Identification of the erythropoietin receptor domain required for calcium channel activation

Barbara A. Miller, Dwayne L. Barber, Laurie L. Bell, Bryan K. Beattie, Min Ying Zhang, Benjamin G. Neel, Monique Yoakim, Lawrence I. Rothblum, Joseph Y. Cheung

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

Abstract

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is dependent on tyrosine phosphorylation. To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovary (CHO) cells were transfected with wild-type or mutant Epo receptors subcloned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) gene, and a cytomegalovirus immediate-early promoter driving expression of the Epo-R. Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca](i)) changes were simultaneously monitored with rhod-2. Transfection of CHO cells with pTracer encoding wild-type Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca](i) increase that was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic tyrosines substituted). Transfection with carboxyl-terminal deletion mutants indicated that removal of the terminal four tyrosine phosphorylation sites, but not the tyrosine at position 479, abolished Epo- induced [Ca](i) increase, suggesting that tyrosines at positions 443, 460, and/or 464 are important. In CHO cells transfected with mutant Epo-R in which phenylalanine was substituted for individual tyrosines, a significant increase in [Ca](i) was observed with mutants Epo-R Y443F and Epo-R Y464F. The rise in [Ca](i) was abolished in cells transfected with Epo-R Y460F. Results were confirmed with CHO cells transfected with plasmids expressing Epo-R mutants in which individual tyrosines were added back to Epo-R F8 and in stably transfected Ba/F3 cells. These results demonstrate a critical role for the Epo-R cytoplasmic tyrosine 460 in Epo-stimulated Ca2+ influx.

Original languageEnglish (US)
Pages (from-to)20465-20472
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number29
DOIs
StatePublished - Jul 16 1999

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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