Characterization of ligand binding to immobilized biotinylated extracellular domains of three growth factor receptors.
For the purpose of developing a cell-free binding assay suitable for the large scale screening of receptor agonists and antagonists, the extracellular ligand binding domains of the low affinity NGF receptor (p75NGFR), a splicing variant of the fibroblast growth factor receptor (FGFR), and the PDGF beta-receptor (PDGFR) were modified by the biotinylation of amino groups. After immobilization in streptavidin-coated microtiter wells, it was determined by ligand binding analysis that all three receptors retained high affinities (0.7-1.8 nM) for their respective ligands. Two receptors, p75NGFR and FGFR, also displayed low affinity ligand binding (45-85 nM) which is likely a result of differential biotinylation. The three assay systems displayed excellent tolerance to several solution variables including organic solvents and high salt, with the exception of the FGF assay's sensitivity to thiols. The consistency obtained by using a coated well protocol, coupled with elimination of cell culture while maintaining natural binding affinities, demonstrates that the immobilized biotinylated extracellular domain creates an excellent system for pharmaceutical screening.
Chiron Corporation, Emeryville, California 94608.
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Kaufman
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Brown
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Stauber
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000648:ultrastructure; D006224:Cricetinae; D005110:Extracellular Space / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009414:Nerve Growth Factors / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D012996:Solutions
D006133:Growth Substances; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D017475:Receptors, Nerve Growth Factor; D012996:Solutions; D016222:Fibroblast Growth Factor 2; D001710:Biotin; D017479:Receptors, Platelet-Derived Growth Factor