RESUMEN
The neurokinin receptor family is known to modulate phospholipase C activity. In order to find new compounds modulating the activity of these receptors we have developed a cellular screening system that measures the biological activity of receptors coupled to the IP3/DAG signal transduction pathway via the transcriptional activation of a reporter gene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the control of a promoter containing TPA response elements (TRE), which did not respond to neurokinin agonists, was used. Stable test cell lines were developed by transfecting the reporter cell line A20 with the genes for the human neurokinin receptors NK1, NK2 or NK3, respectively. In these cell lines, expression of luciferase was inducible by substance P, neurokinin A and neurokinin B, respectively. The order of potency of the three neurokinins substance P, neurokinin A and neurokinin B was consistent with published data and results from ligand binding studies performed with the NK1 and NK2 test cell lines. The agonistic effect of the neurokinins could be inhibited in a dose-dependent manner by simultaneous addition of neurokininspecific antagonists like the non-peptide antagonists CP-99,994 and SR 48968.
Asunto(s)
Bioensayo/métodos , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/metabolismo , Receptores de Neuroquinina-3/metabolismo , Línea Celular , Diglicéridos/metabolismo , Genes Reporteros , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Luciferasas/genética , Ensayo de Unión Radioligante , Receptores de Neuroquinina-1/análisis , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-2/análisis , Receptores de Neuroquinina-2/genética , Receptores de Neuroquinina-3/análisis , Receptores de Neuroquinina-3/genética , Transducción de Señal , Taquicininas/metabolismo , Taquicininas/farmacología , TransfecciónRESUMEN
A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the alpha 2-macroglobulin receptor/LDLR-related protein (alpha 2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptor-associated protein (RAP), providing evidence that alpha 2MR/LRP also acts as a minor group HRV receptor.
Asunto(s)
Receptores de LDL/metabolismo , Receptores Virales/metabolismo , Rhinovirus/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/microbiología , Células HeLa , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Hiperlipoproteinemia Tipo II/microbiología , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Receptores de LDL/clasificación , Receptores de LDL/genética , Receptores Virales/genéticaRESUMEN
Soluble rhinovirus minor group binding activity was found to be shed into the medium upon incubation of HeLa cells at 37 degrees C. Although substantial amounts of this protein were released, no decrease of virus binding to the cell surface was seen. When the membrane-associated receptor was stripped from the cells with trypsin, virus binding was rapidly restored from an intracellular pool even in the absence of de novo protein synthesis. The release of this 85K virus-binding activity was inhibited by metal chelators such as EDTA, EGTA or 1,10-phenanthroline. The potential involvement of a Ca(2+)-dependent protease and/or a phospholipase in this process is discussed.