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Mutational analysis of the alpha subunit of the human interleukin-3
receptor
AP Rapoport, S Luhowskyj, P Doshi and JF DiPersio
Department of Medicine, University of Rochester School of Medicine &
Dentistry, Rochester, NY, USA.
The alpha subunit of the human interleukin-3 receptor (IL-3R alpha) is a
70-kD glycoprotein member of the hematopoietin receptor superfamily. This
protein associates with a beta subunit common to the receptors for IL-5 and
granulocyte-macrophage colony-stimulating factor (GM-CSF) to form a
high-affinity receptor for IL-3. To identify regions of IL-3R alpha
critical for ligand binding and receptor function, cDNAs encoding mutant
receptors were generated and expressed in COS cells along with the beta
subunit. Mutant receptors lacking almost the entire cytoplasmic domain of
IL-3R alpha [IL-3R alpha(CD)] or carrying a substitution of trp for leu in
the membrane proximal leu-ser-x-trp-ser (LSXWS) box bound 125I-IL-3 with
nearly the same affinity as wild-type IL-3R alpha. In contrast, a mutant
lacking the entire "LSXWS" motif failed to bind 125I-IL-3 with high
affinity despite showing surface expression. In addition, hybrid receptors
composed of the first 104 amino acids (aa) of IL-3R alpha joined to aa 118
through 400 of the alpha subunit of the GM-CSF receptor (GM-R alpha) [IL-3R
alpha/GM-R alpha] or the first 118 aa of GM-R alpha joined to aa 104
through 378 of IL-3R alpha [GM-R alpha/IL-3R alpha] failed to bind
125I-IL-3 in the presence of the beta subunit. A third hybrid receptor
composed of the first 281 residues of IL-3R alpha fused to residues 306
through 379 of GM-R alpha [IL-3R alpha/GM-R alpha-DS] also failed to bind
125I-IL-3 in the presence of the beta subunit but, in contrast to the IL-3R
alpha/GM- R alpha hybrid, demonstrated weak surface expression. Mutant
receptors lacking the N-terminal 30 aa and the N-terminal 9 aa also did not
bind 125I-IL-3 with high affinity, although both were expressed on the cell
surface. These data suggest that although the cytoplasmic domain and the
leucine residue of the "LSXWS" box are not critical for ligand binding or
beta-subunit association, the "LSXWS" motif and amino- terminal sequences
are important for these functions.
Volume 87,
Issue 1,
pp. 112-122,
01/01/1996
Copyright © 1996 by The American Society of Hematology
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