Abstract
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.
Highlights
IntroductionFrom structure-function studies on all three ligands, it has been established that a conserved Glu residue in the first ␣-helices of granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin 3 (IL-3), and interleukin 5 (IL-5) is important for high affinity binding and biological activity [7,8,9]
§ To whom reprint requests should be addressed: Div. of Human Immunology, Institute of Medical and Veterinary Science, Box 14 Rundle Mall Post Office, Adelaide, South Australia 5000, Australia
The receptor ␣-chains for granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5), and c belong to the rapidly expanding cytokine receptor superfamily. Within this superfamily several subfamilies are emerging that are characterized by the sharing of a communal receptor subunit by multiple ligands. gp130 acts as an affinity converter and signal transducer for IL-6 [12, 13], IL-11 [14], oncostatin M [15], ciliary neurotrophic factor, leukemia inhibitory factor (LIF) [16], and cardiotrophin-1 [17]; the LIF receptor binds ciliary neurotrophic factor [18], cardiotrophin-1 [17], and oncostatin M in addition to LIF [19]; IL-2R supports affinity conversion and signaling of IL-2 and IL-15 [20]; IL-2R ␥ chain affinity converts IL-2 [21], IL-4 [22], IL-7 [23], IL-9 [24], and
Summary
From structure-function studies on all three ligands, it has been established that a conserved Glu residue in the first ␣-helices of GM-CSF, IL-3, and IL-5 is important for high affinity binding and biological activity [7,8,9] This residue is thought to represent a common motif involved in c interaction and suggests that, reciprocally, the three ligands may use a common binding determinant in c. This study shows for the first time that a single residue in a communal cytokine receptor chain is involved in binding multiple ligands These results may be applicable to other common receptor subunits whose predicted FЈ-GЈ loop is shown to contain hydrophobic residues analogous to Tyr421 in c. The identification of a single residue in c involved in multiple ligand binding and signaling illustrates the profound susceptibility of this region and raises the possibility that a single compound targeted to it could simultaneously interfere with the function of multiple ligands
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