A sensitive and versatile bioassay for ligands that signal through receptor clustering.
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.
Van Ostade
X
X
Flanders' Interuniversity Institute for Biotechnology, Department of Medical Protein Research, University of Ghent, Faculty of Medicine, Belgium.
Schauvliege
L
L
Pattyn
E
E
Verhee
A
A
Vandekerckhove
J
J
Tavernier
J
J
D001681:Biological Assay; D004396:Coloring Agents; D003470:Culture Media / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004921:Erythropoietin / Q000032:analysis* / Q000494:pharmacology; D005354:Fibrosarcoma / Q000473:pathology; D005840:Gentian Violet; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology; D016899:Interferon-beta / Q000494:pharmacology*; D015848:Interleukin-5 / Q000032:analysis* / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D020738:Leptin / Q000494:pharmacology; D008024:Ligands; D008565:Membrane Proteins; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D010430:Pentosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D011940:Receptor Aggregation / Q000187:drug effects; D053633:Receptor, Interferon alpha-beta; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism; D017471:Receptors, Interferon / Q000187:drug effects; D018123:Receptors, Interleukin / Q000235:genetics / Q000378:metabolism; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins; D012641:Selection, Genetic; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D013866:Thioguanine / Q000633:toxicity; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004396:Coloring Agents; D003470:Culture Media; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D015848:Interleukin-5; D015850:Interleukin-6; D020738:Leptin; D008024:Ligands; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D017467:Receptors, Erythropoietin; D017471:Receptors, Interferon; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D004921:Erythropoietin; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D013866:Thioguanine; D005840:Gentian Violet