RESUMEN
Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Regiones Determinantes de Complementariedad/inmunología , Células Germinativas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos/inmunología , Células Clonales , Regiones Determinantes de Complementariedad/química , Simulación por Computador , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Biblioteca de Péptidos , Estabilidad Proteica , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Análisis de Secuencia de Proteína , Proteínas tau/química , Proteínas tau/inmunologíaRESUMEN
Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Anticuerpos/inmunología , Epítopos/inmunología , Proteínas tau/inmunología , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/genética , Encéfalo/metabolismo , Pollos , Epítopos/química , Epítopos/genética , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
Asunto(s)
Complemento C3a/metabolismo , ADN/metabolismo , Interferón gamma/metabolismo , Oligonucleótidos/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Complemento C3a/inmunología , ADN/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Oligonucleótidos/inmunología , Unión Proteica , Receptor para Productos Finales de Glicación Avanzada/inmunología , Resonancia por Plasmón de SuperficieRESUMEN
Oxidatively modified low-density lipoprotein (OxLDL) is a contributing factor of endothelial dysfunction, an early cellular event during atherogenesis. In endothelial cells, OxLDL has been shown to stimulate proinflammatory responses, increase lipid accumulation, and induce the expression of adhesion and extracellular matrix degrading molecules. The primary receptor for OxLDL on endothelial cells has been identified as a member of the scavenger receptor family called lectin-like OxLDL receptor-1 (LOX-1). A number of studies on LOX-1 have implicated its role in multiple cardiovascular diseases including atherosclerosis. To better understand the molecular mechanisms underlying the role of LOX-1 in endothelial cells, we identified interacting proteins in an affinity-purified LOX-1 receptor complex from human aortic endothelial HAECT cells by mass spectrometry. Two molecules involved in Rho signaling pathway, ARHGEF1 and ROCK2, were identified, and their associations with LOX-1 were confirmed in reciprocal immunoprecipitation studies. Particularly, ROCK2 was found to dynamically associate with LOX-1 in the presence of OxLDL. In addition, OxLDL treatment stimulated ROCK2 catalytic activity, and ROCK2 inhibition attenuated NF-kappaB activation and IL-8 production resulting from OxLDL activation of LOX-1. In summary, a functional proteomics approach has enabled us to identify novel LOX-1 interactors that potentially contribute to the cellular and signaling functions of LOX-1.
Asunto(s)
Células Endoteliales/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Receptores Depuradores de Clase E/metabolismo , Quinasas Asociadas a rho/metabolismo , Línea Celular , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Humanos , Lipoproteínas LDL/farmacología , FN-kappa B/genética , FN-kappa B/metabolismo , Quinasas Asociadas a rho/genéticaRESUMEN
Oxidized low-density lipoprotein (OxLDL) has been implicated as a proatherogenic factor with a pathological role in the induction of endothelial dysfunction. Endothelial cells bind and uptake OxLDL primarily through the scavenger receptor lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1), which is believed to mediate critical effects of OxLDL in endothelial cells. To examine the biological events following LOX-1 activation by OxLDL, we used cDNA microarray analysis to globally analyze gene expression changes induced by OxLDL treatment of human aortic endothelial cell line (HAECT) cells overexpressing LOX-1. Consistent with reported functions of OxLDL, in control HAECT cells, OxLDL elicited gene changes in the oxidative stress pathway and other signaling pathways related to OxLDL. With OxLDL treatment, LOX-1-dependent gene expression changes associated with inflammation, cell adhesion, and signal transduction were observed. The transcripts of a number of cytokines and chemokines were induced, which included interleukin-8, CXCL2, CXCL3, and colony-stimulating factor-3. The secretion of these cytokines was confirmed by enzyme-linked immunosorbent assay analysis. In addition, our data revealed a novel link between LOX-1 and a number of genes, including Delta/notch-like epidermal growth factor repeat containing, stanniocalcin-1, cAMP response element modulator, and dual specificity phosphatase 1. Promoter analysis on the genes that changed as a result of LOX-1 activation by OxLDL allowed us to identify early growth response 1 and cAMP response element-binding protein as potential novel transcription factors that function downstream of LOX-1. Our study has enabled us to elucidate the gene expression changes following OxLDL activation of LOX-1 in endothelial cells and discover novel downstream targets for LOX-1.
Asunto(s)
Células Endoteliales/metabolismo , Lipoproteínas LDL/metabolismo , Receptores Depuradores de Clase E/metabolismo , Transcripción Genética , Aorta/citología , Aorta/metabolismo , Adhesión Celular/genética , Línea Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Depuradores de Clase E/genética , Transducción de Señal/genética , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-1, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction.
Asunto(s)
Proteína C-Reactiva/metabolismo , Células Endoteliales/metabolismo , Receptores Depuradores de Clase E/metabolismo , Animales , Arginina , Sitios de Unión , Proteína C-Reactiva/genética , Células CHO , Línea Celular Transformada , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Células Endoteliales/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Interleucina-8/genética , Ligandos , Lipoproteínas LDL/metabolismo , Mutación , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Receptores Depuradores de Clase E/genética , Transfección , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
OBJECTIVE: The present study was conducted to characterize the expression of the cysteine protease legumain in murine and human atherosclerotic tissues, and to explore the molecular mechanisms by which legumain may contribute to the pathophysiology of atherosclerosis. METHODS AND RESULTS: Using microarray analysis, legumain mRNA expression was found to increase with development of atherosclerosis in the aorta of aging Apolipoprotein E deficient mice while expression remained at low level and unchanged in arteries of age-matched C57BL/6 control mice. In situ hybridization and immunohistochemical analysis determined that legumain was predominantly expressed by macrophages in the atherosclerotic aorta, in lesions at the aortic sinus and in injured carotid arteries of Apolipoprotein E deficient mice as well as in inflamed areas in advanced human coronary atherosclerotic plaques. In vitro, M-CSF differentiated human primary macrophages were shown to express legumain and the protein could also be detected in the culture media. When tested in migration assays, legumain induced chemotaxis of primary human monocytes and human umbilical vein endothelial cells. CONCLUSIONS: Legumain is expressed in both murine and human atherosclerotic lesions. The macrophage-specific expression of legumain in vivo and ability of legumain to induce chemotaxis of monocytes and endothelial cells in vitro suggest that legumain may play a functional role in atherogenesis.
Asunto(s)
Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/etiología , Aterosclerosis/enzimología , Aterosclerosis/etiología , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Factores de Edad , Animales , Enfermedades de la Aorta/fisiopatología , Apolipoproteínas E/fisiología , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Femenino , Humanos , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/fisiología , ARN Mensajero/metabolismoRESUMEN
GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.
Asunto(s)
Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Secuencia de Bases , Encéfalo/citología , Línea Celular , AMP Cíclico/biosíntesis , Chaperón BiP del Retículo Endoplásmico , Subunidades alfa de la Proteína de Unión al GTP/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Phosphoinositide lipids generated at the cell membrane are a key component of a variety of signaling pathways. Among several inositol phosphatases that regulate the availability of signaling phosphoinositide lipids, the type II SH2-domain-containing inositol 5-phosphatase (SHIP2; approved gene symbol Inppl1) is believed to have multiple functions, including the regulation of insulin signaling and cytoskeletal functions. To understand the function of SHIP2 in C2C12 muscle cells, we depleted SHIP2 through the use of RNA interference and analyzed the global effect of SHIP2 depletion on gene expression using Affymetrix microarrays containing approximately 45,000 mouse probe sets. Expression of SHIP2-targeting small-hairpin RNA in differentiated C2C12 muscle cells led to >80% decrease in SHIP2 mRNA and 60-80% decrease in SHIP2 protein, which resulted in significant gene expression changes linked to cytoskeletal functions, including altered expression of adducin-alpha, pallidin, stathmin-like-2, and synaptojanin-2 binding protein. Insulin treatment of C2C12 muscle cells caused transcriptional changes associated with known signaling pathways. However, SHIP2 depletion had no discernible effect on insulin-regulated gene expression. Taken together, our results suggest that SHIP2 is involved in the regulation of cytoskeletal functions, but a large reduction of SHIP2 in C2C12 muscle cells is not sufficient to affect insulin-mediated gene expression.
Asunto(s)
Insulina/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Secuencia de Bases , Línea Celular , Citoesqueleto/genética , Endocitosis/genética , Perfilación de la Expresión Génica , Inositol Polifosfato 5-Fosfatasas , Insulina/metabolismo , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Increased hepatic gluconeogenesis is an important contributor to the fasting hyperglycemia found in Type 2 diabetic patients. Low energy states activate the intracellular energy sensor AMP-activated kinase (AMPK). AMPK activation by the AMP mimetic AICAR (5-aminoimidazole-4-carboxamide riboside) has been shown to inhibit hepatic gluconeogenesis. We used transcriptional profiling to search for AICAR-regulated genes in hepatocyte cell lines. We report that a dual specificity phosphatase, Dusp4, is induced by AMPK in AML12, H4IIE, and Fao cells at both mRNA and protein levels. AMPK also induces the immediate early transcription factor Egr1 (early growth response 1), a known transcriptional activator of Dusp4, and it directly binds the Dusp4 promoter at its known binding site. Both reporter gene assays and real time PCR demonstrate that exogenous DUSP4 inhibits the promoter activity and expression of both glucose-6-phosphatase (Glc-6-P) and phosphoenolpyruvate carboxykinase (Pepck) to an extent similar to both AICAR and constitutively active AMPK. Conversely, depletion of EGR1 or DUSP4 using siRNA not only partially abrogates the inhibition of Pepck expression by AICAR, but also importantly affects glucose production by Fao cells. In Fao cells, small interfering RNA targeted EGR1 also depletes DUSP4 expression following treatment with AICAR, further supporting a direct link between EGR1 and DUSP4 activation. Expression of a constitutively active form of p38, a known effector of cAMP-mediated gluconeogenesis, rescues the DUSP4-mediated repression of PEPCK. These results suggest that the inhibition of hepatic gluconeogenesis by AMPK may, in part, be mediated by an immediate early gene response involving EGR1 and its target, DUSP4.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Complejos Multienzimáticos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Activación Transcripcional , Proteínas Quinasas Activadas por AMP , Animales , Línea Celular Tumoral , Fosfatasas de Especificidad Dual , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Gluconeogénesis , Glucosa-6-Fosfatasa/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Regiones Promotoras Genéticas , Ratas , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Galphas protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Expresión Génica/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Northern Blotting/métodos , Encéfalo/metabolismo , Línea Celular , Mapeo Cromosómico/métodos , Clonación Molecular/métodos , AMP Cíclico/metabolismo , Biblioteca de Genes , Genes Reporteros/fisiología , Pruebas Genéticas/métodos , Humanos , Hibridación in Situ/métodos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Transfección/métodos , Técnicas del Sistema de Dos HíbridosRESUMEN
Adiponutrin and a related protein, adipocyte triglyceride lipase (ATGL; also known as Desnutrin), were recently described as adipocyte-specific proteins with lipid hydrolase activity. Using bioinformatics, we identified three additional Adiponutrin family members (GS2, GS2-Like, and PNPLA1). Here, we report on the expression, regulation, and activity of GS2 and GS2-Like compared with Adiponutrin and Desnutrin/ATGL. GS2-Like is expressed and regulated in a manner similar to Adiponutrin; however, the absolute levels of mRNA are significantly lower than those of Adiponutrin or Desnutrin/ATGL. GS2 transcripts were identified only in humans and are highly expressed in adipose as well as other tissues. All four proteins show lipase activity in vitro, which is dependent on the presence of the active site serine for Adiponutrin, Desnutrin/ATGL, and GS2. Overexpression of Desnutrin/ATGL, GS2, and GS2-Like, but not Adiponutrin, decreases intracellular triglyceride levels. This is consistent with a function for Desnutrin/ATGL, GS2, and GS2-Like in lipolysis, but not for Adiponutrin. Consistent with previously reported data, Desnutrin/ATGL is upregulated by fasting in adipose tissue, whereas Adiponutrin is downregulated. Additionally, Adiponutrin and GS2-Like, but not Desnutrin/ATGL, are strongly induced in the liver of ob/ob mice. Our data support distinct functions for Adiponutrin and Desnutrin/ATGL and raise the possibility that GS2 may contribute significantly to lipolysis in human adipose tissue.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Triglicéridos/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Northern Blotting , Biología Computacional/métodos , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Inmunoprecipitación , Lipasa/metabolismo , Lipólisis , Masculino , Cadenas de Markov , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Datos de Secuencia Molecular , Mutación , Ácido Oléico/química , Filogenia , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esterol Esterasa/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Members of the MRG family of G-protein coupled receptors (GPCRs) are expressed predominately in small diameter sensory neurons of the dorsal root ganglia (DRG) suggesting a possible role in nociception. However, the large expansion of this gene family in rodents, combined with the lack of strict rodent orthologs for many of the human MRG genes, limits the usefulness of rodent models to evaluate human MRG involvement in nociception. Furthermore, the high degree of similarity between related rodent Mrg genes suggests that pharmacological approaches to define the function of individual receptors will prove difficult. The creation of an animal model to examine human MRG function will, therefore, require the identification of human MRG orthologs in a non-rodent species. Here we report the identification of MRGD, MRGE, and several MRGX orthologs in the crab-eating macaque, Macaca fascicularis. Similar to their human counterparts, all isolated macaque genes were expressed in dorsal root ganglia neurons. In the case of macaque MrgX2 and MrgD, expression was co-localized with the known nociceptive neuronal markers, IB4, VR1, and SP. Although expression in DRG neurons was the prominent feature of this family, we also found that MrgE was expressed in numerous brain regions of macaque, mouse, and human.
Asunto(s)
Expresión Génica/fisiología , Neuronas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Northern Blotting/métodos , Southern Blotting/métodos , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Clonación Molecular/métodos , ADN Complementario/metabolismo , Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Glicoproteínas/metabolismo , Humanos , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Macaca fascicularis , Ratones , Datos de Secuencia Molecular , Familia de Multigenes/genética , ARN Mensajero/biosíntesis , Receptores de Droga/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia , Especificidad de la Especie , Sustancia P/metabolismo , Factores de Transcripción/clasificaciónRESUMEN
We report here the isolation of a novel gene termed mGluR5R (mGluR5-related). The N-terminus of mGluR5R is highly similar to the extracellular domain of metabotropic glutamate receptor 5 (mGluR5) whereas the C-terminus bears similarity to the testis-specific gene, RNF18. mGluR5R is expressed in the human CNS in a coordinate fashion with mGluR5. Although the sequence suggests that mGluR5R may be a secreted glutamate binding protein, we found that when expressed in HEK293 cells it was membrane associated and not secreted. Furthermore, mGluR5R was incapable of binding the metabotropic glutamate receptor class I selective agonist, quisqualate. Although mGluR5R could not form disulfide-mediated covalent homodimers, it was able to form a homomeric complex, presumably through noncovalent interactions. mGluR5R also formed noncovalent heteromeric associations with an engineered construct of the extracellular domain of mGluR5 as well as with full-length mGluR5 and mGluR1alpha. The ability of mGluR5R to associate with mGluR1alpha and mGluR5 suggests that it may be a modulator of class I metabotropic glutamate receptor function.