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J Biol Chem, Vol. 274, Issue 29, 20465-20472, July 16, 1999

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^¶¶, and Joseph Y. Cheung^¶¶||

From the Departments of  Pediatrics, ^|| Medicine, and ^¶¶ Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, the ^¶ Division of Cellular and Molecular Biology, Ontario Cancer Institute, the  Department of Laboratory Medicine and Pathobiology, The Toronto Hospital, and the ^** Department of Medical Biophysics, University of Toronto, Ontario M5G 2M9, Canada, and the ^§§ Cancer Biology Program and Division of Hematology-Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215

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.

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