Abstract
Dual asymmetric mutagenesis of single-chain interleukin 5 (scIL5) was used to obtain evidence that the normally homodimeric IL5 molecule, which contains two 4-helix bundle domains arranged symmetrically about a 2-fold axis, can recruit receptor alpha and betac subunits asymmetrically. Functionally active scIL5 was constructed using recombinant DNA methods by linking two IL5 monomers with a Gly-Gly linker. Mutants were constructed at residues Arg91, Glu110, and Trp111, previously shown to be involved in IL5 receptor alpha chain binding, and at residue Glu13, known to be involved in signal transduction presumably through interaction with the receptor betac chain. Mutants were examined for receptor alpha chain binding by an optical biosensor assay and for bioactivity using a cell proliferation assay. Substitution of the two binding site residues R91 and W111 in the same 4-helix bundle domain caused a 5-fold greater reduction in receptor binding affinity than when the two substitutions were distributed one in each domain. Substitution of E13 and R91 either in the same or in opposite domains gave comparable IL5Ralpha chain binding kinetics, essentially unchanged from those of scIL5. However, in contrast to the binding affinity patte...Continue Reading
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