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Impaired Recruitment of Grk6 and β-Arrestin2 Causes Delayed Internalization and Desensitization of a WHIM Syndrome-Associated CXCR4 Mutant Receptor  [PDF]
Peter J. McCormick,Marta Segarra,Paola Gasperini,A. Virginia Gulino,Giovanna Tosato
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0008102
Abstract: WHIM (warts, hypogammaglobulinemia, infections, and myelokatexis) syndrome is a rare immunodeficiency syndrome linked to heterozygous mutations of the chemokine receptor CXCR4 resulting in truncations of its cytoplasmic tail. Leukocytes from patients with WHIM syndrome display impaired CXCR4 internalization and enhanced chemotaxis in response to its unique ligand SDF-1/CXCL12, which likely contribute to the clinical manifestations. Here, we investigated the biochemical mechanisms underlying CXCR4 deficiency in WHIM syndrome. We report that after ligand activation, WHIM-associated mutant CXCR4 receptors lacking the carboxy-terminal 19 residues internalize and activate Erk 1/2 slower than wild-type (WT) receptors, while utilizing the same trafficking endocytic pathway. Recruitment of β-Arrestin 2, but not β-Arrestin 1, to the active WHIM-mutant receptor is delayed compared to the WT CXCR4 receptor. In addition, while both kinases Grk3 and Grk6 bind to WT CXCR4 and are critical to its trafficking to the lysosomes, Grk6 fails to associate with the WHIM-mutant receptor whereas Grk3 associates normally. Since β-Arrestins and Grks play critical roles in phosphorylation and internalization of agonist-activated G protein-coupled receptors, these results provide a molecular basis for CXCR4 dysfunction in WHIM syndrome.
Effects of the Dopamine D2 Allosteric Modulator, PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization  [PDF]
Dipannita Basu, Yuxin Tian, Jayant Bhandari, Jian Ru Jiang, Patricia Hui, Rodney L. Johnson, Ram K. Mishra
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0070736
Abstract: The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo?-1-pyrrolidineacetamide(PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA’s effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA’s development into a novel drug for the improved treatment of schizophrenia.
Agonist mediated internalization of M2 mAChR is β-arrestin-dependent
Kymry T Jones, Maria Echeverry, Valerie A Mosser, Alicia Gates, Darrell A Jackson
Journal of Molecular Signaling , 2006, DOI: 10.1186/1750-2187-1-7
Abstract: In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells.In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.Muscarinic acetylcholine receptors belong to the superfamily of G-protein coupled receptors (GPCRs) that are commonly expressed in a variety of tissues and are classified into five known subtypes (M1 -M5 mAChR). M1, M3, and M5 mAChRs are selectively coupled to Gq proteins while M2 and M4 mAChRs are linked to Gi/G0 proteins [1,2]. M2 mAChRs are the primary muscarinic subtype in the heart where their stimulation leads to the regulation of myocardial contractility [3]. As with other GPCRs, M2 mAChR activity is tightly regulated by desensitization and internalization. These regulatory mechanisms are typically associated w
Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells  [PDF]
Katie M. Lowther, Tracy F. Uliasz, Konrad R. G?tz, Viacheslav O. Nikolaev, Lisa M. Mehlmann
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0065365
Abstract: G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.
Co-Expression of GRK2 Reveals a Novel Conformational State of the μ-Opioid Receptor  [PDF]
Sarah A. Nickolls, Sian Humphreys, Mellissa Clark, Gordon McMurray
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083691
Abstract: Agonists at the μ-opioid receptor are known to produce potent analgesic responses in the clinical setting, therefore, an increased understanding of the molecular interactions of ligands at this receptor could lead to improved analgesics. As historically morphine has been shown to be a poor recruiter of β-arrestin in recombinant cell systems and this can be overcome by the co-expression of GRK2, we investigated the effects of GRK2 co-expression, in a recombinant μ-opioid receptor cell line, on ligand affinity and intrinsic activity in both β-arrestin recruitment and [35S]GTPγS binding assays. We also investigated the effect of receptor depletion in the β-arrestin assay. GRK2 co-expression increased both agonist Emax and potency in the β-arrestin assay. The increase in agonist potency could not be reversed using receptor depletion, supporting that the effects were due to a novel receptor conformation not system amplification. We also observed a small but significant effect on agonist KL values. Potency values in the [35S]GTPγS assay were unchanged; however, inverse agonist activity became evident with GRK2 co-expression. We conclude that this is direct evidence that the μ-opioid receptor is an allosteric protein and the co-expression of signalling molecules elicits changes in its conformation and thus ligand affinity. This has implications when describing how ligands interact with the receptor and how efficacy is determined.
High selective long-acting β1-blocker with a vasodilating effect nebivolol in patients with chronic ischemic heart disease  [cached]
V.P. Lupanov
Rational Pharmacotherapy in Cardiology , 2012,
Abstract: Review is devoted to the role of modern beta-blocker nebivolol in the treatment of stable ischemic heart disease (IHD). The mode of nebivolol anti-ischemic activity is considered, as well as its use particularities in IHD patients with different concomitant diseases. Nebivolol effects are compared with these of other beta-blockers and other antianginal drugs in terms of angina symptoms reduction, quality of life improvement and decrease in cardiovascular complications risk.
Quantitative Modeling of GRK-Mediated β2AR Regulation  [PDF]
Sharat J. Vayttaden,Jacqueline Friedman,Tuan M. Tran,Thomas C. Rich,Carmen W. Dessauer,Richard B. Clark
PLOS Computational Biology , 2010, DOI: 10.1371/journal.pcbi.1000647
Abstract: We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.
Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors
Valerie A Mosser, Kymry T Jones, Katie M Hoffman, Nael A McCarty, Darrell A Jackson
Journal of Molecular Signaling , 2008, DOI: 10.1186/1750-2187-3-20
Abstract: Herein we report that agonist activation of M1 mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.There are five subtypes of muscarinic acetylcholine receptors (M1 – M5 mAChR) with distinct yet overlapping tissue distributions. Muscarinic receptors regulate
Phosphorylation of C3a Receptor at Multiple Sites Mediates Desensitization, β-Arrestin-2 Recruitment and Inhibition of NF-κB Activity in Mast Cells  [PDF]
Kshitij Gupta, Hariharan Subramanian, Andreas Klos, Hydar Ali
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0046369
Abstract: Background Phosphorylation of G protein coupled receptors (GPCRs) by G protein coupled receptor kinases (GRKs) and the subsequent recruitment of β-arrestins are important for their desensitization. Using shRNA-mediated gene silencing strategy, we have recently shown that GRK2, GRK3 and β-arrestin-2 promote C3a receptor (C3aR) desensitization in human mast cells. We also demonstrated that β-arrestin-2 provides an inhibitory signal for NF-κB activation. C3aR possesses ten potential phosphorylation sites within its carboxyl terminus but their role on desensitization, β-arrestin recruitment and NF-κB activation has not been determined. Methodology/Principal Findings We utilized a site directed mutagenesis approach in transfected HEK293 cells to determine the role of receptor phosphorylation on β-arrestin-2 recruitment and RBL-2H3 cells for functional studies. We found that although Ala substitution of Ser475/479, Thr480/481 residues resulted in 58±3.8% decrease in agonist-induced C3aR phosphorylation there was no change in β-arrestin-2 binding or receptor desensitization. By contrast, Ala substitution of Thr463, Ser465, Thr466 and Ser470 led to 40±1.3% decrease in agonist-induced receptor phosphorylation but this was associated with 74±2.4% decreases in β-arrestin-2 binding, significantly reduced desensitization and enhanced NF-κB activation. Combined mutation of these Ser/Thr residues along with Ser459 (mutant MT7), resulted in complete loss of receptor phosphorylation and β-arrestin-2 binding. RBL-2H3 cells expressing MT7 responded to C3a for greater Ca2+ mobilization, degranulation and NF-κB activation when compared to the wild-type receptor. Interestingly, co-expression of MT7 with a constitutively active mutant of β-arrestin (R169E) inhibited C3a-induced degranulation by 28±2.4% and blocked NF-κB activation by 80±2.4%. Conclusion/Significance This study demonstrates that although C3a causes phosphorylation of its receptor at multiple sites, Ser459, Thr463, Ser465, Thr466 and Ser470 participate in C3aR desensitization, β-arrestin-2 recruitment and inhibition of NF-κB activity. Furthermore, β-arrestin-2 inhibits C3a-induced NF-κB activation via receptor desensitization-dependent and independent pathways.
Effects of the vasodilating beta-blocker nebivolol on smoking-induced endothelial dysfunction in young healthy volunteers  [cached]
André C Schmidt,Burkhard Flick,Elke Jahn,Peter Bramlage
Vascular Health and Risk Management , 2008,
Abstract: André C Schmidt1, Burkhard Flick1, Elke Jahn2, Peter Bramlage31Charité – Universit tsmedizin Berlin, Institute for Clinical Pharmacology and Toxikology, Berlin, Germany; 2Berlin-Chemie AG, Clinical Research and Medical Information, Berlin, Germany; 3Institute for Clinical Pharmacology, Medical Faculty Carl Gustav Carus, TU Dresden, GermanyObjective: To assess the effect of nebivolol, a highly selective third generation β1-adrenoceptor antagonist with an endothelium-dependent vasodilatory action, on smoking-induced endothelial dysfunction.Research design and methods: This open-label study examined the effect of 14 daily doses of 5 mg nebivolol on forearm blood flow in 21 healthy, young, male, light smokers (≤5 cigarettes/day), measured by plethysmography on Days 1, 7, and 14. The primary endpoint was the difference in forearm blood flow after smoking one standard cigarette from baseline (Day 1) until treatment end on Day 14. Secondary outcomes included the difference in forearm blood flow between Day 1 and Day 7 compared with Day 14 before and after smoking, the effect of nebivolol on blood coagulation parameters, high-sensitive-C-reactive protein (hs-CRP), and the safety and tolerability of nebivolol.Results: Nebivolol for 14 days did not significantly affect forearm blood flow after smoking. On Day 7 of nebivolol treatment, forearm blood flow after smoking was significantly greater than blood flow before smoking (increase of 0.44 mL/min; p = 0.00656). Serum level of hs-CRP showed a marked decrease from Day 1 to Day 14. No changes in coagulation parameters were observed over the course of nebivolol treatment. Nebivolol was well tolerated throughout the study.Conclusions: The increase in forearm blood flow and the marked decrease in hs-CRP over 14 days of treatment suggest that nebivolol has a positive effect on endothelial function in light smokers, but larger studies are required to confirm these observations.Keywords: C-reactive protein, endothelial dysfunction, nebivolol, nitric oxide (NO), smoking
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