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 â¼50% 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 , UbiquitinaçãoRESUMO
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 , UbiquitinaçãoRESUMO
Biased agonism, the ability of different ligands for the same receptor to selectively activate some signaling pathways while blocking others, is now an established paradigm for G protein-coupled receptor signaling. One group of receptors in which endogenous bias is critical is the chemokine system, consisting of over 50 ligands and 20 receptors that bind one another with significant promiscuity. We have previously demonstrated that ligands for the same receptor can cause biased signaling responses. The goal of this study was to identify mechanisms that could underlie biased signaling between different receptor splice variants. The C-X-C motif chemokine receptor 3 (CXCR3) has two splice variants, CXCR3A and CXCR3B, which differ by 51 amino acids at its N-terminus. Consistent with an earlier study, we found that C-X-C motif chemokine ligands 4, 9, 10, and 11 all activated G αi at CXCR3A, while at CXCR3B these ligands demonstrated no measurable G αi or G αs activity. ß-arrestin (ßarr) was recruited at a reduced level to CXCR3B relative to CXCR3A, which was also associated with differences in ßarr2 conformation. ßarr2 recruitment to CXCR3A was attenuated by both G protein receptor kinase (GRK) 2/3 and GRK5/6 knockdown, while only GRK2/3 knockdown blunted recruitment to CXCR3B. Extracellular regulated kinase 1/2 phosphorylation downstream from CXCR3A and CXCR3B was increased and decreased, respectively, by ßarr1/2 knockout. The splice variants also differentially activated transcriptional reporters. These findings demonstrate that differential splicing of CXCR3 results in biased responses associated with distinct patterns of ßarr conformation and recruitment. Differential splicing may serve as a common mechanism for generating biased signaling and provides insights into how chemokine receptor signaling can be modulated post-transcriptionally.
Assuntos
Receptores CXCR3/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Luciferases de Renilla/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores CXCR3/genética , beta-Arrestinas/genéticaRESUMO
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-ArrestinasRESUMO
OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Neuropeptídeos/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Genótipo , Proteínas de Arcabouço Homer/metabolismo , Humanos , Hiperplasia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Fenótipo , Ligação Proteica , Transdução de Sinais , Transfecção , Canais de Potencial de Receptor Transitório/metabolismo , Remodelação VascularRESUMO
OBJECTIVE: Kalirin is a multifunctional protein that contains 2 guanine nucleotide exchange factor domains for the GTPases Rac1 and RhoA. Variants of KALRN have been associated with atherosclerosis in humans, but Kalirin's activity has been characterized almost exclusively in the central nervous system. We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs). APPROACH AND RESULTS: Kalirin-9 protein is expressed abundantly in aorta and bone marrow, as well as in cultured SMCs, endothelial cells, and macrophages. Moreover, arterial Kalirin was upregulated during early atherogenesis in apolipoprotein E-deficient mice. In cultured SMCs, signaling was affected similarly in 3 models of Kalirin loss-of-function: heterozygous Kalrn deletion, Kalirin RNAi, and treatment with the Kalirin Rho-guanine nucleotide exchange factor -1 inhibitor 1-(3-nitrophenyl)-1H-pyrrole-2,5-dione. With reduced Kalirin function, SMCs showed normal RhoA activation but diminished Rac1 activation, assessed as reduced Rac-GTP levels, p21-activated kinase autophosphorylation, and SMC migration. Kalrn(-/+) SMCs proliferated 30% less rapidly than wild-type SMCs. Neointimal hyperplasia engendered by carotid endothelial denudation was ≈60% less in Kalrn(-/+) and SMC-specific Kalrn(-/+) mice than in control mice. CONCLUSIONS: Kalirin functions as a guanine nucleotide exchange factor for Rac1 in SMCs, and promotes SMC migration and proliferation both in vitro and in vivo.
Assuntos
Lesões das Artérias Carótidas/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Neointima , Neuropeptídeos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Artérias Carótidas/enzimologia , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ativação Enzimática , Genótipo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Hiperplasia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , 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 , Fenótipo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais , Transfecção , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
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/metabolismoRESUMO
AIMS: The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation, and pro-inflammatory signalling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis. METHODS AND RESULTS: SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn -/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 weeks, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs were reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. CONCLUSION: We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.
Assuntos
Aterosclerose , Transdiferenciação Celular , Miócitos de Músculo Liso , Neuropeptídeos/genética , Animais , Aterosclerose/genética , Aterosclerose/prevenção & controle , Células Cultivadas , Estudos Transversais , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , NADPH Oxidase 1/genéticaRESUMO
OBJECTIVE: To accelerate vein graft reendothelialization and reduce vein graft thrombosis by infusing human umbilical cord blood-derived endothelial cells (hCB-ECs) because loss of endothelium contributes to vein graft thrombosis and neointimal hyperplasia. METHODS AND RESULTS: Under steady flow conditions in vitro, hCB-ECs adhered to smooth muscle cells 2.5 to 13 times more than ECs derived from peripheral blood or human aorta (P<0.05). Compared with peripheral blood and human aorta ECs, hCB-ECs had 1.4-fold more cell surface α(5)ß(1) integrin heterodimers per cell (P<0.05) and proliferated on fibronectin 4- to 10-fold more rapidly (P<0.05). Therefore, we used hCB-ECs to enhance reendothelialization of carotid interposition vein grafts implanted in NOD.CB17-Prkdc(scid)/J mice. Two weeks postoperatively, vein grafts from hCB-EC-treated mice demonstrated approximately 55% reendothelialization and no luminal thrombosis. In contrast, vein grafts from sham-treated mice demonstrated luminal thrombosis in 75% of specimens (P<0.05) and only approximately 14% reendothelialization. In vein grafts from hCB-EC-treated mice, 33±10% of the endothelium was of human origin, as judged by human major histocompatibility class I expression. CONCLUSIONS: The hCB-ECs adhere to smooth muscle cells under flow conditions in vitro, accelerate vein graft reendothelialization in vivo, and prevent vein graft thrombosis. Thus, hCB-ECs offer novel therapeutic possibilities for vein graft disease.
Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Oclusão de Enxerto Vascular/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Trombose/prevenção & controle , Veias/cirurgia , Animais , Prótese Vascular , Células Cultivadas , Sangue Fetal/citologia , Humanos , Camundongos , Veias/fisiopatologia , Cicatrização/fisiologiaRESUMO
Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.
Assuntos
Aorta/metabolismo , Arrestinas/metabolismo , Aterosclerose/metabolismo , Movimento Celular , Proliferação de Células , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/patologia , Arrestinas/genética , Aterosclerose/genética , Aterosclerose/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Oclusão de Enxerto Vascular/genética , Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/patologia , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Receptores de LDL/genética , Receptores de LDL/metabolismo , beta-ArrestinasRESUMO
Signaling by the platelet-derived growth factor receptor-beta (PDGFRbeta) is diminished when the PDGFRbeta is phosphorylated on seryl residues by G protein-coupled receptor kinase-5 (GRK5), but mechanisms for GRK5 activation by the PDGFRbeta remain obscure. We therefore tested whether the PDGFRbeta is able to tyrosine-phosphorylate and thereby activate GRK5. Purified GRK5 was tyrosine-phosphorylated by the wild-type PDGFRbeta to a stoichiometry of 0.8 mol phosphate/mol GRK5, an extent approximately 5 times greater than observed with a Y857F PDGFRbeta mutant that fails to phosphorylate exogenous substrates but autophosphorylates and activates Src normally. The degree of PDGFRbeta-mediated phosphorylation of GRK5 correlated with GRK5 activity, as assessed by seryl phosphorylation of the PDGFRbeta in purified protein preparations, in intact cells expressing a tyrosine-to-phenylalanine GRK5 mutant, and in GRK5 peptide phosphorylation assays. However, tyrosyl phosphorylation of GRK5 was not necessary for GRK5-mediated phosphorylation of the beta(2)-adrenergic receptor, even though beta(2)-adrenergic receptor activation promoted tyrosyl phosphorylation of GRK5 in smooth muscle cells. Phosphorylation of the PDGFRbeta by GRK5 in smooth muscle cells or in purified protein preparations reduced PDGFRbeta-mediated peptide phosphorylation. In contrast, phosphorylation of GRK5 by the PDGFRbeta enhanced the V(max) of GRK5-mediated peptide phosphorylation, by 3.4-fold, without altering the GRK5 K(M) for peptide. We conclude that GRK5 tyrosyl phosphorylation is required for the activation of GRK5 by the PDGFRbeta, but not by the beta(2)-adrenergic receptor, and that by activating GRK5, the PDGFRbeta triggers its own desensitization.
Assuntos
Quinase 5 de Receptor Acoplado a Proteína G/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/fisiologia , Sequência de Aminoácidos , Animais , Catálise , Bovinos , Linhagem Celular , Quinase 5 de Receptor Acoplado a Proteína G/química , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação/fisiologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Spodoptera , Especificidade por Substrato/fisiologia , Tirosina/metabolismoRESUMO
OBJECTIVE: Inflammation appears intricately linked to vein graft arterialization. We have previously shown that tumor necrosis factor (TNF) receptor-1 (TNFR1, p55) signaling augments vein graft neointimal hyperplasia (NH) and remodeling through its effects on vascular smooth muscle cells (SMCs). In this study we examined the role of TNFR2 (p75) signaling in vein graft arterialization. METHODS AND RESULTS: Inferior vena cava-to-carotid artery interposition grafting was performed between p75-/- and congenic (C57B1/6J) wild-type (WT) mice. Six weeks postoperatively, neointimal and medial dimensions were greater in p75-/- grafts placed into p75-/- recipients (by 42% or 60%, respectively; P<0.05), when compared with WT veins grafted into WT recipients. Relative to WT vein grafts, p75 deficiency augmented early (2-week-old) graft vascular cell adhesion molecule (VCAM)-1 expression (by 2.4-fold, P<0.05), increased endothelial cell apoptosis (2-fold), and delayed graft re-endothelialization. Both cellular proliferation in early, and collagen I content of mature (6-week-old) vein grafts were increased (by 70% and 50%, respectively) in p75-/- grafts. P75 deficiency augmented TNF-induced apoptosis of cultured endothelial cells, but did not affect TNF-stimulated SMC proliferation or migration induced by co-cultured macrophages. CONCLUSIONS: TNF signaling via p75 reduces vein graft neointimal hyperplasia through mechanisms involving reduction of adhesion molecule expression and endothelial cell apoptosis.
Assuntos
Endotélio Vascular/fisiopatologia , Hiperplasia/fisiopatologia , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Túnica Íntima/fisiopatologia , Veia Cava Inferior/transplante , Animais , Apolipoproteínas E/genética , Apoptose , Proliferação de Células , Modelos Animais de Doenças , Masculino , Camundongos , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais/fisiologiaRESUMO
Aims: The actin-binding protein Drebrin is up-regulated in response to arterial injury and reduces smooth muscle cell (SMC) migration and proliferation through its interaction with the actin cytoskeleton. We, therefore, tested the hypothesis that SMC Drebrin inhibits angiotensin II-induced remodelling of the proximal aorta. Methods and results: Angiotensin II was administered via osmotic minipumps at 1000 ng/kg/min continuously for 28 days in SM22-Cre+/Dbnflox/flox (SMC-Dbn-/-) and control mice. Blood pressure responses to angiotensin II were assessed by telemetry. After angiotensin II infusion, we assessed remodelling in the proximal ascending aorta by echocardiography and planimetry of histological cross sections. Although the degree of hypertension was equivalent in SMC-Dbn-/- and control mice, SMC-Dbn-/- mice nonetheless exhibited 60% more proximal aortic medial thickening and two-fold more outward aortic remodelling than control mice in response to angiotensin II. Proximal aortas demonstrated greater cellular proliferation and matrix deposition in SMC-Dbn-/- mice than in control mice, as evidenced by a higher prevalence of proliferating cell nuclear antigen-positive nuclei and higher levels of collagen I. Compared with control mouse aortas, SMC-Dbn-/- aortas demonstrated greater angiotensin II-induced NADPH oxidase activation and inflammation, evidenced by higher levels of Ser-536-phosphorylated NFκB p65 subunits and higher levels of vascular cell adhesion molecule-1, matrix metalloproteinase-9, and adventitial macrophages. Conclusions: We conclude that SMC Drebrin deficiency augments angiotensin II-induced inflammation and adverse aortic remodelling.
Assuntos
Angiotensina II , Doenças da Aorta/metabolismo , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neuropeptídeos/metabolismo , Remodelação Vascular , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Arterial , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células HEK293 , Humanos , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , NADPH Oxidases/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
AIMS: Chronic kidney disease (CKD) is a powerful independent risk factor for cardiovascular events, including vein graft failure. Because CKD impairs the clearance of small proteins, we tested the hypothesis that CKD exacerbates vein graft disease by elevating serum levels of critical cytokines that promote vein graft neointimal hyperplasia. METHODS AND RESULTS: We modelled CKD in C57BL/6 mice with 5/6ths nephrectomy, which reduced glomerular filtration rate by 60%, and we modelled vein grafting with inferior-vena-cava-to-carotid interposition grafting. CKD increased vein graft neointimal hyperplasia four-fold, decreased vein graft re-endothelialization two-fold, and increased serum levels of interleukin-9 (IL-9) five-fold. By quantitative immunofluorescence and histochemical staining, vein grafts from CKD mice demonstrated a â¼two-fold higher prevalence of mast cells, and a six-fold higher prevalence of activated mast cells. Concordantly, vein grafts from CKD mice showed higher levels of TNF and NFκB activation, as judged by phosphorylation of NFκB p65 on Ser536 and by expression of VCAM-1. Arteriovenous fistula veins from humans with CKD also showed up-regulation of mast cells and IL-9. Treating CKD mice with IL-9-neutralizing IgG reduced vein graft neointimal area four-fold, increased vein graft re-endothelialization â¼two-fold, and reduced vein graft total and activated mast cell levels two- and four-fold, respectively. Treating CKD mice with the mast cell stabilizer cromolyn reduced neointimal hyperplasia and increased re-endothelialization in vein grafts. In vitro, IL-9 promoted endothelial cell apoptosis but had no effect on smooth muscle cell proliferation. CONCLUSION: CKD aggravates vein graft disease through mechanisms involving IL-9 and mast cell activation.
Assuntos
Derivação Arteriovenosa Cirúrgica , Artéria Carótida Primitiva/cirurgia , Interleucina-9/metabolismo , Mastócitos/metabolismo , Insuficiência Renal Crônica/complicações , Doenças Vasculares/complicações , Veia Cava Inferior/transplante , Animais , Apoptose , Artéria Carótida Primitiva/imunologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Hiperplasia , Interleucina-9/imunologia , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Neointima , Fosforilação , Insuficiência Renal Crônica/imunologia , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Doenças Vasculares/imunologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Veia Cava Inferior/imunologia , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologiaRESUMO
Despite considerable evidence suggesting that hypertension contributes to the development and progression of atherosclerosis, the causative links remain unclear. We have tested the effects of chronic hypertension induced by suprarenal aortic constriction on the development of atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. Compared with a sham operation, narrowing the aortic luminal diameter by 33% increased blood pressure proximal to the constriction by approximately 15 mm Hg, but the pressures distal to the constriction were unchanged. Kidney renin mRNA and plasma renin activity were also unaffected. Compared with plaque size after the sham operation, atherosclerotic plaque size in the aortic root 8 weeks after coarctation was increased to 245% and 152% in males and females, respectively. Aortic segments at the constriction were free of atherosclerotic deposits, but segments proximal to the constriction were dilated and had atherosclerotic lesions. Thrombi were present immediately below the constriction in Apoe-/- and wild-type vessels. Surprisingly, compared with wild-type mice, the Apoe-/- mice were more susceptible to the cardiac hypertrophy and dysfunction induced by pressure overload. Thus, aortic coarctation exacerbates atherosclerosis in vessels proximal to the constriction without a concomitant increase in the renin-angiotensin system. Our study also suggests that apolipoprotein E plays an important role in modulating cardiac hypertrophy.
Assuntos
Aorta , Apolipoproteínas E/genética , Arteriosclerose/etiologia , Cardiomegalia/etiologia , Hipertensão/complicações , Animais , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Arteriosclerose/patologia , Pressão Sanguínea , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/patologia , Constrição , Progressão da Doença , Feminino , Hipertensão/etiologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sobrevida , UltrassonografiaRESUMO
Lysosomal degradation of ubiquitinated ß(2)-adrenergic receptors (ß(2)ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the ßAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed ß(2)ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-ß(2)ARs in HEK-293 cells. Carvedilol prevented ß(2)AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography-tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound ß(2)AR. The association of MARCH2 with internalized ß(2)ARs was stabilized by carvedilol and did not involve ß-arrestin. Small interfering RNA-mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous ß(2)ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to ß blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface ß(2)AR expression and, consequently, its signaling.
Assuntos
Carbazóis/farmacologia , Proteínas de Transporte/metabolismo , Endocitose , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Animais , Proteínas de Transporte/genética , Carvedilol , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Ratos , Receptores Adrenérgicos beta 2/genética , Ubiquitina-Proteína LigasesRESUMO
Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr(1009) (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr(1021). Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Sequência de Bases , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Primers do DNA/genética , Quinase 5 de Receptor Acoplado a Proteína G , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Músculo Liso Vascular/citologia , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , Coelhos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
G protein-coupled receptor kinase-2 (GRK2) serine-phosphorylates the platelet-derived growth factor receptor-beta (PDGFRbeta), and thereby diminishes signaling by the receptor. Because activation of GRK2 may involve phosphorylation of its N-terminal tyrosines by c-Src, we tested whether the PDGFRbeta itself could tyrosine-phosphorylate and activate GRK2. To do so, we used wild type (WT) and Y857F mutant PDGFRbetas in HEK cells, which lack endogenous PDGFRs. The Y857F PDGFRbeta autophosphorylates normally but does not phosphorylate exogenous substrates. Although PDGF-stimulated Y857F and WT PDGFRbetas activated c-Src equivalently, the WT PDGFRbeta tyrosine-phosphorylated GKR2 60-fold more than the Y857F PDGFRbeta in intact cells. With purified GRK2 and either WT or Y857F PDGFRbetas immunoprecipitated from HEK cells, GRK2 tyrosyl phosphorylation was PDGF-dependent and required the WT PDGFRbeta, even though the WT and Y857F PDGFRbetas autophosphorylated equivalently. This PDGFRbeta-mediated GRK2 tyrosyl phosphorylation enhanced GRK2 activity: GRK2-mediated seryl phosphorylation of the PDGFRbeta was 9-fold greater for the WT than for the Y857F in response to PDGF, but equivalent when GRK2 was activated by sequential stimulation of beta2-adrenergic and PDGF-beta receptors. Furthermore, both PDGFRbeta-mediated GRK2 tyrosyl phosphorylation and GRK2-mediated PDGFRbeta seryl phosphorylation were reduced approximately 50% in intact cells by mutation to phenylalanine of three tyrosines in the N-terminal domain of GRK2. We conclude that the activated PDGFRbeta itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFRbeta.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Bovinos , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática , Quinase 5 de Receptor Acoplado a Proteína G , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Biológicos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Serina/química , Serina/metabolismo , Transdução de Sinais/fisiologia , Tirosina/química , Tirosina/metabolismo , Quinases de Receptores Adrenérgicos beta , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.
Assuntos
Arrestinas/fisiologia , Endocitose , Receptores de LDL/metabolismo , Animais , Arrestinas/deficiência , Arrestinas/genética , Células CHO , Linhagem Celular , Colesterol/sangue , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Clonagem Molecular , Cricetinae , Gorduras na Dieta/administração & dosagem , Embrião de Mamíferos , Fibroblastos , Expressão Gênica , Glutationa Transferase/genética , Humanos , Técnicas de Imunoadsorção , Rim , Lipoproteínas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese , Receptores de LDL/genética , Proteínas Recombinantes de Fusão , Transfecção , beta-ArrestinasRESUMO
Accumulating evidence suggests that receptor protein-tyrosine kinases, like the platelet-derived growth factor receptor-beta (PDGFRbeta) and epidermal growth factor receptor (EGFR), may be desensitized by serine/threonine kinases. One such kinase, G protein-coupled receptor kinase-2 (GRK2), is known to mediate agonist-dependent phosphorylation and desensitization of multiple heptahelical receptors. In testing whether GRK2 could phosphorylate and desensitize the PDGFRbeta, we first found by phosphoamino acid analysis that cells expressing GRK2 could serine-phosphorylate the PDGFRbeta in an agonist-dependent manner. Augmentation or inhibition of GRK2 activity in cells, respectively, reduced or enhanced tyrosine phosphorylation of the PDGFRbeta but not the EGFR. Either overexpressed in cells or as a purified protein, GRK2 demonstrated agonist-promoted serine phosphorylation of the PDGFRbeta and, unexpectedly, the EGFR as well. Because GRK2 did not phosphorylate a kinase-dead (K634R) PDGFRbeta mutant, GRK2-mediated PDGFRbeta phosphorylation required receptor tyrosine kinase activity, as does PDGFRbeta ubiquitination. Agonist-induced ubiquitination of the PDGFRbeta, but not the EGFR, was enhanced in cells overexpressing GRK2. Nevertheless, GRK2 overexpression did not augment PDGFRbeta down-regulation. Like the vast majority of GRK2 substrates, the PDGFRbeta, but not the EGFR, activated heterotrimeric G proteins allosterically in membranes from cells expressing physiologic protein levels. We conclude that GRK2 can phosphorylate and desensitize the PDGFRbeta, perhaps through mechanisms related to receptor ubiquitination. Specificity of GRK2 for receptor protein-tyrosine kinases, expressed at physiologic levels, may be determined by the ability of these receptors to activate heterotrimeric G proteins, among other factors.