RESUMO
Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.
Assuntos
Quiasma Óptico , Células Ganglionares da Retina , Animais , Camundongos , Axônios/metabolismo , Diencéfalo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Vias VisuaisRESUMO
Biallelic mutations in DONSON, an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterized Donson expression and consequences of conditional Donson deletion in the mouse telencephalon. Donson was widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease. Emx1-Cre-mediated Donson deletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage using Tbr2-Cre evoked no apparent malformations, whereas Nkx2.1-Cre-mediated Donson deletion in subpallial progenitors ablated 75% of Nkx2.1-derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals with DONSON mutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. Targeting Donson using an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.
Assuntos
Proteínas de Ciclo Celular/genética , Diferenciação Celular , Córtex Cerebral/metabolismo , Neurônios GABAérgicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Córtex Cerebral/citologia , Neurônios GABAérgicos/citologia , Hipocampo/metabolismo , Camundongos , Neocórtex , Células-Tronco Neurais/citologia , Neurogênese/genética , Neurônios/citologia , Telencéfalo/citologia , Telencéfalo/metabolismoRESUMO
The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.
Assuntos
Inflamação/imunologia , Interleucina-33/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos/imunologia , Mastócitos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Psoríase/imunologia , Pele/imunologia , Animais , Movimento Celular , Células Cultivadas , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
The CXCL12/CXCR4 signaling pathway is involved in the development of numerous neuronal and non-neuronal structures. Recent work established that the atypical second CXCL12 receptor, CXCR7, is essential for the proper migration of interneuron precursors in the developing cerebral cortex. Two CXCR7-mediated functions were proposed in this process: direct modulation of ß-arrestin-mediated signaling cascades and CXCL12 scavenging to regulate local chemokine availability and ensure responsiveness of the CXCL12/CXCR4 pathway in interneurons. Neither of these functions has been proven in the embryonic brain. Here, we demonstrate that migrating interneurons efficiently sequester CXCL12 through CXCR7. CXCR7 ablation causes excessive phosphorylation and downregulation of CXCR4 throughout the cortex in mice expressing CXCL12, but not in CXCL12-deficient animals. Cxcl12(-/-) mice lack activated CXCR4 in embryonic brain lysates and display a similar interneuron positioning defect as Cxcr4(-/-), Cxcr7(-/-) and Cxcl12(-/-);Cxcr7(-/-) animals. Thus, CXCL12 is the only CXCR4-activating ligand in the embryonic brain and deletion of one of the CXCL12 receptors is sufficient to generate a migration phenotype that corresponds to the CXCL12-deficient pathway. Our findings imply that interfering with the CXCL12-scavenging activity of CXCR7 causes loss of CXCR4 function as a consequence of excessive CXCL12-mediated CXCR4 activation and degradation.
Assuntos
Movimento Celular , Córtex Cerebral/citologia , Quimiocina CXCL12/metabolismo , Regulação para Baixo , Interneurônios/citologia , Interneurônios/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Animais , Embrião de Mamíferos/citologia , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
CXCL12 signaling through G protein-coupled CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. The second CXCL12-receptor CXCR7 modulates the CXCL12/CXCR4 pathway by acting as a CXCL12 scavenger and exerts G protein-independent functions. Given the distinct properties of CXCR4 and CXCR7, we hypothesized that the distinct C-terminal domains differently regulate receptor trafficking and stability. Here, we examined epitope-tagged wild type and C-terminal mutant receptors in human embryonic kidney cells (HEK293) with respect to trafficking, stability, (125)I-CXCL12 degradation, and G protein-coupling. The 24 CXCR7 C-terminal residues were sufficient to promote rapid spontaneous internalization. Replacement of the CXCR7 C terminus with that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization and phosphorylation at the heterologous domain. The reverse tail-swap caused ligand-independent internalization of the resulting CXCR4-7tail mutant. Receptor-mediated (125)I-CXCL12 uptake and release of (125)I-CXCL12 degradation products were accelerated with receptors bearing the CXCR7 C terminus and impaired after conversion of CXCR7 C-terminal serine/threonine residues into alanines. C-terminal lysine residues were dispensable for plasma membrane targeting and the CXCL12 scavenger function but involved in constitutive degradation of CXCR7. Although the CXCR7 C terminus abolished G protein coupling in the CXCR4-7tail mutant, replacement of the CXCR7 C terminus, CXCR7 second intracellular loop, or both domains with the corresponding CXCR4 domain did not result in a G protein-coupled CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C terminus influences the ligand-uptake/degradation rate, G protein coupling, and receptor stability. Regulatory pathways targeting CXCR7 C-terminal serine/threonine sites may control the CXCL12 scavenger activity of CXCR7.
Assuntos
Quimiocina CXCL12/metabolismo , Proteólise , Receptores CXCR/metabolismo , Animais , Quimiocina CXCL12/genética , Células HEK293 , Humanos , Camundongos , Mutação , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores CXCR/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMO
Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one-third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4-GFP expression in NSCs, neuronal-committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12-promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2-positive neural stem/early progenitor cells, NeuroD-positive neuronal-committed progenitors, and DCX-positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one-third of the GCL as differentiation area for immature granule neurons.
Assuntos
Giro Denteado/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Receptores CXCR4/metabolismo , Fatores Etários , Animais , Fármacos Anti-HIV/farmacologia , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Benzilaminas , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/farmacologia , Ciclamos , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Regulação da Expressão Gênica no Desenvolvimento/genética , Compostos Heterocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Receptores CXCR4/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The function of the cerebral cortex depends on various types of interneurons (cortical interneurons [cINs]) and their appropriate allocation to the cortical layers. Caudal ganglionic eminence-derived cINs (cGE-cINs) are enriched in superficial layers. Developmental mechanisms directing cGE-cINs toward superficial layers remain poorly understood. We examine how developmental and final positioning of cGE-cINs are influenced by the Cxcl12, Cxcr4, Ackr3 module, the chief attractant system guiding medial ganglionic eminence-derived cINs (mGE-cINs). We find that Cxcl12 attracts cGE-cINs through Cxcr4 and supports their layer-specific positioning in the developing cortex. This requires the prevention of excessive Cxcr4 stimulation by Ackr3-mediated Cxcl12 sequestration. Postnatally, Ackr3 confines Cxcl12 action to the marginal zone. Unlike mGE-cINs, cGE-cINs continue to express Cxcr4 at early postnatal stages, which permits cGE-cINs to become positioned in the forming layer 1. Thus, chemoattraction by Cxcl12 guides cGE-cINs and holds them in superficial cortical layers.
Assuntos
Córtex Cerebral , Interneurônios , Movimento Celular/fisiologia , Córtex Cerebral/fisiologia , Interneurônios/fisiologia , Eminência Mediana , MesodermaRESUMO
Cxcl12-null embryos have dysplastic, misaligned, and hyperplastic semilunar valves (SLVs). In this study, we show that CXCL12 signaling via its receptor CXCR4 fulfills distinct roles at different stages of SLV development, acting initially as a guidance cue to pattern cellular distribution within the valve primordia during the endocardial-to-mesenchymal transition (endoMT) phase and later regulating mesenchymal cell proliferation during SLV remodeling. Transient, anteriorly localized puncta of internalized CXCR4 are observed in cells undergoing endoMT. In vitro, CXCR4+ cell orientation in response to CXCL12 requires phosphatidylinositol 3-kinase (PI3K) signaling and is inhibited by suppression of endocytosis. This dynamic intracellular localization of CXCR4 during SLV development is related to CXCL12 availability, potentially enabling activation of divergent downstream signaling pathways at key developmental stages. Importantly, Cxcr7-/- mutants display evidence of excessive CXCL12 signaling, indicating a likely role for atypical chemokine receptor CXCR7 in regulating ligand bioavailability and thus CXCR4 signaling output during SLV morphogenesis.
Assuntos
Quimiocina CXCL12/metabolismo , Morfogênese/fisiologia , Organogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CXCR/deficiência , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/genéticaRESUMO
Monocyte-derived and tissue-resident macrophages are ontogenetically distinct components of the innate immune system. Assessment of their respective functions in pathology is complicated by changes to the macrophage phenotype during inflammation. Here we find that Cxcr4-CreER enables permanent genetic labeling of hematopoietic stem cells (HSCs) and distinguishes HSC-derived monocytes from microglia and other tissue-resident macrophages. By combining Cxcr4-CreER-mediated lineage tracing with Cxcr4 inhibition or conditional Cxcr4 ablation in photothrombotic stroke, we find that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue. After transient focal ischemia, Cxcr4 deficiency reduces monocyte infiltration and blunts the expression of pattern recognition and defense response genes in monocyte-derived macrophages. This is associated with an altered microglial response and deteriorated outcomes. Thus, Cxcr4 is essential for an innate-immune-system-mediated defense response after cerebral ischemia. We further propose Cxcr4-CreER as a universal tool to study functions of HSC-derived cells.
Assuntos
Isquemia Encefálica/imunologia , Células-Tronco Hematopoéticas/imunologia , Microglia/imunologia , Monócitos/imunologia , Receptores CXCR4/metabolismo , Acidente Vascular Cerebral/imunologia , Animais , Isquemia Encefálica/patologia , Linhagem da Célula , Infarto Cerebral/imunologia , Infarto Cerebral/patologia , Células-Tronco Hematopoéticas/patologia , Imunidade Inata/genética , Ataque Isquêmico Transitório/imunologia , Ataque Isquêmico Transitório/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Monócitos/patologia , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Acidente Vascular Cerebral/patologia , Trombose/patologia , Resultado do TratamentoRESUMO
The atypical chemokine receptor 3 (ACKR3) plays a pivotal role in directing the migration of various cellular populations and its over-expression in tumors promotes cell proliferation and invasiveness. The intracellular signaling pathways transducing ACKR3-dependent effects remain poorly characterized, an issue we addressed by identifying the interactome of ACKR3. Here, we report that recombinant ACKR3 expressed in HEK293T cells recruits the gap junction protein Connexin 43 (Cx43). Cx43 and ACKR3 are co-expressed in mouse brain astrocytes and human glioblastoma cells and form a complex in embryonic mouse brain. Functional in vitro studies show enhanced ACKR3 interaction with Cx43 upon ACKR3 agonist stimulation. Furthermore, ACKR3 activation promotes ß-arrestin2- and dynamin-dependent Cx43 internalization to inhibit gap junctional intercellular communication in primary astrocytes. These results demonstrate a functional link between ACKR3 and gap junctions that might be of pathophysiological relevance.
Assuntos
Astrócitos/metabolismo , Comunicação Celular/fisiologia , Conexina 43/metabolismo , Junções Comunicantes/patologia , Receptores CXCR/metabolismo , Animais , Proliferação de Células , Conexina 43/efeitos dos fármacos , Conexinas/metabolismo , Técnicas de Introdução de Genes , Glioblastoma/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR/agonistas , Receptores CXCR/genética , Transdução de Sinais/fisiologiaRESUMO
Phosphorylation of heptahelical receptors is thought to regulate G protein signaling, receptor endocytosis, and non-canonical signaling via recruitment of ß-arrestins. We investigated chemokine receptor functionality under phosphorylation-deficient and ß-arrestin-deficient conditions by studying interneuron migration in the embryonic cortex. This process depends on CXCL12, CXCR4, G protein signaling and on the atypical CXCL12 receptor ACKR3. We found that phosphorylation was crucial, whereas ß-arrestins were dispensable for ACKR3-mediated control of CXCL12 levels in vivo. Cortices of mice expressing phosphorylation-deficient ACKR3 exhibited a major interneuron migration defect, which was accompanied by excessive activation and loss of CXCR4. Cxcl12-overexpressing mice mimicked this phenotype. Excess CXCL12 caused lysosomal CXCR4 degradation, loss of CXCR4 responsiveness, and, ultimately, similar motility defects as Cxcl12 deficiency. By contrast, ß-arrestin deficiency caused only a subtle migration defect mimicked by CXCR4 gain of function. These findings demonstrate that phosphorylation regulates atypical chemokine receptor function without ß-arrestin involvement in chemokine sequestration and non-canonical signaling.
Assuntos
Movimento Celular , Interneurônios/metabolismo , Receptores CXCR/metabolismo , Animais , Células CHO , Quimiocina CXCL12/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Humanos , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Receptores CXCR/genética , beta-Arrestinas/metabolismoRESUMO
NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases.
Assuntos
Quinase I-kappa B/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Mastócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Ativação Enzimática , Genótipo , Células HEK293 , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Interleucina-33/metabolismo , MAP Quinase Quinase Quinases/genética , Mastócitos/enzimologia , Mastócitos/patologia , Camundongos Knockout , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Fenótipo , Cultura Primária de Células , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Fatores de Tempo , Transfecção , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis - probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose-response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.
Assuntos
Quimiocinas CXC/metabolismo , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Células Jurkat , Sistema de Sinalização das MAP Quinases , Transporte Proteico , Receptores CXCR4/químicaRESUMO
The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven ß-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and ß-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail.
Assuntos
Endocitose/efeitos dos fármacos , Octreotida/farmacologia , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Sequência de Aminoácidos , Antineoplásicos Hormonais/farmacologia , Arrestinas/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Dados de Sequência Molecular , Tumores Neuroendócrinos/tratamento farmacológico , Fosforilação , Transporte Proteico/efeitos dos fármacos , Somatostatina/farmacologia , beta-ArrestinasRESUMO
The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.
Assuntos
Quimiocina CXCL12/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Receptores CXCR4/metabolismo , Sítios de Ligação/genética , Cálcio/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/genética , Células HEK293 , Humanos , Immunoblotting , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Cinética , Mutação , Fosforilação , Doenças da Imunodeficiência Primária , Interferência de RNA , Receptores CXCR4/genética , Serina/genética , Serina/metabolismo , Verrugas/genética , Verrugas/metabolismoRESUMO
Munc-18 interacts with the SNARE protein syntaxin and is supposed to influence transmitter release by controlling the formation of exocytosis-relevant SNARE complexes. Here, we used combined biochemical and physiological analyses to study the role of the Munc-18/syntaxin interaction in large dense core vesicle (LDCV) exocytosis of neuroendocrine PC12 cells. We compared two Munc-18 mutants carrying mutations in the syntaxin-binding region and show that Munc-18's membrane association depends on direct binding to syntaxin. The data suggest that perturbation of syntaxin binding inhibits neurotransmitter release upstream of the individual fusion event implying an essential role of the Munc-18/syntaxin complex leading to exocytosis. Furthermore, we show that a Munc-18 mutant lacking any syntaxin binding has a stimulatory effect on secretion, and provide evidence that the Munc-18/Mint1 interaction may constitute a second pathway for Munc-18 to regulate exocytosis. We propose that Munc-18 represents a dynamic link between syntaxin-related and Mint1-related mechanisms, both involved in the control of LDCV exocytosis in neuroendocrine cells.