O012 Structural and molecular basis of IL-3/GM-CSF/IL-5 receptor signalling

Abstract

Interleukin (IL)-3, granulocyte–macrophage colony stimulating factor (GM-CSF) and IL-5 are important cytokines that bridge innate and adaptive immunity, and play a significant role in cancer. They control the production and function of myeloid cells and dendritic cells, and are recognised key players in crippling immunopathological conditions such as rheumatoid arthritis and asthma. All three cytokines signal through heterodimeric specific receptors consisting of major binding and ligand-specific α chains, and a signalling subunit β c which is shared amongst all three receptors. The IL-3, GM-CSF and IL-5 receptors are also themselves important determinants of disease. For example, we have established a functional role for the IL-3 receptor α chain (CD123) in promoting the survival, homing, proliferation and engraftment of acute myeloid leukaemic stem cells using our blocking monoclonal antibody 7G3 in vivo and demonstrated a number of patients in which IL-3 autocrine signalling occurs in vitro in leukaemic stem cell populations [1] . In addition, our genetic studies on β c showed that this receptor subunit is required for the development of asthma [2] . We have used a combination of molecular biology, cell biology, biochemistry, crystallization and computer modelling to obtain 3D structures of cytokine receptors. To understand how these receptors signal and to help in the rational design and pre-clinical testing of future biologicals and small molecules that can selectively eliminate cancer stem cells or block inflammatory cells requires detailed 3D structural knowledge of druggable targets. Towards this aim we have recently solved the crystal structure of the human GM-CSF receptor ternary complex that revealed a novel mode of cytokine receptor activation involving a higher-order dodecamer complex [3] ; however, details of some specific receptor: ligand interactions were missing. We have now solved the structure of the binary GM-CSF: GM-CSF receptor α chain complex based on X ray diffraction data collected to 2.8 Å resolution which reveals for the first time all three extra cellular domains of the GM-CSF α receptor chain. In particular, this new structure suggests a mechanism of binding involving the N-terminal domain that differs from the related IL-3 receptor. Intriguingly, docking of the complete GM-CSF receptor α chain onto the dodecamer complex places the N-terminal domains from adjacent hexamers in close proximity to each other suggesting a direct role in receptor activation. We have now assembled and obtained crystals of IL-3 receptor complexes in solution comparable to those obtained for GM-CSF representing binary (IL-3 receptor α chain: IL-3) and ternary (IL-3 receptor α chain+ β c +IL-3 ) complexes. Importantly, we have also crystallised and solved the structure of the IL-3 receptor α chain in complex with an antagonist monoclonal antibody. This provides unique information on the IL-3 binding site in the IL-3 receptor α chain, and optimisation opportunities for our antagonistic monoclonal antibody towards its more effective use. Details of these structures and their implications for receptor signalling and the design of antagonists will be discussed.