Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation.

Abstract

Interleukin 4 (IL‐4) exerts a decisive role in the coordination of protective immune responses against parasites, particularly helminths. A disregulation of IL‐4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human IL‐4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4‐helix‐bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL‐4 variants deficient in binding to the extracellular domain of the IL‐4 receptor (IL‐4R(ex)). In parallel, up to 1000‐fold increased concentrations of this type of variant were required to induce T‐cell proliferation and B‐cell CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild‐type). IL‐4 variants affected at site 2 exhibited partial agonist activity during T‐cell proliferation; however, they still bound with high affinity to IL‐4R(ex). [The generation of an IL‐4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO J., 11, 3237‐3244)]. These findings indicate that IL‐4 functions by binding IL‐4R(ex) via site 1 which is constituted by residues on helices A and C.(ABSTRACT TRUNCATED AT 250 WORDS)

Global and Local Determinants for the Kinetics of Interleukin‐4/Interleukin‐4 Receptor α Chain Interaction

“…It is unclear how this might influence the interaction kinetics. (Kruse et al, 1993), which is similar to the values determined in the present biosensor experiments. The data obtained with IL4-BP and IL-4 receptors in situ are therefore consistent.…”

“…The contribution of the whole side chains as revealed by alanine substitutions appears to be even higher (Gampfer, J. and Sebald, W., unpublished results). Other side chains appear to contribute much less to the total binding affinity (Kruse et al, 1993;Ramanathan et al, 1993; Gampfer, J. and Sebald, W., unpublished results). T h e biosensor technology will be able to reliably measure small differences as demonstrated in this communication, and therefore will be a valuable tool for mapping the whole IL-4-binding site for lL4-BP.…”

“…In previous experiments (Kruse et al, 1993), the substitution of Glu9 of 1L-4 by glutamine was found to cause a more than 100-fold decrease in radioligand-binding affinity. not yet been analysed.…”

Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13

“…These mutants have clarified the crucial role of a particular residue in the ligand-receptor interaction. Among them, various antagonistic muteins including mGM-CSF (38,39), hIL-5 (40), human IL-6 (41, 42), human leukemia inhibitory factor (43,44), human IL-15 (45), human and murine IL-2, and human IL-4 (46,47) have been produced. However, no antagonist of murine or human IL-13 has been produced.…”

“…For example, when Glu-21, located in ␣-helix A of the mGM-CSF molecule, was mutated to Ala, a mGM-CSF with decreased bioactivity was produced. Similarly, when Glu-9, also located in ␣-helix A of the IL-4 molecule, was mutated to Lys (IL-4E9K), a dramatic inhibition in binding to IL-4R␣ chain was observed (47). When amino acid residues in ␣-helix A of IL-13, IL-4, mGM-CSF, and hIL-5 were aligned, Glu-13 in IL-13 was identified, which is located at the equivalent position in IL-4, mGM-CSF, and hIL-5 (Fig.…”