Identification of the erythropoietin receptor domain required for calcium channel activation

Erythropoietin (Epo) activates a voltage-independent Ca2+ channel that is d ependent on tyrosine phosphorylation, To identify the domain(s) of the Epo receptor (Epo-R) required for Epo-induced Ca2+ influx, Chinese hamster ovar y (CHO) cells were transfected with wildtype or mutant Epo receptors subclo ned into pTracer-cytomegalovirus vector. This vector contains an SV40 early promoter, which drives expression of the green fluorescent protein (GFP) g ene, and a cytomegalovirus immediate-early Promoter driving expression of t he Epo-R, Successful transfection was verified in single cells by detection of GFP, and intracellular Ca2+ ([Ca](i)) changes were simultaneously monit ored with rhod-2, Transfection of CHO cells with pTracer encoding wildtype Epo-R, but not pTracer alone, resulted in an Epo-induced [Ca], increase tha t was abolished in cells transfected with Epo-R F8 (all eight cytoplasmic t yrosines 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) incre ase, suggesting that tyrosines at positions 443, 460, and/or 464 are import ant. 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 wi th CHO cells transfected with plasmids expressing Epo-R mutants in which in dividual 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 cytoplas mic tyrosine 460 in Epo-stimulated Ca2+ influx.