Article

A sensitive and versatile bioassay for ligands that signal through receptor clustering.

Flanders' Interuniversity Institute for Biotechnology, Department of Medical Protein Research, University of Ghent, Faculty of Medicine, Belgium.
Journal of Interferon & Cytokine Research (impact factor: 3.06). 02/2000; 20(1):79-87. DOI:10.1089/107999000312757 pp.79-87
Source: PubMed

ABSTRACT The induced expression of xanthine-guanine phosphoribosyl transferase (XGPRT) by low concentrations (-2 pg/ml) of interferon-alpha (IFN-alpha) or IFN-beta, in the 2fTPGH cell line caused a 50% cytotoxicity when these cells were grown in medium containing 6-thioguanine. We extended the application of this sensitive, reliable, and easy bioassay to other members of the cytokine family. To activate the IFN signaling pathway, we made receptor chimeras, consisting of the IFN type I receptor intracellular and transmembrane domains, fused to either the interleukin-5 (IL-5) receptors or erythropoietin (Epo) receptor extracellular domains as model systems. 2fTGH cells, stably transfected with these receptor chimeras, responded to very low concentrations of IL-5 or Epo (IC50 values of approximately 15 pg and 3 pg/ml, respectively) and thus can be used as a very sensitive bioassay for both ligands. Background activity of IL-5, Epo, tumor necrosis factor (TNF), IL-6, or leptin on cells that did not carry the receptor chimeras was very low. This methodology can in principle be extended to any ligand that acts via clustering of its receptors.

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    Article: Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.
    Irma Lemmens, Sven Eyckerman, Lennart Zabeau, Dominiek Catteeuw, Els Vertenten, Kristin Verschueren, Danny Huylebroeck, Joël Vandekerckhove, Jan Tavernier
    [show abstract] [hide abstract]
    ABSTRACT: We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.
    Nucleic Acids Research 08/2003; 31(14):e75. · 8.03 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

2fTGH cells
 
2fTPGH cell line
 
acts
 
Background activity
 
cytokine family
 
IC50 values
 
IFN signaling pathway
 
IFN type
 
IFN-alpha
 
IFN-beta
 
IL-5
 
induced expression
 
interferon-alpha
 
ligands
 
low concentrations
 
model systems
 
reliable
 
transmembrane domains
 
tumor necrosis factor
 
xanthine-guanine phosphoribosyl transferase