Human IL-4 (IL-4), one of the small four-helix-bundle cytokines, uses the specific IL-4 receptor a chain together with a promiscuous subunit, the common gamma chain (gamma(c)) for transmembrane signaling. The ligand-binding properties of gamma(c), which are presently poorly understood, were analysed by biosensor techniques employing recombinant ectodomains of gamma(c) and a receptor chains (IL4-BP). The formation of a ternary complex between solute gamma(c) ectodomain and IL-4 saturated IL4-BP could be established to exhibit a high dissociation constant Kd = 3 microM and a short half life tau1/2 = 7 s. This binding affinity resulted to the major part from the interaction of gamma(c) ectodomain with IL-4 and not from a direct contact of the ectodomains, since binding between solute gamma(c) ectodomain and IL-4 could be established (Kd about 150 microM), whereas no binding was found between the gamma(c) ectodomain and IL4-BP in the absence of IL-4. The IL-4 epitope involved in gamma(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 gamma(c) is too low. This site-2 affinity seems to be sufficient, however, to promote, in a second step, a productive association of gamma(c) to an IL-4/alpha chain complex in the membrane.