CDB15:0000377 CSF2 — CSF2RB

ABSTRACT: The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.

ABSTRACT: High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.