The objective of this proposal is to map the receptor recognition sites on human interleukin 5. Human IL-5 (hIL-5) is the major hematopoietin responsible for differentiation, proliferation, migration and activation of eosinophils. IL-5 is a homodimeric protein dominated by 2 four-helix bundle units and acts on eosinophils through a cell surface receptor. The IL-5R contains alpha and beta chains, with alpha being primarily responsible for ligand binding and beta for signal transduction. The proposed project will focus on defining structural elements in IL-5 which control binding to the alpha subunit and the biological activity that IL-5 exerts through the beta subunit. This project has several specific aims: (1) to map receptor recognition residues in hIL-5 by site directed mutagenesis and quantitative characterization of the binding and bioactivity properties of key mutants of both wild type IL-5 and a newly designed, biologically active single chain construct of hIL-5; (2) to determine the three dimensional structures of key mutants of wild type and single chain IL-5 and correlate these with solution and cell proliferation properties in order to determine structure-activity relationships in IL-5 and their relatedness to those for other cytokines; and (3) to establish conditions for crystallization of the hIL-5R alpha chain and its complex with hIL-5 and then determine their structures by X-ray diffraction in order to define at atomic resolution, the structure of the IL-5R interface and conformational transitions which may occur in the IL-5R interaction process. Long-term, this project will lead to a fundamental understanding of hIL-5R interaction, and will enhance our knowledge of processes as they occur in normal and disease states such as asthma. It will identify topological and conformational motifs in hIL-5 responsible for both receptor recognition and signal transduction. And, it will provide strategies to investigate the structural basis of biological activity of other 4-helix bundle growth factor proteins, such as the closely related granulocyte-macrophage colony stimulating factor (GM-CSF). Both topological and conformational motifs in hIL-5 responsible for receptor recognition and signal transduction will be defined. As such, it will provide strategies for potential development of agonist/antagonist of IL-5 activity.
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