RESUMO
G protein-coupled receptor kinases (GRKs) phosphorylate the activated form of G protein-coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6(-/-)) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6(+/+)). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because ß-arrestin-2-deficient (ßarr2(-/-)) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6(-/-), we developed a double GRK6(-/-)/ßarr2(-/-) mouse model. Surprisingly, GRK6(-/-)/ßarr2(-/-) mice exhibited faster tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6(-/-) and GRK6(-/-)/ßarr2(-/-) animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6(-/-)/ßarr2(-/-) showed greater tumor growth relative to GRK6(-/-) or ßarr2(-/-) mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.
Assuntos
Arrestinas/genética , Carcinoma Pulmonar de Lewis/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Neutrófilos/imunologia , Receptores de Interleucina-8B/metabolismo , Animais , Arrestinas/deficiência , Arrestinas/metabolismo , Linhagem Celular Tumoral , Quimiotaxia , Progressão da Doença , Regulação para Baixo , Quinases de Receptores Acoplados a Proteína G/deficiência , Genótipo , Neoplasias Pulmonares/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Compostos de Fenilureia/farmacologia , Fosforilação , Receptores de Interleucina-8B/antagonistas & inibidores , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima , Cicatrização , beta-Arrestina 2 , beta-ArrestinasRESUMO
Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.
Assuntos
Apoptose , Doenças Autoimunes/enzimologia , Doenças Autoimunes/patologia , Quinases de Receptores Acoplados a Proteína G/deficiência , Fagocitose , Animais , Doenças Autoimunes/sangue , Proteínas de Ciclo Celular/metabolismo , Senescência Celular , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Quinases de Receptores Acoplados a Proteína G/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Fosforilação , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.
Assuntos
Quinases de Receptores Acoplados a Proteína G/metabolismo , Neutrófilos/imunologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Animais , Linhagem Celular Tumoral , Exocitose/genética , Exocitose/imunologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/deficiência , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/antagonistas & inibidores , Quinases de Receptores Acoplados a Proteína G/deficiência , Humanos , Interleucina-8/fisiologia , Leucemia Basofílica Aguda/genética , Leucemia Basofílica Aguda/imunologia , Leucemia Basofílica Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/imunologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosforilação/genética , Ratos , Receptores de Interleucina-8A/fisiologia , Receptores de Interleucina-8B/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting a powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed a reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. A reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/deficiência , Quinase 3 de Receptor Acoplado a Proteína G/deficiência , Quinase 5 de Receptor Acoplado a Proteína G/deficiência , Quinases de Receptores Acoplados a Proteína G/deficiência , Córtex Pré-Frontal/metabolismo , Transtornos Psicóticos/genética , Esquizofrenia/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Arrestinas/biossíntese , Arrestinas/deficiência , Arrestinas/genética , Estudos de Coortes , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/biossíntese , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/biossíntese , Quinase 3 de Receptor Acoplado a Proteína G/genética , Quinase 5 de Receptor Acoplado a Proteína G/biossíntese , Quinase 5 de Receptor Acoplado a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/biossíntese , Quinases de Receptores Acoplados a Proteína G/genética , Humanos , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/fisiopatologia , Transtornos Psicóticos/metabolismo , Transtornos Psicóticos/fisiopatologia , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Adulto JovemRESUMO
BACKGROUND: The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca(2+) mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation. CONCLUSION/SIGNIFICANCE: This study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.
Assuntos
Complemento C3a/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Mastócitos/citologia , Transdução de Sinais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/genética , Linhagem Celular , Quinases de Receptores Acoplados a Proteína G/deficiência , Quinases de Receptores Acoplados a Proteína G/genética , Técnicas de Silenciamento de Genes , Humanos , Lentivirus/genética , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , Receptores de Complemento/agonistas , Receptores de Complemento/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Heart failure (HF) is the end stage of many underlying cardiovascular diseases and is among the leading causes of morbidity and mortality in industrialized countries. One of the striking characteristics of HF is the desensitization of G protein-coupled receptor (GPCR) signaling, particularly the beta-adrenergic receptor (betaAR) system. GPCR desensitization is initiated by phosphorylation by GPCR kinases (GRKs), followed by downregulation and functional uncoupling from their G proteins. In the heart, the major GRK isoforms, GRK2 and GRK5, undergo upregulation due to the heightened sympathetic nervous system activity that is characteristic of HF as catecholamine levels increase in an effort to drive the failing pump. This desensitization leads to the distinctive loss of inotropic reserve and functional capacity of the failing heart. Moreover, GRK2 and GRK5 have an increasing non-GPCR interactome, which may play critical roles in cardiac physiology. In the current review, the canonical GPCR kinase function of GRKs and the novel non-GPCR kinase activity of GRKs, their contribution to the pathogenesis of cardiac hypertrophy and HF, and the possibility of GRKs serving as future drug targets will be discussed.