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.

The beta-chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptors functions as a communal receptor subunit and is often referred to as beta common (betac). Analogous to other shared receptor subunits including gp130 and the IL-2R gamma chain, betac mediates high affinity binding and signal transduction of all of its ligands. It is not clear, however, how these common receptor subunits can recognize several ligands and indeed whether they exhibit a common binding pocket to accomplish this. We have performed molecular modeling of betac based on the known structures of the growth hormone and prolactin receptors and targeted the putative F'-G' loop for mutagenesis. Substitution of this whole predicted loop region with alanines completely abrogated high affinity binding of GM-CSF, IL-3, and IL-5. Individual alanine substitutions across the loop revealed that a single residue, Tyr421, is critical for high affinity binding of GM-CSF, IL-3, and IL-5, whereas alanine substitution of adjacent residues has little or no effect on high affinity binding. Significantly, reintroducing Tyr421 into the polyalanine-substituted mutant restored high affinity ligand binding of GM-CSF, IL-3, and IL-5, indicating that within this region the tyrosine residue alone is sufficient for high affinity ligand interaction. Functional studies measuring STAT5 activation revealed that alanine substitution of Tyr421 severely impaired the ability of betac to signal. These results show for the first time that a single residue in a shared receptor subunit acts as a binding determinant for different ligands and may have implications for other receptor systems where communal receptor subunits exhibit hydrophobic residues in their putative F'-G' loops. These results also raise the possibility that a single compound targeted to this region may simultaneously inhibit the binding and function of multiple cytokines.

Cytokine receptor dimerization is a common theme in receptor activation (1). Following the binding of the cognate ligand to cytokine receptors, a sequential process takes place whereby receptor subunits associate and recruit cytoplasmic signaling molecules leading to receptor activation and cellular signaling (2). The general process of receptor dimerization exhibits variations among the cytokine receptor superfamily and may involve homodimerization or heterodimerization events depending on receptor subunit composition (3, 4). In the case of the growth hormone receptor, growth hormone binds initially to one receptor subunit and induces its homodimerization with a second, identical subunit (5). A similar process probably takes place with erythropoietin and granulocyte colony-stimulating factor, leading, in both cases, to receptor homodimerization and activation (6, 7).With cytokine receptors that comprise multiple subunits, receptor activation is accompanied by homodimerization or heterodimerization of the signaling subunits. For example, in the IL-6 1 receptor system, IL-6 induces dimerization of IL-6R␣ with gp130 (8), homodimerization of gp130, and receptor activation (9). On the other hand, the binding of CNTF to CNTFR␣ induces its association with gp130 and the LIF receptor, and the heterodimerization of gp130 and the LIF receptor is accompanied by receptor activation (10). Similarly, heterodimerization of IL-2R␤ and IL-2R␥ subunits is necessary for IL-2 receptor activation (11,12). Interestingly, in these cases, each receptor ␣ chain constitutes the major binding subunit but does not seem to form part of the signaling receptor complex.The mechanism of activation of the GM-CSF/IL-3/IL-5 receptor system exhibits features similar to the mechanism employed by the above receptors, although some unique features are becoming evident. One of the most important differences is the contribution that each receptor ␣ chain makes to signaling. This is manifested in two ways: first, unlike IL-6R␣, CNTFR␣, and the IL-2R␣, the cytoplasmic domains of GM-CSFR␣, IL-3R␣, and IL-5R␣ are all required for full receptor activation and signaling (13-16). Second, IL-3R␣ and GM-CSFR␣ form disulfide-linked dimers with the common ␤ chain (␤ c ) of their receptor (4, 17). The disulfide-mediated dimerization of IL-3R␣ with ␤ c and of GM-CSFR␣ with ␤ c is accompanied by tyrosine phosphorylation of ␤ c (4). In all of these cases, however, tyrosine phosphorylation is observed in the disulfide-linked dimers as well as in the monomeric molecules, and hence it is not clear which is the critical species for receptor activation. Furthermore, the location of the cysteines involved in disulfide linkage is not known, nor is it apparent whether they constitute a functionally conserved motif in the cytokine receptor superfamily. We have now performed single alanine substitutions