Soluble interleukin-5 receptor alpha-chain binding assays: use for screening and analysis of interleukin-5 mutants.
Interleukin-5 (IL-5) is a key cytokine for the production, differentiation, and activation of eosinophils. IL-5 is a member of the four helical bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a ligand binding alpha-chain and a signal-transducing beta-chain. We have established two receptor/ligand binding assays based on the extracellular domain of the receptor alpha-chain which we have produced as a fusion protein. One assay is based on scintillation proximity fluoromicrospheres and radiolabeled ligand and the other on detection of biotinylated ligand binding to immobilized receptor using a chemiluminescent substrate in a 96-well microtiter plate format. Both receptor binding assays have been optimized for high throughput screening for receptor antagonists. These assays were also used for analytical purposes and the binding of ligand to the receptor alpha-chain was compared directly to receptor binding assays performed on TF-1 cells which express the receptor alpha beta-heterodimer. These three assays have been used to study site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. Graber et al. (1995) J. Biol. Chem. 270, 15762-15769).
Banks
M
M
Department of New Lead Discovery, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom.
Graber
P
P
Proudfoot
A E
AE
Arod
C Y
CY
Allet
B
B
Bernard
A R
AR
Sebille
E
E
McKinnon
M
M
Wells
T N
TN
Solari
R
R
D000818:Animals; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D013329:Structure-Activity Relationship
D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5; D011993:Recombinant Fusion Proteins