RESUMEN
A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células CHO , Cricetinae , Femenino , Genes Supresores de Tumor , Humanos , Cinética , Kisspeptinas , Ligandos , Melanoma/genética , Datos de Secuencia Molecular , Nephropidae , Neuronas/metabolismo , Especificidad de Órganos , Fragmentos de Péptidos/farmacología , Hipófisis/metabolismo , Placenta/metabolismo , Embarazo , Proteínas/química , Ratas , Receptores de Superficie Celular/química , Receptores Acoplados a Proteínas G , Receptores de Kisspeptina-1 , Receptores de Neuropéptido/química , Receptores de Neuropéptido/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Anémonas de Mar , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Proteínas Supresoras de TumorRESUMEN
Truncated peptide analogues of orexin-A were prepared and their biological activity assesed at the orexin-1 receptor. Progressive N-terminal deletions identified the minimum C-terminal sequence required for maintaining a significant agonist effect, whilst an alanine scan and other pertinent substitutions identified key side-chain and stereochemical requirements for receptor activation.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/farmacología , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/química , Neuropéptidos/farmacología , Receptores de Neuropéptido/agonistas , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Portadoras/síntesis química , Cricetinae , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neuropéptidos/síntesis química , Receptores de Orexina , Orexinas , Unión Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R: BB1), the GRP-preferring/bombesin receptor (GRP-R: BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered. which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3.
Asunto(s)
Bombesina/farmacología , Péptido Liberador de Gastrina/farmacología , Receptores de Bombesina/agonistas , Receptores de Bradiquinina/agonistas , Alanina/química , Alanina/metabolismo , Sustitución de Aminoácidos , Animales , Bombesina/química , Bombesina/metabolismo , Calcio/metabolismo , Línea Celular , Péptido Liberador de Gastrina/química , Humanos , Riñón/metabolismo , Leucemia/patología , Conformación Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacología , Ratas , Receptor de Bradiquinina B1 , Receptor de Bradiquinina B2 , Receptores de Bombesina/metabolismo , Receptores de Bradiquinina/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell-permeable peptide consisting of a 16-amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion-fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 microg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose-dependent fashion. Doses of 1 microg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 microg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/farmacocinética , Sistemas de Liberación de Medicamentos , Proteínas de Homeodominio/farmacocinética , Neuronas/metabolismo , Factores de Edad , Secuencia de Aminoácidos , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/citología , Proteínas Portadoras/toxicidad , Péptidos de Penetración Celular , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Relación Dosis-Respuesta a Droga , Drosophila , Encefalitis/inducido químicamente , Proteínas de Homeodominio/toxicidad , Humanos , Inyecciones Intravenosas , Inyecciones Intraventriculares , Riñón/citología , Microinyecciones , Datos de Secuencia Molecular , Neurobiología/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Death receptors are associated with the homeostatic and pathologic induction of cell death. TR3 is a recently characterised member of the death receptor family that is expressed in the adult brain. In order to establish the role of TR3 in acute CNS disease and chronic neurodegeneration, we analysed brain regions from Alzheimer's disease (AD), stroke and neurotrauma patients, using a novel anti-peptide antibody generated to an exposed epitope in the extracellular domain of the receptor. We show a statistically significant increase in TR3 protein levels in AD brain samples but not in stroke, neurotrauma or control samples. The increase observed for TR3 was specific to neurons in regions associated with AD pathology. This is the first report describing the neuron-specific regulation of a death receptor in chronic disease and may indicate that a TR3 receptor-mediated signalling pathway is involved in AD-associated neuronal loss.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Apoptosis/fisiología , Encéfalo/patología , Encéfalo/fisiopatología , Proteínas de Unión al ADN/análisis , Neuronas/patología , Neuronas/fisiología , Receptores de Esteroides , Receptores de Hormona Tiroidea , Factores de Transcripción/análisis , Anciano , Anciano de 80 o más Años , Traumatismos Craneocerebrales/fisiopatología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Two electrophoretic techniques have been developed for the analysis of synthetic beta-A4 related peptides. The first is an acetic acid--urea--polyacrylamide gel electrophoresis denaturing system while the second employs capillary zone electrophoresis at pH 7. Both methods were calibrated to identify the state (monomeric or aggregate) of the longer amyloid fragments beta-A4(1-40) and beta-A4(1-43) during analysis.