Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor

Akihiko Yoshimura, Teresa Zimmers, Drorit Neumann, Gregory Longmore, Yuko Yoshimura, Harvey F. Lodish

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Abstract

The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-A-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-S)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1500 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 WSAWS 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 WSAWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSAWS motif is critical for protein folding, ligand-binding, and signal transduction.

Original languageEnglish
Pages (from-to)11619-11625
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number16
Publication statusPublished - 1992 Jun 5
Externally publishedYes

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Erythropoietin Receptors
Erythropoietin
Chemical activation
Ligands
Mutation
Processing
Interleukin-3
Cells
Point Mutation
INDEL Mutation
Protein folding
Signal transduction
Cytokine Receptors
Protein Folding
Viruses
Endoplasmic Reticulum
Signal Transduction
Glycoproteins
Binding Sites
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. / Yoshimura, Akihiko; Zimmers, Teresa; Neumann, Drorit; Longmore, Gregory; Yoshimura, Yuko; Lodish, Harvey F.

In: Journal of Biological Chemistry, Vol. 267, No. 16, 05.06.1992, p. 11619-11625.

Research output: Contribution to journalArticle

Yoshimura, Akihiko ; Zimmers, Teresa ; Neumann, Drorit ; Longmore, Gregory ; Yoshimura, Yuko ; Lodish, Harvey F. / Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 16. pp. 11619-11625.
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abstract = "The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-A-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-S)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1500 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 WSAWS 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 WSAWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSAWS motif is critical for protein folding, ligand-binding, and signal transduction.",
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