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1.
Cell ; 182(5): 1362-1362.e1, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32888497

RESUMO

The arrestins are ubiquitously expressed adaptor proteins that orchestrate transmembrane signaling cascades triggered by the 7-transmembrane G protein-coupled receptors. While originally discovered as proteins that block receptor-G protein coupling, arrestins are now appreciated for their expanding repertoire of dynamic protein interactions and cellular functions.


Assuntos
Arrestinas/metabolismo , Membrana Celular/metabolismo , Mapas de Interação de Proteínas/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia
2.
J Biol Chem ; 299(5): 104690, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37037304

RESUMO

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.


Assuntos
Glucagon , Receptores de Glucagon , Ubiquitinação , Glucagon/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Humanos , Células HEK293
3.
J Biol Chem ; 299(7): 104911, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37311534

RESUMO

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.


Assuntos
Aterosclerose , Inflamação , Quinases Associadas a Receptores de Interleucina-1 , Interleucina-1beta , Músculo Liso Vascular , Miócitos de Músculo Liso , Fosfosserina , Ubiquitina Tiolesterase , Animais , Humanos , Camundongos , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Hiperplasia/metabolismo , Hiperplasia/patologia , Inflamação/metabolismo , Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/química , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Fosforilação , Fosfosserina/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , NF-kappa B/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Interleucina-1beta/metabolismo , Ubiquitinação
4.
J Biol Chem ; 299(8): 105045, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37451484

RESUMO

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.


Assuntos
Glucagon , Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Fígado , Animais , Camundongos , Glucagon/metabolismo , Gluconeogênese , Glucose/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-39365672

RESUMO

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well-characterized. To define if the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4-weeks of TAC compared to WT cohorts. Both USP20-TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume-overload states and decompensated heart failure. In contrast, WT-TAC mice displayed increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis and mouse mortality were augmented in USP20-KO-TAC compared to WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.

6.
J Biol Chem ; 298(5): 101837, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35307348

RESUMO

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of ß-arrestin2 (ß-arr2) and blocks its association with activated G protein-coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of ß-arr2-GPCR complexes. ß-arr2 Phe116Ala mutant has negligible effect on blunting ß2-adrenergic receptor-induced cAMP generation unlike ß-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6-activated forms of bovine ß-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of ß-arr2. Surprisingly, Phe116 is dispensable for the association of ß-arr2 with its non-GPCR partners. ß-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with ß-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent ß-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that ß-arr2 Phe116Ala interaction with activated ß2-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of ß-arr2, regulates the formation of ß-arr2-GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent ß-arr2 localization and trafficking.


Assuntos
Fenilalanina , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 2 , Animais , Bovinos , Ubiquitina/metabolismo , beta-Arrestina 2/química , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
7.
J Biol Chem ; 295(49): 16630-16642, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-32967969

RESUMO

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endossomos/metabolismo , Receptores de Glucagon/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Linhagem Celular , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glucagon/farmacologia , Humanos , Monensin/farmacologia , Mutagênese , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Glucagon/agonistas , Receptores de Glucagon/genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
8.
J Biol Chem ; 294(7): 2500-2518, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30538132

RESUMO

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial ß1-adrenergic receptors (ß1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the ß1AR. ß1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of ß1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the ß1AR and failed to deubiquitinate the ß1AR. USP20-KO mice showed normal baseline systolic function but impaired ß1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on ß1AR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial ß1AR expression in USP20-KO was drastically reduced, whereas ß2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in ß1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates ß1AR signaling in vitro and in vivo Additionally, ß1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize ß1AR expression and signaling during pathological insults to the myocardium.


Assuntos
Endopeptidases/biossíntese , Regulação Enzimológica da Expressão Gênica , Ativação do Canal Iônico , Miocárdio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Substituição de Aminoácidos , Animais , Endopeptidases/genética , Ventrículos do Coração , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Fosforilação , Receptores Adrenérgicos beta 1/genética , Ubiquitina Tiolesterase , Ubiquitinação
9.
Arterioscler Thromb Vasc Biol ; 38(10): 2295-2305, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30354204

RESUMO

Objective- Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results- To address this question, we used male Ldlr-/- mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1ß (interleukin-1ß). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions- USP20 attenuates TNF- and IL-1ß-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.


Assuntos
Aortite/prevenção & controle , Aterosclerose/prevenção & controle , Endopeptidases/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/patologia , Aortite/enzimologia , Aortite/genética , Aortite/patologia , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Modelos Animais de Doenças , Endopeptidases/genética , Feminino , Hiperplasia , Interleucina-1beta/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Neointima , Placa Aterosclerótica , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de LDL , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitina Tiolesterase , Ubiquitinação
10.
J Biol Chem ; 291(14): 7450-64, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26839314

RESUMO

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ß-arrestin2. Although ß-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ß-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of ß-arrestin2 activity could be dictated by ß-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ß-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that ß-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ß-arrestin2 was augmented when ß-arrestin2 ubiquitination was prevented and reduced when ß-arrestin2 ubiquitination was rendered constitutive. Constitutive ß-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of ß-arrestin2 are determined by ß-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ß-arrestin2.


Assuntos
Arrestinas/metabolismo , Endopeptidases/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Ubiquitinação/fisiologia , Animais , Arrestinas/genética , Linhagem Celular , Endopeptidases/genética , Camundongos , Camundongos Knockout , NF-kappa B/genética , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/genética , Ubiquitina Tiolesterase , beta-Arrestinas
11.
J Cardiovasc Pharmacol ; 70(3): 142-158, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28328745

RESUMO

ß-arrestin1 (or arrestin2) and ß-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that ß-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by ß-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted ß-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that ß-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by ß-arrestins and highlight the physiologic effects of ß-arrestin-dependent signaling.


Assuntos
Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo , Animais , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Humanos , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/farmacologia
12.
Nature ; 477(7364): 349-53, 2011 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-21857681

RESUMO

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates ß(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated ß(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of ß-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which ß-adrenergic catecholamines, acting through both Gs-PKA and ß-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, ß-arrestin-1 (ARRB1), activated via ß(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.


Assuntos
Arrestinas/metabolismo , Dano ao DNA , Receptores Adrenérgicos beta 2/metabolismo , Estresse Fisiológico/fisiologia , Animais , Arrestinas/deficiência , Arrestinas/genética , Catecolaminas/farmacologia , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Testículo/metabolismo , Timo/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , beta-Arrestina 1 , beta-Arrestinas
13.
J Biol Chem ; 290(14): 8888-903, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25666616

RESUMO

Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated ß2 adrenergic receptors (ß2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated ß2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a ß2AR-dependent manner. Upon ß2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated ß2AR complex, and facilitates trafficking of the ubiquitinated ß2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the ß2AR to autophagosomes while promoting plasma membrane recycling of internalized ß2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose , Fagossomos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Células HEK293 , Humanos , Fosforilação , Transporte Proteico , Serina/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/química
14.
J Cell Physiol ; 231(10): 2071-80, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26790995

RESUMO

The non-visual arrestins, ß-arrestin1, and ß-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of ß-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of ß-arrestins in protein ubiquitination is evolutionarily conserved. ß-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of ß-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on ß-arrestin plays a critical role in stabilizing ß-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as ß-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Movimento Celular/fisiologia , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , beta-Arrestinas/metabolismo , Animais , Humanos , Ubiquitinação/fisiologia
15.
EMBO Rep ; 14(2): 164-71, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23208550

RESUMO

ß-arrestin 1 and 2 (also known as arrestin 2 and 3) are homologous adaptor proteins that regulate seven-transmembrane receptor trafficking and signalling. Other proteins with predicted 'arrestin-like' structural domains but lacking sequence homology have been indicated to function like ß-arrestin in receptor regulation. We demonstrate that ß-arrestin2 is the primary adaptor that rapidly binds agonist-activated ß(2) adrenergic receptors (ß(2)ARs) and promotes clathrin-dependent internalization, E3 ligase Nedd4 recruitment and ubiquitin-dependent lysosomal degradation of the receptor. The arrestin-domain-containing (ARRDC) proteins 2, 3 and 4 are secondary adaptors recruited to internalized ß(2)AR-Nedd4 complexes on endosomes and do not affect the adaptor roles of ß-arrestin2. Rather, the role of ARRDC proteins is to traffic Nedd4-ß(2)AR complexes to a subpopulation of early endosomes.


Assuntos
Arrestinas/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Ubiquitinação , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Células COS , Chlorocebus aethiops , Endocitose , Células HEK293 , Humanos , Isoproterenol/farmacologia , Microscopia de Fluorescência , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , beta-Arrestina 1 , beta-Arrestinas
16.
Handb Exp Pharmacol ; 219: 187-203, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24292831

RESUMO

Arrestins constitute a small family of four homologous adaptor proteins (arrestins 1-4), which were originally identified as inhibitors of signal transduction elicited by the seven-transmembrane G protein-coupled receptors. Currently arrestins (especially arrestin2 and arrestin3; also called ß-arrestin1 and ß-arrestin2) are known to be activators of cell signaling and modulators of endocytic trafficking. Arrestins mediate these effects by binding to not only diverse cell-surface receptors but also by associating with a variety of critical signaling molecules in different intracellular compartments. Thus, the functions of arrestins are multifaceted and demand interactions with a host of proteins and require an array of selective conformations. Furthermore, receptor ligands that specifically induce signaling via arrestins are being discovered and their physiological roles are emerging. Recent evidence suggests that the activity of arrestin is regulated in space and time by virtue of its dynamic association with specific enzymes of the ubiquitination pathway. Ubiquitin-dependent, arrestin-mediated signaling could serve as a potential platform for developing novel therapeutic strategies to target transmembrane signaling and physiological responses.


Assuntos
Arrestinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Desenho de Fármacos , Humanos , Ligantes , Terapia de Alvo Molecular , Transdução de Sinais/fisiologia
17.
Curr Opin Physiol ; 372024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38094036

RESUMO

The lethality of heart failure (HF), particularly in the context of post-acute sequelae SARS-CoV-2 infection (PASC)-related myocarditis, necessitates the discovery of the cellular pathways implicated in cardiovascular disease (CVD). We summarize the signaling mechanisms of the catecholamine-binding ß-adrenergic receptors (ß-ARs), with an emphasis on the role of ß-arrestins. ß-ARs, a subset of G protein-coupled receptors (GPCRs), canonically propagate signals through heterotrimeric G proteins. However, since their discovery in the late 1980s, ß-arrestins have been shown to, both (i) quench G protein signaling and (ii) initiate their own independent signaling cascades, which is influenced by post-translational modifications. ß-arrestin-biased agonism by the beta-blocker carvedilol and its allosteric modulation can serve a cardioprotective role. The increasingly labyrinthine nature of GPCR signaling suggests that ligand-dependent ß-AR signaling, either stimulated by an agonist or blocked by an antagonist, is selectively enhanced or suppressed by allosteric modulations, which are orchestrated by novel drugs or endogenous post-translational modifications.

18.
EMBO J ; 28(12): 1684-96, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19424180

RESUMO

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.


Assuntos
Endocitose , Receptores Adrenérgicos beta 2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Animais , Arrestinas/metabolismo , Domínio Catalítico , Bovinos , Compartimento Celular , Endossomos/enzimologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Lisossomos/enzimologia , Proteínas Mutantes/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Ratos , Ubiquitinação , beta-Arrestinas
19.
Arterioscler Thromb Vasc Biol ; 32(2): 308-16, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22095977

RESUMO

OBJECTIVE: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. METHODS AND RESULTS: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe(-/-) mice, carotid interposition grafts from Grk5(-/-) mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5(-/-) with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-ß in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-κB-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-κB activity in macrophages. CONCLUSIONS: GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-κB activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-ß.


Assuntos
Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores CCR2/metabolismo , Transdução de Sinais/fisiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/fisiopatologia , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Quinase 5 de Receptor Acoplado a Proteína G/deficiência , Quinase 5 de Receptor Acoplado a Proteína G/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptor 4 Toll-Like/metabolismo
20.
J Biol Chem ; 286(14): 12785-95, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21330366

RESUMO

Agonist stimulation of the ß2-adrenergic receptors (ß2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length ß2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized ß2ARs in nonlysosomal compartments. Additionally, a lysine-less ß2AR (0K-ß2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT ß2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated ß2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the ß2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited ß-arrestin2, and internalized just as the WT ß2AR. However, only loop 3 and carboxyl tail ß2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the ß2AR. We overexpressed and purified the ß2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved ß2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the ß2AR. These findings introduce a new concept that two distinct domains in the ß2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.


Assuntos
Lisossomos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Isoproterenol/farmacologia , Espectrometria de Massas , Microscopia Confocal , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Proteômica , Receptores Adrenérgicos beta 2/química , Espectrometria de Massas em Tandem , Ubiquitinação
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