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Abstract

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.