RESUMEN
AIMS: The trace amine-associated receptor (Taar) family displays high species- and subtype-specific pharmacology. Several trace amines such as ß-phenylethylamine (ß-PEA), p-tyramine and tryptamine are agonists at TA(1) but poorly activate rat and mouse Taar4. PRINCIPAL RESULTS: Using rat TA(1) and Taar4 chimera, we identified determinants in transmembrane helices 3 and 6, which, when replaced by the corresponding portion of rat TA(1) , can rescue cell surface expression of rat Taar4. When expressed at the cell surface, rat Taar4 pharmacology was very similar to that of TA(1) and coupled to the Gα(s) -protein/AC pathway. Our data suggest that binding pockets of Taar for surrogate agonists overlap between paralogs. CONCLUSIONS: This implicates that the repertoire of Taar ensures functional redundancy, tissue- and cell-specific expression and/or different downstream signalling rather than different agonist specificity.
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Receptores Acoplados a Proteínas G/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Fenetilaminas/farmacología , Transporte de Proteínas , Ratas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Triptaminas/farmacología , Tiramina/farmacologíaRESUMEN
The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.
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Agresión , Sitios de Carácter Cuantitativo , Selección Genética , Alelos , Animales , Secuencia de Bases , Femenino , Variación Genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Polimorfismo de Nucleótido Simple , Ratas , Análisis de Secuencia de ADNRESUMEN
Mesenchymal stem cells (MSCs) can ameliorate symptoms in several neurodegenerative diseases. However, the toxic environment of a degenerating central nervous system (CNS) characterized by hypoxia, glutamate (Glu) excess and amyloid beta (Abeta) pathology may hamper the survival and regenerative/replacing capacities of engrafted stem cells. Indeed, human MSC (hMSC) exposed to hypoxia were disabled in (i) the capacity of their muscarinic receptors (mAChRs) to respond to acetylcholine (ACh) with a transient increase in intracellular [Ca(2+)], (ii) their capacity to metabolize Glu, reflected by a strong decrease in glutamine synthetase activity, and (iii) their survival on exposure to Glu. Cocultivation of MSC with PC12 cells expressing the amyloid precursor protein gene (APPsw-PC12) increased the release of IL-6 from MSC. HMSC exposed to erythropoietin (EPO) showed a cholinergic neuron-like phenotype reflected by increased cellular levels of choline acetyltransferase, ACh and mAChR. All their functional deficits observed under hypoxia, Glu exposure and APPsw-PC12 cocultivation were reversed by the application of EPO, which increased the expression of Wnt3a. EPO also enhanced the metabolism of Abeta in MSC by increasing their neprilysin content. Our data show that cholinergic neuron-like differentiation of MSC, their functionality and resistance to a neurotoxic environment is regulated and can be improved by EPO, highlighting its potential for optimizing cellular therapies of the CNS.
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Diferenciación Celular , Eritropoyetina/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Adolescente , Adulto , Anciano , Péptidos beta-Amiloides/metabolismo , Animales , Calcio/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Neprilisina/metabolismo , Neuronas/metabolismo , Ratas , Receptores Colinérgicos/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A , Adulto JovenRESUMEN
CASE HISTORY AND CLINICAL FINDINGS: A 4-year-old, slightly overweight (BMI 18 kg/m2, BMI SDS 1.29 approximately 90.1 (th) percentile) and otherwise healthy girl presented with accelerated linear growth (99.6 (th) percentile) and hyperphagia due to loss of satiety feeling. These findings are consistent with melanocortin-4 receptor ( MC4R) mutations. GENETIC ANALYSIS AND DIAGNOSIS: We found the partially inactivating mutation Arg (165)Trp by direct sequencing of the MC4R coding region. Interestingly, the patient's mother is also heterozygous for this mutation, but lean (BMI 19 kg/m2). TREATMENT AND COURSE: Carriers of MC4R mutations develop hyperphagia due to lack of satiety feeling as a result of central dysregulation. The reduction of energy intake and the encouragement of physical activity can be successful to control the body weight. CONCLUSION: Early diagnosis can promote lifestyle intervention to prevent the development of obesity even in the presence of a genetic predisposition.
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Hiperfagia/genética , Obesidad/genética , Mutación Puntual , Receptor de Melanocortina Tipo 4/genética , Respuesta de Saciedad/fisiología , Preescolar , Ingestión de Energía , Femenino , Humanos , Hiperfagia/prevención & control , Estilo de Vida , Obesidad/prevención & control , LinajeRESUMEN
Restitution of single-cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time-lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single-cell loss from an HT-29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 +/- 0.05 microS 2 min post lesion to 0.17 +/- 0.02 microS 8 min post lesion, n = 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO-1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin-ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML-7. The Rho-associated protein kinase inhibitor Y-27632 did not affect restitution. After loosening of intercellular contacts in low Ca(2+) Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12-fold higher in low Ca(2+) Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single-cell defects comprises rapid closure by an actinomyosin 'purse-string' mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.
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Actinas/fisiología , Células Epiteliales/fisiología , Miosinas/fisiología , Uniones Estrechas/fisiología , Actinas/metabolismo , Algoritmos , Calcio/fisiología , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , ADN/biosíntesis , Electrofisiología , Células Epiteliales/ultraestructura , Células HT29 , Humanos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Ocludina , Técnicas de Placa-Clamp , Uniones Estrechas/ultraestructuraRESUMEN
The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.
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Proteínas de la Membrana/análisis , Membrana Nuclear/química , Proteoma/análisis , Animales , Células COS , Fraccionamiento Celular , Chlorocebus aethiops , Detergentes , Electroforesis en Gel Bidimensional/métodos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Octoxinol , Fracciones Subcelulares , Transfección , Células Tumorales CultivadasRESUMEN
Sporadic and familial nonautoimmune hyperthyroidism are very rarely occurring diseases. Within the last years constitutively activating TSH receptor mutations were identified as one possible pathomechanism. Except for S281N in the extracellular N-terminal domain, all other germline mutations are located in the transmembrane domains 2, 3, 5, 6, and 7 of the TSH receptor, whereas no mutation was reported in transmembrane domains 1 and 4 to date. Here we report the first family with a constitutively active TSHR mutation in transmembrane domain 1 resulting in a substitution of the conserved Gly(431) for Ser. This mutation was found in the investigated patient, his father, and the paternal grandmother. As known from other familial cases of nonautoimmune hyperthyroidism, the age of onset of the disease was variable, ranging from early childhood in the patient and his father to adolescence in the grandmother. Functional characterization of this mutation showed a constitutive activation of the G(s)/adenylyl cyclase system. Moreover, this germline mutation also activates the G(q/11)/phospholipase C pathway. The importance of Gly(431) for receptor quiescence is supported further by introduction of other mutations at this position, all leading to constitutive receptor activity. Our data show now that constitutively activating mutations can be found in the entire transmembrane domain region of the TSH receptor, indicating the important role of all parts of the transmembrane domain region for maintaining the inactive receptor conformation.
Asunto(s)
ADN/genética , Hipertiroidismo/genética , Mutación Missense/genética , Receptores de Tirotropina/genética , Adenilil Ciclasas/genética , Animales , Células COS , Preescolar , AMP Cíclico/metabolismo , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Genoma , Humanos , Hipertiroidismo/sangre , Masculino , Linaje , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Hormonas Tiroideas/sangre , Tirotropina/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Activation of caspases results in the disruption of structural and signaling networks in apoptotic cells. Recent biochemical and cell biological studies have shown that components of the cadherin-catenin adhesion complex in epithelial adherens junctions are targeted by caspases during apoptosis. In epithelial cells, desmosomes represent a second type of anchoring junctions mediating strong cell-cell contacts. Using antibodies directed against a set of desmosomal proteins, we show that desmosomes are proteolytically targeted during apoptosis. Desmogleins and desmocollins, representing desmosome-specific members of the cadherin superfamily of cell adhesion molecules, are specifically cleaved after onset of apoptosis. Similar to E-cadherin, the desmoglein-3 cytoplasmic tail is cleaved by caspases. In addition the extracellular domains of desmoglein-3 and desmocollin-3 are released from the cell surface by a metalloproteinase activity. In the presence of caspase and/or metalloproteinase inhibitors, both cleavage reactions are almost completely inhibited. As reported previously, the desmosomal plaque protein plakoglobin is cleaved by caspase-3 during apoptosis. Our studies now show that plakophilin-1 and two other major plaque proteins, desmoplakin-1 and -2, are also cleaved by caspases. Immunofluorescence analysis confirmed that this cleavage results in the disruption of the desmosome structure and thus contributes to cell rounding and disintegration of the intermediate filament system.
Asunto(s)
Apoptosis , Desmosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Línea Celular , Hidrólisis , Microscopía Confocal , Microscopía FluorescenteRESUMEN
beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.
Asunto(s)
Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Integrina beta1/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Factor Natriurético Atrial/farmacología , Canales de Calcio Tipo L/metabolismo , Compartimento Celular , Células Cultivadas , Citocalasina D/farmacología , Adhesiones Focales , Integrina beta1/genética , Isoproterenol/farmacología , Ratones , Antagonistas Muscarínicos/farmacología , Miocardio/citología , Óxido Nítrico/farmacología , Canales de Potasio/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: The glycoprotein hormone TSH (thyroid-stimulating hormone) and its receptor, the TSH-receptor (TSHR), play a crucial role in thyroid growth and function. Constitutively activating germline mutations within the TSHR gene were identified in patients with sporadic or familial non-autoimmune hyperthyroidism. Inheritance of these mutations is autosomal dominant. PATIENTS AND METHODS: We investigated two patients with neonatal onset of non-autoimmune hyperthyroidism and two families in whom the child and one parent are affected. RESULTS: Hyperthyroidism was difficult to treat in all of these patients and was complicated by premature craniosynostosis. Sequencing of all exons of the TSHR gene in one family with hyperthyroidism revealed a mutation in exon 10 (T6321), which was first identified in toxic adenomas and found to constitutively activate the TSHR. In the other family, we identified a new mutation in the first membrane spanning segment (G431S). In both patients with sporadic hyperthyroidism, a heterozygous mutation in exon 9 (S281N) was detected. The functional characterization of S281N and G431S demonstrated that both mutants were constitutively active. Therefore, these mutations are the molecular cause of non-autoimmune hyperthyroidism in the patients. CONCLUSIONS: For patients suffering from non-autoimmune hyperthyroidism, screening for mutations and their functional characterization is recommended. In case of an ineffective hyperthyroidism treatment, thyroidectomy should be performed to prevent lengthy anti-thyroid drug treatment and complications like premature craniosynostosis.
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Mutación de Línea Germinal , Hipertiroidismo/fisiopatología , Receptores de Tirotropina/genética , Niño , Preescolar , Exones/genética , Glicina/genética , Humanos , Hipertiroidismo/genética , Lactante , Serina/genéticaRESUMEN
The V2 vasopressin receptor (V2R) plays a key role in the maintenance of a normal body water balance. To generate an in vivo model that allows the physiological and molecular analysis of the role of V2Rs in kidney function, we have created mouse lines that lack functional V2Rs by using targeted mutagenesis in mouse embryonic stem cells. Specifically, we introduced a nonsense mutation known to cause X-linked nephrogenic diabetes insipidus (XNDI) in humans (Glu242stop) into the mouse genome. V2R-deficient hemizygous male pups showed a decrease in basal urine osmolalities and were unable to concentrate their urine. These pups also exhibited an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Interestingly, female mice heterozygous for the V2R mutation showed normal growth but displayed an XNDI-like phenotype, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis and immunoelectron microscopic studies showed that the loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1). The V2R mutant mice described here should serve as highly useful tools for the development of novel therapeutic strategies for the treatment of XNDI.
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Codón sin Sentido , Receptores de Vasopresinas/genética , Animales , Animales Recién Nacidos , Acuaporina 2 , Acuaporina 6 , Acuaporinas/genética , Acuaporinas/metabolismo , Cruzamiento , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diabetes Insípida/genética , Diabetes Insípida/mortalidad , Diabetes Insípida/patología , Femenino , Expresión Génica , Ingeniería Genética/métodos , Ligamiento Genético , Genotipo , Riñón/química , Riñón/patología , Riñón/ultraestructura , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Microscopía Inmunoelectrónica , Fenotipo , Receptores de Vasopresinas/deficiencia , Simportadores de Cloruro de Sodio-Potasio , Tasa de Supervivencia , Cromosoma XRESUMEN
Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.
Asunto(s)
Receptores de HL/metabolismo , Receptores de Tirotropina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Asparagina/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Receptores de HL/química , Receptores de Tirotropina/química , Homología de Secuencia de AminoácidoRESUMEN
X-Linked nephrogenic diabetes insipidus (NDI) is a rare inherited disorder characterized by the excretion of abnormal large volumes of diluted urine mainly caused by mutations in the V2 vasopressin receptor (AVPR2) gene. By screening NDI patients for mutations within the AVPR2 gene we have identified three novel (I46K, F105V, I130F) and four recurrent (D85N, R106C, R113W, Q225X) mutations. In addition, a recurrent missense mutation (A147T) within the aquaporin-2 gene was identified in a female patient with autosomal recessive NDI associated with sensorineural deafness. Selected clinical data of the NDI patients were compared with the results from the in vitro studies. Functional analysis of I46K and I130F revealed reduced maximum agonist-induced cAMP responses as a result of an improper cell surface targeting. In contrast, the F105V mutation is delivered to the cell surface and displayed an unchanged maximum cAMP response, but impaired ligand binding abilities of F105V were reflected in a shifted concentration-response curve toward higher vasopressin concentrations. As the extracellularly located F105 is highly conserved among the vasopressin/oxytocin receptor family, functional analysis of this residue implicates an important role in high affinity agonist binding.
Asunto(s)
Acuaporinas/genética , Diabetes Insípida Nefrogénica/genética , Mutación , Receptores de Vasopresinas/genética , Cromosoma X , Secuencia de Aminoácidos , Animales , Acuaporina 2 , Acuaporina 6 , Arginina Vasopresina/metabolismo , Células COS , Niño , Preescolar , Análisis Mutacional de ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Femenino , Ligamiento Genético , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Missense , Linaje , Reacción en Cadena de la Polimerasa , Receptores de Vasopresinas/química , Alineación de Secuencia , TransfecciónRESUMEN
The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.
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Proteínas de Unión al GTP/metabolismo , Receptores LHRH/metabolismo , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Células COS , Calmodulina/metabolismo , Cricetinae , AMP Cíclico/biosíntesis , Humanos , Mutagénesis , Unión Proteica , Receptores LHRH/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Previous studies have established that G-protein-coupled receptors (GPCRs) are composed of independent folding domains. Based on this findings we attempted to rescue the function of clinically relevant missense mutations (R137H, S167L, and R181C) within the N-terminal domain of the V2 vasopressin receptor (V2-R), by coexpressing mutated full-length (Y280C) and C-terminally truncated (E242X) receptor constructs in COS-7 cells. Coimmunoprecipitation and enzyme-linked immunosorbent assay studies demonstrated a specific association of E242X with full-length V2-Rs even in the presence of missense mutations. Systematic analysis of the structural requirements for the observed receptor/fragment association showed that N-terminal fragments containing at least transmembrane regions 1-3 interact with the full-length V2-R. Despite this specific interaction, no functional reconstitution was achieved for mutant V2-Rs following coexpression with E242X and Y280C. However, functional activity of R137H and R181C upon coexpression with E242X was regained by mutational disruption of the extracellular disulfide bond, which is highly conserved among GPCRs. Our data with the V2-R are consistent with a structural model in which class I GPCRs form contact oligomers by lateral interaction rather than by a domain-swapping mechanism.
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Receptores de Vasopresinas/química , Secuencia de Aminoácidos , Animales , Biopolímeros , Células COS , Disulfuros/química , Datos de Secuencia Molecular , Mutagénesis , Receptores de Vasopresinas/genéticaRESUMEN
Based on structural similarities of an expressed sequence tag with the platelet-activating factor (PAF) receptor a cDNA clone encoding a novel G-protein-coupled receptor (GPCR), named GPR34, was isolated from a human fetal brain cDNA library. Genomic DNA analyses revealed the receptor to be encoded by an intronless single-copy gene at Xp11. 3-11.4. The predicted 381-amino-acid protein disclosed all structural features characteristic of a member of the class I GPCR family. Except an obvious sequence homology in transmembrane domain 6, no further similarities to the PAF receptor or any other known GPCR were found. The corresponding mouse receptor DNA was isolated from a genomic P1 library displaying a 90% amino acid identity compared to the human receptor. Phylogenetic studies showed that GPR34 is preserved among vertebrates, and the existence of GPR34 subtypes was demonstrated. The receptor mRNA is abundantly expressed in human and mouse tissues. In addition to the major 2-kb transcript, a 4-kb transcript was found only in mouse liver and testis. Expression of the human GPR34 in COS-7 cells followed by Western blot studies revealed specific bands of a highly glycosylated protein between 75 and 90 kDa. A number of potential ligands including phospholipids, leukotrienes, hydroxy-eicosatetraenoic acids, nucleotides and peptides were tested in functional assays. Since none of the applied substances led to significant changes in second messenger levels (cAMP and inositol phosphates), the natural ligand and coupling profile of this novel GPCR subgroup remains unknown.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/clasificación , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , Células COS , Clonación Molecular , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Filogenia , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Lisofosfolípidos , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Alineación de SecuenciaRESUMEN
The function of small GTPases is fine-tuned by a complex network of regulatory proteins such as GTPase-activating proteins. The C1 gene at Xq28 encodes a protein assumed to function as a Rho GTPase-activating protein (rhoGAP). Characterization of the molecular defect causing X-linked nephrogenic diabetes insipidus (NDI) in a patient revealed a submicroscopic deletion of a 21.5-kb genomic fragment encompassing the entire arginine-vasopressin V2 receptor gene (AVPR2) and most of the C1 gene locus. In the absence of detailed information about the physiological relevance and specific functions of rhoGAP C1, a thorough clinical and laboratory investigation of the patient was performed. Besides clearly defined NDI symptoms caused by deletion of the AVPR2 gene, no major morphological abnormalities as determined by physical examination, radiography, ultrasound, and computed tomographic scan were detected. Extensive analysis of blood chemical, enzyme, and hormone values over a period of 16 years showed no deviations from normal ranges. On the basis of our observations, the rhoGAP C1 protein is not essential for normal development in the human. Because of a predominant expression pattern of the C1 gene in hematopoietic cells, we focused on immunologic and hematologic laboratory parameters of the affected boy and the mother who was found to be heterozygous. Differential white cell counts, including lymphocyte typing, determination of lymphokines, cytokines, and immunoglobulins, as well as numerous leukocyte function tests, showed no pathological findings. Therefore, we postulate that the loss of rhoGAP C1 function is most likely compensated by other members of the GAP family.
Asunto(s)
Diabetes Insípida Nefrogénica/genética , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa , Eliminación de Gen , Receptores de Vasopresinas/genética , Adolescente , Envejecimiento/sangre , Recuento de Células Sanguíneas , Análisis Mutacional de ADN , Diabetes Insípida Nefrogénica/sangre , Sistema Hematopoyético/metabolismo , Humanos , Inmunofenotipificación , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Cromosoma X/genéticaRESUMEN
The vast majority of extracellular signaling molecules, like hormones and neurotransmitters, interact with a class of membranous receptors characterized by a uniform molecular architecture of seven transmembrane alpha-helices linked by extra- and intracelluar peptide loops. In a reversible manner, binding of diverse agonists to heptahelical receptors leads to activation of a limited repertoire of heterotrimeric guanine nucleotide-binding proteins (G proteins) forwarding the signal to intracellular effectors such as enzymes and ion channels. Proper functioning of a G protein-coupled receptor is based on a complex interplay of structural determinants which are ultimately responsible for receptor folding, trafficking and transmembrane signaling. Applying novel biochemical and molecular biological methods interesting insights into receptor structure/function relationships became available. These studies have a significant impact on our understanding of the molecular basis of human diseases and may eventually lead to novel therapeutic strategies.