Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor.
The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-X-Trp-Ser (WSXWS), near the transmembrane domain is highly conserved in this family. To determine the function of this motif, we constructed deletion and insertion mutations in this part of the EPOR and introduced them into an interleukin-3 (IL-3)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1,500 erythropoietin (EPO)-binding sites/cell with a single affinity of about 300 pM and proliferate in the presence of IL-3 or EPO. Ba/F3 cells expressing receptors mutated in the WSXWS motif displayed little EPO binding on the cell surface and did not grow in the presence of EPO. The mutant receptors were retained in the endoplasmic reticulum (ER) and, as such, were unable to bind EPO. A single Gly insertion between the two WS sequences caused defects in receptor structure and function similar to mutations lacking all or part of the WSXWS motif. The EPOR can be activated, resulting in proliferation independent of EPO either by an Arg129 to Cys point mutation or by association with the Friend spleen focus-forming virus (SFFV) envelope glycoprotein gp55. Introduction of the point mutation (Arg129 to Cys) did not activate any of the receptors mutated in the WSXWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSXWS motif is critical for protein folding, ligand-binding, and signal transduction.
Yoshimura
A
A
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
Zimmers
T
T
Neumann
D
D
Longmore
G
G
Yoshimura
Y
Y
Lodish
H F
HF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D004921:Erythropoietin / Q000378:metabolism*; D007377:Interleukin-3 / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017467:Receptors, Erythropoietin
D007377:Interleukin-3; D008024:Ligands; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D004247:DNA