Identification of the erythropoietin receptor domain required for calcium channel activation.
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.
Department of Pediatrics, The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA. bmiller2@psghs.edu
B A
BA
Miller
D L
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Barber
L L
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Bell
B K
BK
Beattie
M Y
MY
Zhang
B G
BG
Neel
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Yoakim
L I
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Rothblum
J Y
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Cheung
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D049452:Green Fluorescent Proteins; D017136:Ion Transport; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014443:Tyrosine / Q000378:metabolism
D015220:Calcium Channels; D008164:Luminescent Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D002118:Calcium