ABSTRACT
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.
Subject(s)
Gene Products, tat/metabolism , Growth Substances/metabolism , HIV-1/metabolism , HIV-2/metabolism , Intercellular Signaling Peptides and Proteins , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Conserved Sequence/genetics , Cysteine/genetics , Cysteine/metabolism , Exons/genetics , Gene Library , Gene Products, tat/genetics , Granulins , Growth Substances/genetics , HIV-1/genetics , HIV-2/genetics , Humans , Leukocytes, Mononuclear/metabolism , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , RNA, Messenger/analysis , RNA-Binding Proteins , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Deletion , Serine-Arginine Splicing Factors , Tumor Cells, Cultured , Viral Proteins/genetics , Viral Proteins/metabolism , tat Gene Products, Human Immunodeficiency VirusABSTRACT
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.
