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Truncated erythropoietin receptor in a murine erythroleukemia cell line
T Bittorf1, S J Busfield, S P Klinken
1Department of Biochemistry, University of Western Australia, Nedlands.
Abstract:
The Friend spleen focus forming virus produces a 55 kDa envelope glycoprotein which associates with the erythropoietin receptor. We compared the erythropoietin receptor in Friend virus transformed murine erythroleukemic F4N and 707 cell lines with the J2E erythroid line generated by the J2 retrovirus. Reverse transcriptase PCR was used to determine transcript size. Erythropoietin receptor cDNAs were then sequenced and protein products analysed by Western blotting and immunoprecipitation. We show here that the F4N murine erythroleukemic cell line had an enlarged erythropoietin receptor mRNA. In contrast, the 707 and J2E cell line had normal sized transcripts for the receptor. Sequence analysis of the receptor in F4N cells revealed that introns which separate the exons coding for the cytoplasmic domain of the receptor were retained in these transcripts. As a consequence, a premature stop codon had been introduced, leaving only four amino acids in the intracellular portion of the receptor molecule. The normal erythropoietin receptor is approx. 66-70 kDa, but immunoprecipitation of [35S]methionine/cysteine labelled cell lysates with an antibody to the amino-terminus of the erythropoietin receptor identified a truncated 37 kDa protein in F4N cells. Despite the severe carboxy-terminal truncation of the erythropoietin receptor, F4N cells continued to proliferate like the other murine erythroleukemia cell lines. This study shows that failure to remove introns from the erythropoietin receptor mRNA in F4N cells has resulted in the production of a smaller protein with virtually no cytoplasmic domain.
Insights
Friend spleen focus forming virus infection causes enlarged erythropoietin receptor mRNA in F4N cells. This results in a truncated protein, yet F4N cells proliferate, indicating altered erythropoietin receptor function.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- The Friend spleen focus forming virus (SFFV) produces an envelope glycoprotein that interacts with the erythropoietin receptor (EpoR).
- Murine erythroleukemic cell lines F4N and 707, and the J2E erythroid line, were utilized to study EpoR in Friend virus-transformed cells.
Purpose of the Study:
- To investigate the molecular alterations of the erythropoietin receptor in Friend virus-transformed murine erythroleukemic cell lines.
- To compare the erythropoietin receptor mRNA and protein products in F4N cells with those in 707 and J2E cell lines.
Main Methods:
- Reverse transcriptase PCR (RT-PCR) was employed to determine erythropoietin receptor transcript size.
- Erythropoietin receptor cDNAs were sequenced.
- Protein products were analyzed using Western blotting and immunoprecipitation.
Main Results:
- The F4N cell line exhibited an enlarged erythropoietin receptor mRNA, unlike the normal-sized transcripts in 707 and J2E cells.
- Sequence analysis revealed intron retention in the F4N erythropoietin receptor mRNA, leading to a premature stop codon and a truncated protein (37 kDa) lacking most of its cytoplasmic domain.
- Despite the truncated receptor, F4N cells continued to proliferate.
Conclusions:
- Intron retention in erythropoietin receptor mRNA processing in F4N cells results in a non-functional protein with a severely truncated cytoplasmic domain.
- The proliferation of F4N cells despite the defective erythropoietin receptor suggests alternative signaling pathways or mechanisms contributing to erythroleukemia.