The objective of this continuation application is to elucidate the mechanism of human interleukin 5 receptor recognition at the molecular and structural levels. Human IL5 is the major hematopoietin responsible for differentiation, proliferation, migration and activation of eosinophils. IL5 is a homodimeric protein dominated by 2 four-helix bundle units and acts on eosinophils through a cell surface receptor. The IL5 receptor contains alpha and beta c chains, with alpha primarily responsible for ligand binding and beta c for signal transduction. Recent major advances in our molecular understanding of this system, along with recombinant tools and methods for mutagenic, structural, molecular interaction and cellular analysis, together provide an important opportunity to identify the structural determinants in IL5 and receptor subunit ectodomains that drive recruitment of alpha and beta c receptor subunits into a functional complex. This application has three specific aims: (1) Using a combination of mutagenic and biophysical methods, elucidate the complementary structural determinants in IL5 and receptor alpha chain that lead to formation of the IL5-R alpha specificity complex; work on this goal will help interpret the co-crystal structure obtained in aim 3; (2) Identify the structural determinants and conformational organization in IL5 and receptor that lead to functional beta c recruitment into a three way IL5-alpha-beta c complex, including through use of IL5 mutants and assays with recombinant beta c in both soluble and cellular forms; (3) Obtain the co-crystal structure of the IL5-receptor a complex and correlate this structure with determinants for alpha and beta c recognition mapped from mutagenic/solution studies; deduce the structural mechanism of receptor subunit recruitment and assembly by IL5. Long term, this project will lead to a fundamental understanding of hIL5-receptor recruitment and assembly mechanism and will enhance our knowledge of these processes as they occur in normal and disease states such as asthma. It will identify topological and conformational motifs in hIL5 responsible for receptor recruitment and assembly into an IL5-alpha-beta c heterocomplex. And, it will provide strategies to investigate the structural basis of biological activity of other 4-helix bundle growth factor proteins, including the cytokine family that encompasses the structurally- and receptor-related granulocyte-macrophage colony stimulating factor and interleukin 3.
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