The interleukin-4 site-2 epitope determining binding of the common receptor γ chain

Abstract

Human IL-4 (IL-4), one of the small four-helix-bundle cytokines, uses the specific IL-4 receptor α chain together with a promiscuous subunit, the common 7 chain (γ(c)) for transmembrane signaling. The ligand-binding properties of γ(c), which are presently poorly understood, were analysed by biosensor techniques employing recombinant ectodomains of γ(c) and a receptor chains (IL4-BP). The formation of a ternary complex between solute γ(c) ectodomain and IL-4 saturated IL4-BP could be established to exhibit a high dissociation constant K(d) = 3 μM and a short half life τ(1/2) = 7 s. This binding affinity resulted to the major part from the interaction of γ(c) ectodomain with IL-4 and not from a direct contact of the ectodomains, since binding between solute γ(c) ectodomain and IL-4 could be established (K(d) about 150 μM), whereas no binding was found between the γ(c) ectodomain and IL4-BP in the absence of IL-4. The IL-4 epitope involved in γ(c) ectodomain interaction (site 2) was identified by means of an alanine- scanning mutational approach. The IL-4 site 2 comprised residues I11 and N15 on helix A together with Y124 on helix D as major binding determinants. The IL-4 alanine variants at site 2 generally showed only moderate defects in biological activity. Even the most affected variant [A124]IL-4 retained a partial agonist activity of about 50% during a T-cell proliferation assay. The dosis leading to a half-maximal response (EC50) was not altered by site-2 substitutions. The present results are in accordance with a two-step- dimerisation mechanism for IL-4 receptor activation, where solute IL-4 at physiological concentrations binds first via the high-affinity site 1 to the a chain only, since the affinity of IL-4 site 2 for γ(c) is too low. This site-2 affinity seems to be sufficient, however, to promote, in a second step, a productive association of γ(c) to an IL-4/α chain complex in the membrane.