Mapping of human granulocyte-macrophage-colony-stimulating-factor domains interacting with the human granulocyte-macrophage-colony-stimulating-factor-receptor alpha-subunit.
The high-affinity granulocyte-macrophage-colony-stimulating-factor (GM-CSF) receptor (R) is composed of at least two subunits, termed alpha and beta. The alpha subunit is crucial for initiating ligand/receptor interaction and for ligand specificity. The experiments reported in this study sought to identify the domains of human (h)GM-CSF which are responsible for interaction with hGM-CSFR alpha. Anti-(human-GM-CSF) mAb were used as competitors in a 125I-GM-CSF receptor-binding assay on cells expressing the recombinant hGM-CSFR alpha chain. Inhibition of 125I-GM-CSF binding to the GM-CSFR alpha chain was demonstrated by mAb which mapped to the middle third of the third alpha helix (amino acids 78-83), the distal two-thirds of the third alpha helix and the initial 7 residues of the loop between the third and fourth helix (amino acids 78-94), and the extreme carboxy terminus. No inhibition of binding occured with an antibody whose domain begins in the first beta-pleated sheet (amino acids, 39-43), continues through the second helix (amino acids 55-64) and into the proximal third of the third helix (to amino acid 77). mAb which mapped to the distal half of the fourth helix and the carboxy-terminal tail (amino acids 110-127) increased the binding of 125I-GM-CSF to GM-CSFR alpha. Due to the known discontinous epitopes the possibility that the distal portion of the fourth helix contributes to binding cannot be eliminated. However, the domains of hGM-CSF most clearly involved in binding to the hGM-CSFR alpha are the distal two-thirds of the third alpha helix, the immediate downstream residues and the extreme carboxy terminus of hGM-CSF.
Cancer Biology Research Group, University of Calgary, Alberto, Canada.
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Brown
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Pihl
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Kaushansky
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D006224:Cricetinae; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D011487:Protein Conformation; D011869:Radioligand Assay; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013006:Growth Hormone