A single tyrosine residue in the membrane-proximal domain of the granulocyte-macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 receptor common beta-chain is necessary and sufficient for high affinity binding and signaling by all three ligands.
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.
Woodcock
J M
JM
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia.
Bagley
C J
CJ
Zacharakis
B
B
Lopez
A F
AF
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D015848:Interleukin-5 / Q000378:metabolism; D019169:Jurkat Cells; D008894:Milk Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000737:chemistry*; D016185:Receptors, Interleukin-3 / Q000737:chemistry / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D015848:Interleukin-5; D008894:Milk Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D018123:Receptors, Interleukin; D016185:Receptors, Interleukin-3; D053649:Receptors, Interleukin-5; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D000409:Alanine