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

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 γ 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 α 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 (EC 50 ) 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 α 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.