The Ig-like domain of human GM-CSF receptor α plays a critical role in cytokine binding and receptor activation

Abstract

GM-CSF (granulocyte/macrophage colony-stimulating factor) is an important mediator of inducible haemopoiesis and inflammation, and has a critical role in the function of alveolar macrophages. Its clinical applications include the mobilization of haemopoietic progenitors, and a role as an immune stimulant and vaccine adjuvant in cancer patients. GM-CSF signals via a specific alpha receptor (GM-CSFRalpha) and the shared hbetac (human common beta-subunit). The present study has investigated the role of the Ig-like domain of GM-CSFRalpha in GM-CSF binding and signalling. Deletion of the Ig-like domain abolished direct GM-CSF binding and decreased growth signalling in the presence of hbetac. To locate the specific residues in the Ig-like domain of GM-CSFRalpha involved in GM-CSF binding, a structural alignment was made with a related receptor, IL-13Ralpha1 (interleukin-13 receptor alpha1), whose structure and mode of interaction with its ligand has recently been elucidated. Mutagenesis of candidate residues in the predicted region of interaction identified Val51 and Cys60 as having critical roles in binding to the alpha receptor, with Arg54 and Leu55 also being important. High-affinity binding in the presence of hbetac was strongly affected by mutation of Cys60 and was also reduced by mutation of Val51, Arg54 and Leu55. Of the four key residues, growth signalling was most severely affected by mutation of Cys60. The results indicate a previously unrecognized role for the Ig-like domain, and in particular Cys60, of GM-CSFRalpha in the binding of GM-CSF and subsequent activation of cellular signalling.

Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

“…By identifying a naturally occurring splice variant of the IL-3 ␣-subunit termed SP2 that lacks the N-terminal Ig-like domain that is otherwise present in the full-length receptor (termed SP1) in addition to the membrane-proximal cytokine receptor homology module (16), we have uncovered additional complexity within the IL-3 receptor system that would enable IL-3 to activate distinct signaling outcomes. Interestingly, whereas the IL-3R␣ subunits in both mouse and humans exist as either SP1 or SP2 splice variants, no such naturally occurring isoforms have been identified in the related GM-CSF or IL-5 ␣ receptors (38), consistent with the essential roles of the N-terminal Ig-like domains within these ␣-subunits for receptor activation (38,40).…”

“…Overall, we conclude that despite being properly transported to the cell surface, the hIL-3R␣ SP2 subunit exhibits a lower propensity to engage in high affinity hIL-3 binding in CTLL2 cells. This situation clearly differs from the Ig-like domain deletion mutants of the mouse and human IL-6␣ receptor, which fail to localize to the cell surface (37), and the human GM-CSF recep- tor ␣-subunit, which requires its Ig-like domain for high affinity GM-CSF binding and normal receptor activation (38).…”

Murine Interleukin-3: Structure, Dynamics, and Conformational Heterogeneity in Solution

“…Additionally, homology modeling and mutagenesis have indicated that the surface of human GM-CSF that mediates R-receptor binding will be larger than that revealed by the complex crystal structure, as no electron density was observed for the GM-CSFRR N-terminal Ig-like domain: a domain since shown to play an important role in GM-CSF binding. 49 ' DISCUSSION Although mIL-3 conveys essential biological signals in blood cell development and inflammatory responses, structural studies of this important protein have been confounded by its intrinsic poor solubility and strong tendency to aggregate in solution. A recently developed soluble variant of mIL-3, mIL-3 , which retains wild-type biological activity and exhibits significantly improved solution behavior, 11 has enabled us to perform a comprehensive characterization of its 3D solution structure, backbone dynamics, and hydrodynamic properties in two different buffer solutions over the temperature range 283-303 K. We begin by comparing the structure of mIL-3 with structures of the hIL-3 analogue SC-55494, GM-CSF, and IL-5 and then evaluate the novel features of mIL-3 33-156 revealed in the current study.…”

High Yield Production of a Soluble Human Interleukin-3 Variant from E. coli with Wild-Type Bioactivity and Improved Radiolabeling Properties

“…Purified hIL-3 (13-125; W13Y) was radio-iodinated using Pierce Pre-Coated Iodination tubes (Thermo Scientific) according to established protocols [22,23]. COS cells were transfected by electroporation with expression plasmids encoding the human IL-3Rα subunit (pSG5: IL‑3Rα) and the human βc subunit (pSG5: βc).…”