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1.
Development ; 151(11)2024 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-38682276

RESUMEN

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.


Asunto(s)
Barrera Hematoencefálica , Encéfalo , Neovascularización Fisiológica , Receptores Acoplados a Proteínas G , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/embriología , Neovascularización Fisiológica/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Ratones , Encéfalo/metabolismo , Encéfalo/embriología , Dominios Proteicos , Ratones Noqueados , Transducción de Señal , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Humanos , Células Endoteliales/metabolismo , Angiogénesis , Proteínas Ligadas a GPI
2.
Int J Mol Sci ; 23(5)2022 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-35269767

RESUMEN

Transforming growth factor ß (TGFß) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFß receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFß signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFß signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFß signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFß signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.


Asunto(s)
Degeneración Retiniana , Factor de Crecimiento Transformador beta , Animales , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-34208383

RESUMEN

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-ß regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-ß, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-ß, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.


Asunto(s)
Perfilación de la Expresión Génica , Neuroprotección/genética , Retinitis Pigmentosa/genética , Transcripción Genética , Regulación hacia Arriba/genética , Animales , Quimiocina CCL2/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Redes Reguladoras de Genes , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuroglía/metabolismo , Degeneración Retiniana/complicaciones , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Rodopsina/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
4.
Pharmacol Rev ; 67(2): 338-67, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25713288

RESUMEN

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential.


Asunto(s)
Moléculas de Adhesión Celular/metabolismo , AMP Cíclico/fisiología , Modelos Moleculares , Receptores Acoplados a Proteínas G/metabolismo , Sistemas de Mensajero Secundario , Animales , Adhesión Celular , Moléculas de Adhesión Celular/química , Membrana Celular/enzimología , Membrana Celular/metabolismo , Movimiento Celular , Humanos , Agencias Internacionales , Ligandos , Farmacología/tendencias , Farmacología Clínica/tendencias , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/clasificación , Transducción de Señal , Sociedades Científicas , Terminología como Asunto
5.
Cancer Sci ; 107(5): 576-82, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26934061

RESUMEN

The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.


Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Neovascularización Fisiológica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
6.
Cell Mol Life Sci ; 71(18): 3489-506, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24760128

RESUMEN

Angiogenesis, the formation of new blood vessels from pre-existing vessels, in the central nervous system (CNS) is seen both as a normal physiological response as well as a pathological step in disease progression. Formation of the blood-brain barrier (BBB) is an essential step in physiological CNS angiogenesis. The BBB is regulated by a neurovascular unit (NVU) consisting of endothelial and perivascular cells as well as vascular astrocytes. The NVU plays a critical role in preventing entry of neurotoxic substances and regulation of blood flow in the CNS. In recent years, research on numerous acquired and hereditary disorders of the CNS has increasingly emphasized the role of angiogenesis in disease pathophysiology. Here, we discuss molecular mechanisms of CNS angiogenesis during embryogenesis as well as various pathological states including brain tumor formation, ischemic stroke, arteriovenous malformations, and neurodegenerative diseases.


Asunto(s)
Sistema Nervioso Central/irrigación sanguínea , Neovascularización Patológica , Neovascularización Fisiológica , Enfermedades Neurodegenerativas/patología , Malformaciones Arteriovenosas/patología , Barrera Hematoencefálica , Neoplasias Encefálicas/irrigación sanguínea , Desarrollo Embrionario , Humanos , Modelos Biológicos , Factor A de Crecimiento Endotelial Vascular/metabolismo
7.
Biochem J ; 441(3): 937-44, 2012 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-22013897

RESUMEN

TEM5 (tumour endothelial marker 5; also known as GPR124) is an adhesion G-protein-coupled receptor containing a cryptic RGD motif in its extracellular domain. TEM5 is expressed in endothelial cells and pericytes during angiogenesis. In the present paper, we report that thrombin mediates shedding of an N-terminal TEM5 fragment of 60 kDa (termed N60) containing the RGD motif in an open conformation. Thrombin directly cleaved rsTEM5 (recombinant soluble TEM5) 5 and 34 residues downstream of the RGD motif, resulting in formation of N60 and its C-terminal counterpart (termed C50). Interestingly, N60 derived from thrombin cleavage of rsTEM5 was covalently linked to C50 by disulfide bonds, whereas N60 shed from thrombin-treated cells was not associated with its membrane-bound C-terminal counterpart. Inhibition of the reducing function of cell-surface PDI (protein disulfide-isomerase) abrogated thrombin-induced N60 shedding. Conversely, addition of reduced PDI enhanced N60 shedding. Furthermore, thrombin cleavage of rsTEM5 was increased by reduced PDI and resulted in dissociation of the N60-C50 heterodimer. We conclude that PDI regulates thrombin-induced shedding of N60 and exposure of the TEM5 RGD motif by catalysing the reduction of crucial disulfide bonds of TEM5 on the cell surface. Binding of N60 to RGD-dependent integrins may modulate cellular functions such as adhesion and migration during angiogenesis.


Asunto(s)
Proteína Disulfuro Isomerasas/fisiología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Trombina/farmacología , Células Cultivadas , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Modelos Biológicos , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/fisiología , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Proteolisis/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Transfección
8.
Eur J Nucl Med Mol Imaging ; 39(12): 1886-97, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22872310

RESUMEN

PURPOSE: Targeted therapy with α-particle emitting radionuclides is a promising new option in cancer therapy. Stable conjugates of the vascular tumour-homing peptide F3 with the α-emitter (213)Bi specifically target tumour cells. The aim of our study was to determine efficacy of combined (213)Bi-diethylenetriaminepentaacetic acid (DTPA)-F3 and paclitaxel treatment compared to treatment with either (213)Bi-DTPA-F3 or paclitaxel both in vitro and in vivo. METHODS: Cytotoxicity of treatment with (213)Bi-DTPA-F3 and paclitaxel, alone or in combination, was assayed towards OVCAR-3 cells using the alamarBlue assay, the clonogenic assay and flow cytometric analyses of the mode of cell death and cell cycle arrest. Therapeutic efficacy of the different treatment options was assayed after repeated treatment of mice bearing intraperitoneal OVCAR-3 xenograft tumours. Therapy monitoring was performed by bioluminescence imaging and histopathologic analysis. RESULTS: Treatment of OVCAR-3 cells in vitro with combined (213)Bi-DTPA-F3 and paclitaxel resulted in enhanced cytotoxicity, induction of apoptosis and G2/M phase arrest compared to treatment with either (213)Bi-DTPA-F3 or paclitaxel. Accordingly, i.p. xenograft OVCAR-3 tumours showed the best response following repeated (six times) combined therapy with (213)Bi-DTPA-F3 (1.85 MBq) and paclitaxel (120 µg) as demonstrated by bioluminescence imaging and histopathologic investigation of tumour spread on the mesentery of the small and large intestine. Moreover, mean survival of xenograft mice that received combined therapy with (213)Bi-DTPA-F3 and paclitaxel was significantly superior to mice treated with either (213)Bi-DTPA-F3 or paclitaxel alone. CONCLUSION: Combined treatment with (213)Bi-DTPA-F3 and paclitaxel significantly increased mean survival of mice with peritoneal carcinomatosis of ovarian origin, thus favouring future therapeutic application.


Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma/terapia , Quimioradioterapia , Compuestos Organometálicos/uso terapéutico , Paclitaxel/uso terapéutico , Neoplasias Peritoneales/terapia , Radiofármacos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Células HEK293 , Proteína HMGN2/química , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Compuestos Organometálicos/farmacología , Paclitaxel/farmacología , Estructura Terciaria de Proteína , Radiofármacos/farmacología , Resultado del Tratamiento
9.
Exp Cell Res ; 316(3): 412-21, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19853600

RESUMEN

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.


Asunto(s)
Capilares/crecimiento & desarrollo , Inhibición de Contacto , Células Endoteliales/citología , Células Endoteliales/enzimología , Morfogénesis , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Capilares/citología , Capilares/enzimología , Proliferación Celular , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Solubilidad , Regulación hacia Arriba
10.
Biomolecules ; 11(9)2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34572573

RESUMEN

Ischemic insults to the heart and brain, i.e., myocardial and cerebral infarction, respectively, are amongst the leading causes of death worldwide. While there are therapeutic options to allow reperfusion of ischemic myocardial and brain tissue by reopening obstructed vessels, mitigating primary tissue damage, post-infarction inflammation and tissue remodeling can lead to secondary tissue damage. Similarly, ischemia in retinal tissue is the driving force in the progression of neovascular eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), which eventually lead to functional blindness, if left untreated. Intriguingly, the easily observable retinal blood vessels can be used as a window to the heart and brain to allow judgement of microvascular damages in diseases such as diabetes or hypertension. The complex neuronal and endocrine interactions between heart, retina and brain have also been appreciated in myocardial infarction, ischemic stroke, and retinal diseases. To describe the intimate relationship between the individual tissues, we use the terms heart-brain and brain-retina axis in this review and focus on the role of transforming growth factor ß (TGFß) and neurotrophins in regulation of these axes under physiologic and pathologic conditions. Moreover, we particularly discuss their roles in inflammation and repair following ischemic/neovascular insults. As there is evidence that TGFß signaling has the potential to regulate expression of neurotrophins, it is tempting to speculate, and is discussed here, that cross-talk between TGFß and neurotrophin signaling protects cells from harmful and/or damaging events in the heart, retina, and brain.


Asunto(s)
Encéfalo/metabolismo , Miocardio/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Retina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Humanos , Unión Proteica
11.
Cell Stem Cell ; 27(5): 840-851.e6, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-32818433

RESUMEN

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.


Asunto(s)
Receptores Frizzled , Organoides , Hepatocitos , Células Madre , Vía de Señalización Wnt
12.
Nat Struct Mol Biol ; 26(6): 407-414, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31086346

RESUMEN

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.


Asunto(s)
Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/metabolismo , Simulación del Acoplamiento Molecular , Animales , Ancirinas/química , Ancirinas/metabolismo , Línea Celular , Cristalografía por Rayos X , Descubrimiento de Drogas , Duodeno/citología , Duodeno/metabolismo , Receptores Frizzled/química , Humanos , Ratones Endogámicos C57BL , Unión Proteica , Conformación Proteica , Células Madre/citología , Células Madre/metabolismo
13.
Ann N Y Acad Sci ; 1456(1): 5-25, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31168816

RESUMEN

The adhesion class of G protein-coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N-terminal region that is linked to a C-terminal seven transmembrane (7TM) domain via a GPCR-autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N-terminal fragment (NTF) bound to the 7TM of the C-terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell-cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs.


Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Humanos , Receptores Acoplados a Proteínas G/química
14.
Cell Rep ; 25(2): 339-349.e9, 2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30304675

RESUMEN

WNT7A and WNT7B control CNS angiogenesis and blood-brain barrier formation by activating endothelial Wnt/ß-catenin signaling. The GPI-anchored protein RECK and adhesion G protein-coupled receptor GPR124 critically regulate WNT7-specific signaling in concert with FZD and LRP co-receptors. Here, we demonstrate that primarily the GPR124 ectodomain, but not its transmembrane and intracellular domains, mediates RECK/WNT7-induced canonical Wnt signaling. Moreover, RECK is the predominant binding partner of GPR124 in rat brain blood vessels in situ. WNT7A and WNT7B, but not WNT3A, directly bind to purified recombinant soluble RECK, full-length cell surface RECK, and the GPR124:RECK complex. Chemical cross-linking indicates that RECK and WNT7A associate with 1:1 stoichiometry, which stabilizes short-lived, active, monomeric, hydrophobic WNT7A. In contrast, free WNT7A rapidly converts into inactive, hydrophilic aggregates. Overall, RECK is a selective WNT7 receptor that mediates GPR124/FZD/LRP-dependent canonical Wnt/ß-catenin signaling by stabilizing active cell surface WNT7, suggesting isoform-specific regulation of Wnt bioavailability.


Asunto(s)
Receptores Frizzled/metabolismo , Proteínas Ligadas a GPI/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3A/metabolismo , Animales , Disponibilidad Biológica , Barrera Hematoencefálica , Femenino , Receptores Frizzled/genética , Proteínas Ligadas a GPI/genética , Células HEK293 , Humanos , Masculino , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Proteínas Wnt/genética , Proteína Wnt3A/genética , beta Catenina/genética , beta Catenina/metabolismo
15.
Nat Med ; 23(4): 450-460, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28288111

RESUMEN

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-ß-catenin signaling. Constitutive activation of Wnt-ß-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Glioblastoma/genética , Infarto de la Arteria Cerebral Media/genética , Hemorragias Intracraneales/genética , Receptores Acoplados a Proteínas G/genética , Uniones Estrechas/metabolismo , Animales , Barrera Hematoencefálica/ultraestructura , Modelos Animales de Enfermedad , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glioblastoma/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Hemorragias Intracraneales/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Microvasos , Pericitos/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Uniones Estrechas/ultraestructura , Vía de Señalización Wnt
16.
Oncotarget ; 6(7): 4692-703, 2015 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-25576914

RESUMEN

In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter ²¹³Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of ²¹³Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with ²¹³Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. ²¹³Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of ²¹³Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with ²¹³Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of ²¹³Bi-induced toxicity. Preclinical treatment of MM with ²¹³Bi-anti-CD38-MAb turned out as an effective therapeutic option.


Asunto(s)
ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Bismuto/farmacología , Inmunoconjugados/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Mieloma Múltiple/inmunología , Mieloma Múltiple/radioterapia , Radioinmunoterapia , ADP-Ribosil Ciclasa 1/inmunología , Partículas alfa/uso terapéutico , Animales , Apoptosis/inmunología , Apoptosis/efectos de la radiación , Western Blotting , Ciclo Celular/inmunología , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Citometría de Flujo , Humanos , Glicoproteínas de Membrana/inmunología , Ratones , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
17.
Nat Med ; 19(10): 1331-1337, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24037094

RESUMEN

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas Sustrato del Receptor de Insulina/fisiología , Insulina/metabolismo , Hígado/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Diabetes Mellitus Tipo 2/terapia , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa
18.
PLoS One ; 4(5): e5715, 2009 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-19479088

RESUMEN

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology.


Asunto(s)
Bismuto/uso terapéutico , Núcleo Celular/metabolismo , Sistemas de Liberación de Medicamentos , Ácido Pentético/uso terapéutico , Péptidos/administración & dosificación , Péptidos/uso terapéutico , Neoplasias Peritoneales/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Bismuto/administración & dosificación , Bismuto/efectos adversos , Bismuto/farmacocinética , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Humanos , Marcaje Isotópico , Ratones , Ácido Pentético/administración & dosificación , Ácido Pentético/efectos adversos , Ácido Pentético/farmacocinética , Péptidos/efectos adversos , Péptidos/farmacocinética , Radioisótopos , Distribución Tisular , Ensayos Antitumor por Modelo de Xenoinjerto
19.
J Biol Chem ; 281(45): 34179-88, 2006 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-16982628

RESUMEN

Tumor endothelial marker (TEM) 5 is a member of the adhesion family of G-protein-coupled receptors and up-regulated in endothelial cells during tumor and physiologic angiogenesis. Here, we report that TEM5 is expressed on the surface of endothelial cells. A soluble TEM5 (sTEM5) fragment is shed by endothelial cells during capillary-like network formation and upon growth factor stimulation. We found that sTEM5 binds to several glycosaminoglycans. Furthermore, sequence analysis and functional and biochemical studies revealed that sTEM5 contains a cryptic RGD-binding site for integrin alpha(v)beta3. Matrix metalloprotease 9-processed, but not full-length, sTEM5 mediated endothelial cell adhesion by direct interaction with integrin alpha(v)beta3. Adhesion to proteolytically processed sTEM5 (ppsTEM5) or glycosaminoglycan-bound ppsTEM5 promoted survival of growth factor deprived endothelial cells. ppsTEM5-mediated cell survival was inhibited by a function blocking integrin alpha(v)beta3 antibody. Based on our results we conclude that sTEM5 is shed by endothelial cells during angiogenesis and binds to glycosaminoglycans present on extracellular matrix and cell surface proteoglycans. Further proteolytic processing of sTEM5 leads to exposure of its RGD motif mediating endothelial cell survival by linking integrin alpha(v)beta3 to glycosaminoglycans.


Asunto(s)
Supervivencia Celular , Endotelio Vascular/metabolismo , Glicosaminoglicanos/metabolismo , Integrina alfaVbeta3/metabolismo , Neovascularización Fisiológica/fisiología , beta-Lactamasas/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Biomarcadores de Tumor/metabolismo , Adhesión Celular , Células Cultivadas , Colágeno/metabolismo , Combinación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Integrina alfaVbeta3/antagonistas & inhibidores , Integrina alfaVbeta3/inmunología , Laminina/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Oligopéptidos/metabolismo , Plásmidos , Proteoglicanos/metabolismo , Receptores Acoplados a Proteínas G , Transfección , Venas Umbilicales
20.
J Biol Chem ; 278(41): 39822-9, 2003 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-12860988

RESUMEN

Toll-like receptors (TLRs) mediate microbial pattern recognition in vertebrates. A broad variety of agonists has been attributed to TLR2 and three TLRs, TLR4, TLR2, and TLR5, have been demonstrated to bind microbial products. Distinct agonists might interact with different subdomains of the TLR2 extracellular domain. The TLR2 extracellular domain sequence includes 10 canonical leucine-rich repeat (LRR) motifs and 8-10 additional and potentially functionally relevant LRR-like motifs. Thus, the transfection of TLR2 LRR/LRR-like motif deletion constructs in human embryonic kidney 293 cells and primary TLR2-deficient mouse fibroblasts was performed for analysis of the role of the regarding domains in specific pattern recognition. Preparations applied as agonists were highly purified soluble peptidoglycan, lipoteichoic acid, outer surface protein A from Borrelia burgdorferi, synthetic mycoplasmal macrophage-activating lipoprotein-2, tripalmitoyl-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4), dipalmitoyl-CSK4 (P2-CSK4), and monopalmitoyl-CSK4 (PCSK4) as well as lipopolysaccharide and inactivated bacteria. We found that a block of the N-terminal seven LRR/LRR-like motifs was not involved in TLR2-mediated cell activation by P3CSK4 and P2CSK4 ligands mimicking triacylated and diacylated bacterial polypeptides, respectively. In contrast, the integrity of the TLR2 holoprotein was compulsory for effective cellular recognition of other TLR2 agonists applied, including PCSK4. The formation of a functionally relevant subdomain by a region including the N-terminal seven LRR/LRR-like motifs rather than by single LRRs is suggested by our results. They further imply that TLR2 contains multiple binding domains for ligands that may contribute to the characterization of its promiscuous molecular pattern recognition.


Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Péptidos/química , Péptidos/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , ADN/genética , ADN/metabolismo , Humanos , Técnicas In Vitro , Sistema de Señalización de MAP Quinasas , Glicoproteínas de Membrana/genética , Mutagénesis , FN-kappa B/metabolismo , Ácido Palmítico/química , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptores Toll-Like
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