A Single Tyrosine Residue in the Membrane-proximal Domain of the Granulocyte-Macrophage Colony-stimulating Factor, Interleukin (IL)-3, and IL-5 Receptor Common β-Chain Is Necessary and Sufficient for High Affinity Binding and Signaling by All Three Ligands

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.

Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

“…Accordingly, the low affinity site K d was fixed at 100 nM during analysis in LIGAND in order to obtain a more accurate estimate of the high affinity site (K d , 107 pM). The dissociation constants we obtained are consistent with those reported in previous studies (28,30) on the hIL-3 receptor. The h␤c subunit used in these studies was derived from HL-60 eosinophils and contained a six-amino acid insertion in the C-D loop of domain 3 (34).…”

“…6B). The above results together with previous mutagenesis studies on domain 4 (30,38) suggest that the functional epitope of the h␤c subunit for high affinity hIL-3 binding consists of Tyr 15 and Phe 79 in domain 1 and Tyr 403 in domain 4, with secondary contributions from Tyr 347 , His 349 , and Ile 350 . The reductions in high affinity binding and receptor activation by hIL-3 observed for the Y15A and F79A mutant h␤c subunits are not a consequence of impaired receptor translation, folding, or cell-surface expression, as previously these mutants were shown to be expressed at wild-type levels in COS7 cells (31).…”

“…Residues (28,29), and Tyr 403 (FЈ-GЈ loop) (30). By analogy with human ␤c, we prepared alanine substitution mutants of residues in the relevant loops of the ␤ IL-3 subunit, and we examined their capacity to bind mIL-3 directly.…”

“…the high affinity hIL-3 complex, but the functional epitope based on this and previous mutagenesis studies (30,38) The residues in the murine ␤ IL-3 receptor critical for mIL-3 binding were also determined. This receptor provides the opportunity to compare residues involved in both low and high affinity binding.…”

IL-3 signaling and the role of Src kinases, JAKs and STATs: a covert liaison unveiled

“…In addition, the cytoplasmic portion of the achains of IL-3, GM-CSF and IL-5 receptors also appear to be involved in the activation of JAK2 kinase (Quelle et al, 1994;Cornelis et al, 1995;Kouro et al, 1996;Matsuguchi et al, 1997;. Activation of JAK2 leads to phosphorylation of the IL-3R b c chain on multiple tyrosine residues (Y577, Y612, Y695 and Y750) which in turn serve as docking sites for other signal transducing proteins, the most important of which are the STATs Inhorn et al, 1995;van Dijk et al, 1997;Pazdrak et al, 1995;Chin et al, 1996;Woodcock et al, 1996). IL-3 activation of hematopoietic cells appears to lead to the activation of multiple STATs, which include STAT-1, STAT-3, STAT-5 and STAT-6.…”