RESUMEN
Pharmacoperones correct the folding of otherwise misfolded protein mutants, restoring function (i.e., providing "rescue") by correcting their trafficking. Currently, most pharmacoperones possess intrinsic antagonist activity because they were identified using methods initially aimed at discovering such functions. Here, we describe an ultra-high-throughput homogeneous cell-based assay with a cAMP detection system, a method specifically designed to identify pharmacoperones of the vasopressin type 2 receptor (V2R), a GPCR that, when mutated, is associated with nephrogenic diabetes insipidus. Previously developed methods to identify compounds capable of altering cellular trafficking of V2R were modified and used to screen a 645,000 compound collection by measuring the ability of library compounds to rescue a mutant hV2R [L83Q], using a cell-based luminescent detection system. The campaign initially identified 3734 positive modulators of cAMP. The confirmation and counterscreen identified only 147 of the active compounds with an EC50 of ≤5 µM. Of these, 83 were reconfirmed as active through independently obtained pure samples and were also inactive in a relevant counterscreen. Active and tractable compounds within this set can be categorized into three predominant structural clusters, described here, in the first report detailing the results of a large-scale pharmacoperone high-throughput screening campaign.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas/aislamiento & purificación , Diabetes Insípida Nefrogénica/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores de Vasopresinas/genética , Antagonistas de los Receptores de Hormonas Antidiuréticas/uso terapéutico , Diabetes Insípida Nefrogénica/genética , Predisposición Genética a la Enfermedad , Humanos , Mutación , Pliegue de Proteína , Transporte de Proteínas , Receptores de Vasopresinas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Vasopresinas/genética , Vasopresinas/metabolismoRESUMEN
The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R224â274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224â274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹5N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Receptores de Vasopresinas/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Escherichia coli/metabolismo , Humanos , Cuerpos de Inclusión , Espacio Intracelular , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Vasopresinas/aislamiento & purificación , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The distribution of three types of arginine vasotocin (AVT) receptors in the brain and pituitary of the newt Cynops pyrrhogaster, namely, the V1a-, V2-, and V3/V1b-type receptors, was studied by means of in situ hybridization and immunohistochemistry. mRNA signals and immunoreactive cells for the V1a-type receptor were observed in the telencephalon (mitral layer of the olfactory bulb, dorsal and medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali, bed nucleus of the stria terminalis), diencephalon (anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, ventral thalamus, dorsal and ventral hypothalamic nucleus), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (median reticular formation, nucleus motorius tegmenti). Cells expressing the V2-type receptor were found in the telencephalon (medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali), and mesencephalon (tegmentum trigemini and facialis). In the paraphysis (possibly the main site of cerebrospinal fluid production), only V2-type receptor mRNA signal and immunoreactivity were detected. V3/V1b-type receptor mRNA was expressed in the diencephalon (dorsal hypothalamic nucleus, nucleus tuberculi posterioris), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (raphe nucleus), whereas V3/V1b-type-receptor-like immunoreactivity was scarcely detectable in the entire brain. The V3/V1b-type receptor was predominantly expressed in the anterior pituitary. V3/V1b-type receptor and proopiomelanocortin mRNAs were co-localized in the distal lobe of the pituitary. This is the first report of the distribution of three types of AVT receptor in the brain and pituitary of non-mammalian vertebrates.
Asunto(s)
Química Encefálica , Adenohipófisis/química , Adenohipófisis/citología , Receptores de Vasopresinas/análisis , Salamandridae/metabolismo , Animales , Diencéfalo/química , Diencéfalo/citología , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Bulbo Raquídeo/química , Bulbo Raquídeo/citología , Mesencéfalo/química , Mesencéfalo/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero , Receptores de Vasopresinas/aislamiento & purificación , Transducción de Señal , Telencéfalo/química , Telencéfalo/citologíaRESUMEN
The expression of mRNAs encoding vasopressin V1a, V2, and ANP-B receptors in the rat cochlea was examined by PCR and in situ hybridization. After reverse-transcription of rat cochlear RNA, cDNA was amplified by PCR using pairs of primers specific to these receptors. After subcloning of the PCR products, clones with sequences identical to those cloned previously from the rat liver (V1a receptor), kidney (V2 receptor) and brain (ANP-B receptor) were obtained. The localization of expression of those receptors in the developing and adult rat cochlea was examined by in situ hybridization using 35S-labeled cRNA probes. The V1a and V2 receptors were expressed throughout the whole of the neonatal rat cochlea, while no expression was detected in the adult cochlea. The ANP-B receptor was expressed throughout the whole of the neonatal cochlea. In the adult cochlea, expression was observed in the spiral ganglion and the spiral ligament. These results suggest that vasopressin may play a role in the development of the cochlea, and that natriuretic peptide may play a role in the function of the spiral ganglion and the spiral ligament.
Asunto(s)
Envejecimiento , Arginina Vasopresina/genética , Cóclea/química , Regulación de la Expresión Génica , Guanilato Ciclasa , ARN Mensajero/análisis , Receptores del Factor Natriurético Atrial/genética , Receptores de Vasopresinas/genética , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Arginina Vasopresina/aislamiento & purificación , Secuencia de Bases , Cóclea/embriología , ADN Complementario/análisis , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/aislamiento & purificación , Ganglio Espiral de la Cóclea/metabolismo , Ganglio Espiral de la Cóclea/fisiologíaRESUMEN
The human V2 vasopressin receptor belongs to the superfamily of G protein-coupled receptors believed to be anchored to the plasma membrane by seven transmembrane regions. The extracellular portion of the human V2 vasopressin receptor contains one site susceptible to N-linked glycosylation. Metabolic labeling and immunoprecipitation of the receptor expressed in transfected cells were applied to examine whether the protein was indeed glycosylated. The V2 vasopressin receptor expressed transiently was glycosylated, but glycosidase treatment to test the complexity of the sugar moiety linked to asparagine revealed that the majority of the receptor protein lacked complex carbohydrates, an indication of an improperly processed protein. This immature protein displayed a tendency to form aggregates. In contrast with these data, testing of the sugar complexity of the receptor protein synthesized in stably transfected cells identified the predominant form as an appropriately processed receptor protein. Mutagenesis of asparagine 22 to glutamine produced on expression in transfected cells a nonglycosylated receptor with ligand binding affinity and coupling characteristics almost identical to those of the wild-type form. After exposure to elevated concentrations of AVP (100 nM), the nonglycosylated form desensitized to the same extent as the wild-type receptor.
Asunto(s)
Arginina Vasopresina/farmacología , Receptores de Vasopresinas/biosíntesis , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Arginina Vasopresina/metabolismo , Línea Celular , Chlorocebus aethiops , Epítopos , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Glicósido Hidrolasas , Glicosilación , Humanos , Riñón , Cinética , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Receptores de Vasopresinas/química , Receptores de Vasopresinas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , TransfecciónRESUMEN
To purify the renal V2 receptor and identify domains involved in hormone binding, photoaffinity labeling of the membrane-bound bovine V2 receptor with a tritium-labeled photoreactive vasopressin agonist was performed. The labeled 30,000 M(r) protein was purified to homogeneity by anion-exchange chromatography, isoelectric focusing, gel filtration, gel electrophoresis, and reversed-phase HPLC. N-terminal sequencing showed that the isolated protein which contains the covalently bound hormonal ligand, represents an N-terminal truncated bovine V2 receptor. The purified labeled V2 vasopressin receptor protein was digested with V8 protease, and peptide fragments were isolated. Protein microsequencing and comparison with the cDNA sequence of a cloned PCR product identified two extra- and two intracellular peptides of the bovine V2 receptor. Radioactivity was incorporated into two amino acid residues localized in the second extracellular domain. Our results indicate that this extracellular domain is involved in peptide agonist binding of the V2 receptor.
Asunto(s)
Médula Renal/química , Receptores de Vasopresinas/aislamiento & purificación , Marcadores de Afinidad , Animales , Secuencia de Bases , Bovinos , Membrana Celular/química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Lipresina/análogos & derivados , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fotoquímica , Reacción en Cadena de la Polimerasa , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Análisis de Secuencia , Serina Endopeptidasas/metabolismo , SolubilidadRESUMEN
We used several approaches to identify the G protein coupled to V1 vascular arginine vasopressin (AVP) receptors of human platelets. In purified platelet membranes, high-affinity specific binding of [3H]AVP but not that of the V1 vascular antagonist [3H]d(CH2)5Tyr(Me)AVP was modulated by guanosine 5'-O-(3-thiotriphosphate) or sodium fluoride both in the presence and absence of MgCl2. AVP failed to modify the [alpha-32P]GTP labeling pattern or the cytosolic translocation of the 24- to 27-kDa GTP-binding proteins. AVP-stimulated GTPase activity of platelet membranes was blocked by antibodies specific for the COOH-terminal of the Gq alpha protein. AVP increased labeling of a 42-kDa platelet membrane protein by the photoreactive GTP analogue [alpha-32P]azidoanilido GTP. Immunoblotting of platelet proteins with various G protein-specific antibodies revealed that the 42-kDa protein labeled with [alpha-32P]azidoanilido GTP was immunoblotted only by antibodies specific for the alpha-subunit of GQ-11. Thus V1 vascular AVP receptors of human platelets are coupled in a divalent cation-dependent manner to a G protein belonging to the Gq-11 family.