Abstract
We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-gamma(2), whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-gamma(2) activation.
Highlights
We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-␥2 in FDC-P1 cells transfected with the wild-type erythropoietin-receptor
By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor
Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-␥2, whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects
Summary
Epo-R, erythropoietin receptor; ␣-MEM, ␣-minimum essential medium; GPI, glycosylphosphatidylinositol; SH2, Src homology domain 2; IPG, inositol phosphoglycan; EGF, epidermal growth factor; PLC, phospholipase C; PI-PLC, phosphatidylinositol-phospholipase C; WT, wild type; HPTLC, high performance thin layer chromatography; AHM, 2,5-anhydromannitol; PIP, phosphatidylinositol monophosphate; PC, phosphatidylcholine. We show that erythropoietin induces a rapid and transient hydrolysis of GPI and tyrosine phosphorylation of PLC-␥2 Both effects require the presence of the same tyrosine residues on the intracellular domain of Epo receptor. Our results suggest that tyrosine Y1 (Tyr343), Y2 (Tyr401), Y7, and/or Y8 (Tyr464 and/or Tyr479) are implicated in Epo-induced GPI hydrolysis and tyrosine phosphorylation of PLC-␥2, whereas tyrosines Y3 and/or Y4 (Tyr429 and/or Tyr431) seem to be involved in Epo-induced inhibition of GPI hydroly-. Our results strongly suggest that Epo regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-␥2 activation
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