The Ig-like domain of human GM-CSF receptor alpha plays a critical role in cytokine binding and receptor activation.
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.
Mirza
Shamaruh
S
Structural Biology Program, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia.
Walker
Andrew
A
Chen
Jinglong
J
Murphy
James M
JM
Young
Ian G
IG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053648:Cytokine Receptor Common beta Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D053661:Interleukin-13 Receptor alpha1 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018121:Receptors, Cytokine / Q000235:genetics / Q000378:metabolism*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D053648:Cytokine Receptor Common beta Subunit; D016207:Cytokines; D053661:Interleukin-13 Receptor alpha1 Subunit; D018121:Receptors, Cytokine; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003545:Cysteine