High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.
The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.
Shanafelt
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AB
Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304-1104.
Kastelein
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RA
D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D004921:Erythropoietin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013006:Growth Hormone / Q000494:pharmacology; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D007378:Interleukins / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D007378:Interleukins; D008024:Ligands; D046911:Macromolecular Substances; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013006:Growth Hormone