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Volume 271, Number 50, Issue of December 13, 1996 pp. 31922-31928
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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A Discontinuous Eight-Amino Acid Epitope in Human Interleukin-3 Binds the -Chain of Its Receptor

(Received for publication, April 15, 1996, and in revised form, September 18, 1996)

From the Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5001, Australia

We have previously reported that, within the first helix of human interleukin (IL)-3, residues Asp²¹ and Glu²² are important for interaction with the - and -chains of the IL-3 receptor, respectively. In order to define more precisely the sites of interaction with the receptor, we have performed molecular modeling of the helical core of IL-3 and single amino acid substitution mutagenesis of residues predicted to lie on the surfaces of the A, C, and D helices. The resulting analogues were characterized for their abilities to stimulate proliferation of TF-l cells and for binding to the high affinity (- and -chain; IL-3R/R) or low affinity (-chain alone; IL-3R) IL-3 receptor. We found that in addition to Asp²¹, residues Ser¹⁷, Asn¹⁸, and Thr²⁵ within the A helix and Arg¹⁰⁸, Phe¹¹³, Lys¹¹⁶, and Glu¹¹⁹ within the D helix of IL-3 were important for biological activity. Analysis of their binding characteristics revealed that these analogues were deficient in binding to both the IL-3R/R and the IL-3R forms of the receptor, consistent with a selective impairment of interaction with IL-3R. Molecular modeling suggests that these eight amino acid residues are adjacent in the tertiary structure, consistent with a discontinuous epitope interacting selectively with IL-3R. On the other hand, Glu²² of IL-3 was found to interact preferentially with the -chain with bulky and positively charged substitutions causing greater than 10,000-fold reduction in biological activity. These results show fundamental differences between IL-3 and granulocyte-macrophage colony-stimulating factor in the structural basis for recognition of their receptors that has implications for the construction of novel analogues and our understanding of receptor activation.

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