RESUMEN
Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids. Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function. Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay. Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1. In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays. Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors.
Asunto(s)
Productos del Gen tat/metabolismo , Sustancias de Crecimiento/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Secuencia Conservada/genética , Cisteína/genética , Cisteína/metabolismo , Exones/genética , Biblioteca de Genes , Productos del Gen tat/genética , Granulinas , Sustancias de Crecimiento/genética , VIH-1/genética , VIH-2/genética , Humanos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Mensajero/análisis , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Factores de Empalme Serina-Arginina , Células Tumorales Cultivadas , Proteínas Virales/genética , Proteínas Virales/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Specific receptors for angiotensin II (AII) were expressed in albino Xenopus laevis oocytes co-injected with poly(A)+ mRNA isolated from rat adrenal cortex and the calcium-specific photoprotein, aequorin. In such oocytes, AII elicited rapid, dose-dependent rises in cytosolic free calcium with light emission responses up to 100-fold above basal levels. Ligand-induced light emission was also observed in oocytes injected with rat brain mRNA and stimulated with acetylcholine and glutamate. These findings demonstrate that mammalian AII receptors expressed in Xenopus oocytes are functionally linked to intracellular Ca2+ mobilization, and indicate the potential value of aequorin-injected oocytes for rapid and specific screening of mRNAs transcribed from expression libraries containing cloned receptor cDNAs.