RESUMEN
Activation of adhesion receptor GPR110 by the endogenous ligand synaptamide promotes neurogenesis, neurite growth, and synaptogenesis in developing brains through cAMP signal transduction. However, interacting partners of GPR110 and their involvement in cellular function remain unclear. Here, we demonstrate using chemical crosslinking, affinity purification, and quantitative mass spectrometry that GPR110 interacts with the tight junction adhesion protein occludin. By removing non-specific partners by comparing the binding proteins of GPR110 WT and an inactive mutant exhibiting impaired surface expression, occludin was distinguished as a true binding partner which was further confirmed by reciprocal co-immunoprecipitation assay. Deletion of GPR110 in mice led to the disruption of blood-brain barrier (BBB) and reduced occludin phosphorylation at Y285 in the brain. The Y285 phosphorylation increased upon the ligand-induced activation of GPR110. These data suggest an important role of GPR110-occludin interaction in BBB function and association of previously unknown GPR110-dependent occludin phosphorylation at Y285 with BBB integrity.
RESUMEN
It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.
Asunto(s)
Traumatismos del Nervio Óptico , Animales , Ratones , Axones , Ligandos , Ratones Noqueados , Compresión Nerviosa , Regeneración Nerviosa/fisiología , Receptores Acoplados a Proteínas G/genética , Retina , Células Ganglionares de la Retina/fisiologíaRESUMEN
Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.
Asunto(s)
Axones/metabolismo , Etanolaminas/farmacología , Regeneración Nerviosa , Receptores Acoplados a Proteínas G/metabolismo , Animales , Femenino , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microfluídica , Simulación del Acoplamiento Molecular , Compresión Nerviosa , Neurogénesis , Neuronas/metabolismo , Nervio Óptico/metabolismo , Retina/metabolismoRESUMEN
Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.
Asunto(s)
Etanolaminas/farmacología , Proteínas Oncogénicas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Sitios de Unión , Etanolaminas/metabolismo , Células HEK293 , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.
Asunto(s)
Cognición/fisiología , Ácidos Docosahexaenoicos/metabolismo , Endocannabinoides/fisiología , Neurogénesis/fisiología , Proteínas Oncogénicas/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Encéfalo/citología , Línea Celular , AMP Cíclico/metabolismo , Endocannabinoides/metabolismo , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Neuronas/fisiología , Proteínas Oncogénicas/metabolismo , Cultivo Primario de Células , Proteómica/métodos , Ratas , Receptores Acoplados a Proteínas G/metabolismo , Reconocimiento en Psicología/fisiología , Transducción de Señal/fisiología , Memoria Espacial/fisiologíaRESUMEN
It is well documented that mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. However, the functions of wild-type myocilin are still not well understood. To study the functions of human myocilin and its two proteolytic fragments, these proteins were expressed in HEK293 cells. Conditioned medium from myocilin-expressing cells, as well as purified myocilin, induced the formation of stress fibers in primary cultures of human trabecular meshwork or NIH 3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin, as well as secreted inhibitors of Wnt signaling, secreted Frizzled-related protein 1 (sFRP1) or sFRP3, and beta-catenin small interfering RNA. Interaction of myocilin with sFRP1, sFRP3, and several Frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different Frizzled and sFRPs. Treatment of NIH 3T3 cells with myocilin and its fragments induced intracellular redistribution of beta-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of beta-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of beta-catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, since myocilin modulates Wnt signaling by interacting with components of this signaling pathway.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Proteínas del Ojo/fisiología , Glicoproteínas/fisiología , Proteínas Wnt/metabolismo , Animales , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Glicoproteínas/metabolismo , Humanos , Ratones , Ratones Transgénicos , beta Catenina/metabolismoRESUMEN
Protein transduction (PT) is a method for delivering proteins into mammalian cells. PT is accomplished by linking a small peptide tag--called a PT domain (PTD)--to a protein of interest, which generates a functional fusion protein that can penetrate efficiently into mammalian cells. In order to study the functions of a transcription factor (TF) of interest, expression plasmids that encode the TF often are transfected into mammalian cells. However, the efficiency of DNA transfection is highly variable among different cell types and is usually very low in primary cells, stem cells and tumor cells. Zinc-finger transcription factors (ZF-TFs) can be tailor-made to target almost any gene in the human genome. However, the extremely low efficiency of DNA transfection into cancer cells, both in vivo and in vitro, limits the utility of ZF-TFs. Here, we report on an artificial ZF-TF that has been fused to a well-characterized PTD from the human immunodeficiency virus-1 (HIV-1) transcriptional activator protein, Tat. This ZF-TF targeted the endogenous promoter of the human VEGF-A gene. The PTD-attached ZF-TF was delivered efficiently into human cells in vitro. In addition, the VEGF-A-specific transcriptional repressor retarded the growth rate of tumor cells in a mouse xenograft experiment.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción/genética , Dedos de Zinc , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Desnudos , Neoplasias/patología , Neoplasias/terapia , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.