) following agonist binding and receptor phosphorylation by G-protein-coupled receptor kinases. Binding of β-arrestin to the 7TMR cytoplasmic domain interrupts further heterotrimeric G-protein interaction with the receptor, causing signal termination. β-arrestin also mediates endocytosis and receptor sequestration, further enhancing desensitization of receptor signaling (1, 17, 19-21, 27). β-Arrestin can also play a role as a 7TMR signal transducer by recruiting activated Src to the receptor complex, leading to mitogen-activated protein kinase activation (6, 7, 18, 19). We have also reported that β-arrestin can play an important role in the process of heterologous desensitization of receptor tyrosine kinases (RTKs), as well as 7TMRs. Thus, insulin treatment leads to β-arrestin-1 Ser412 phosphorylation, ubiquitination, and degradation, all of which impair mitogen-activated protein kinase phosphorylation mediated by Gα i-coupled receptors, such as the lysophosphatidic acid (LPA) receptor, β2-adrenergic receptor (β2-AR), and the insulin-like growth factor I (IGF-I) receptor (4). Since β-arrestin is involved in homologous G-protein-coupled receptor (GPCR) desensitization, as well as insulin-induced heterologous desensitization of Gα i-coupled receptor signaling, we wondered whether β-arrestin could also function in the process of insulin-induced homologous desensitization. It is well known that chronic insulin treatment leads to subsequent desensitization of insulin signaling at several steps and that an important component of this involves insulin-induced ubiquitination and degradation of one of its major substrates, insulin receptor substrate (IRS) (9, 23-26, 28-30, 33). While β-arrestin-1 does not appear to function in the normal process of insulin signaling, since insulin treatment causes β-arrestin phosphorylation as well as β-arrestin degradation, we sought to determine whether β-arrestin might play a role in the normal desensitization process of insulin signaling induced by chronic insulin treatment. The present studies show that β-arrestin-1 can regulate the process of insulin-mediated IRS-1 ubiquitination and degradation by competing with IRS-1 for Mdm2, an E3 ubiquitin ligase. As such, these studies identify a novel role for β-arrestin-1 as a regulation locus modulating the overall state of cellular insulin sensitivity.

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation.

Abstract: G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, ...

Modified trafficking patterns for arrestin and g-protein-coupled receptors via arrestin-ubiquitin chimera

Abstract: The present invention relates to a modified arrestin which includes an arrestin molecule and a ubiquitin molecule. This modified arrestin has an increased affinity for a GPCR, and traffics with the GPCR into endosomes. The present invention further relates to a method of screening compounds and sample solutions for a GPCR agonist, antagonist, inverse agonist, or desensitization active compound. The modified arrestin is useful in the methods of the present invention.

Resonating to the music of ubiquitination

Abstract: Beyond protein destruction: a new real-time assay to measure dynamic changes in ubiquitination promises to highlight the many other roles of this critical protein modification.

Modeles de trafic modifies pour l'arrestine et les recepteurs couples a la proteine g par l'intermediaire d'une chimere d'arrestine-ubiquitine

Abstract: La presente invention concerne une arrestine modifiee comprenant une molecule d'arrestine et une molecule d'ubiquitine. Cette arrestine modifiee presente une affinite accrue pour un GPCR, et se deplace avec ce GPCR dans des endosomes. La presente invention concerne en outre une methode de criblage de composes et de solutions d'echantillon en vue de l'identification d'un agoniste, d'un antagoniste ou d'un agoniste inverse du GPCR, ou d'un compose actif de desensibilisation. L'arrestine modifiee est utile dans les methodes de la presente invention.