The role of interchain heterodisulfide formation in activation of the human common beta and mouse betaIL-3 receptors.
The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Department of Structural Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 0200, Australia.
Shamaruh
S
Mirza
Jinglong
J
Chen
James M
JM
Murphy
Ian G
IG
Young
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D016185:Receptors, Interleukin-3 / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction
D004220:Disulfides; D007377:Interleukin-3; D020033:Protein Isoforms; D016185:Receptors, Interleukin-3