To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].
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. 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. Erythropoietin is a hematopoietic growth factor that regulates the proliferation, differentiation, and viability of erythroid progenitors and precursors (1Klingmuller U. Eur. J. Biochem. 1997; 249: 637-647Crossref PubMed Scopus (98) Google Scholar). The erythropoietin receptor is a member of the superfamily of cytokine receptors (2Ihle G.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (549) Google Scholar, 3Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1144) Google Scholar), which is characterized by the conservation of cysteines and a WSXWS motif in the extracellular domain (1Klingmuller U. Eur. J. Biochem. 1997; 249: 637-647Crossref PubMed Scopus (98) Google Scholar, 2Ihle G.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (549) Google Scholar, 3Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1144) Google Scholar, 4Noguchi C.T. Bae K.S. Chin K. Wada Y. Schechter A.N. Hankins W.D. Blood. 1991; 78: 2548-2556Crossref PubMed Google Scholar, 5Maouche L. Tournamille C. Hattab C. Boffa G. Cartron J.-P. Chretien S. Blood. 1991; 78: 2557-2563Crossref PubMed Google Scholar, 6D'Andrea A.D. Lodish H.F. Wong G.G. Cell. 1989; 57: 277-285Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 7Middleton S.A. Johnson D.L. Jin R. McMahon F.J. Collins A. Tullai J. Gruninger R.H. Jolliffe L.K. Mulcahy L.S. J. Biol. Chem. 1996; 271: 14045-14054Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Hilton D.J. Watowich S.S. Katz L. Lodish H.F. J. Biol. Chem. 1996; 271: 4699-4708Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), as well as limited similarities in the cytoplasmic domain, including Box 1 and Box 2 in the membrane proximal region (2Ihle G.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (549) Google Scholar, 3Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1144) Google Scholar). No kinase or other enzyme motif has been identified in the cytoplasmic domains of members of the cytokine receptor superfamily. For erythropoietin, interaction with its receptor rapidly induces dimerization/oligomerization (9Watowich S.S. Hilton D.J. Lodish H.F. Mol. Cell. Biol. 1994; 14: 3535-3549Crossref PubMed Scopus (187) Google Scholar, 10Miura O. Ihle J.N. Arch. Biochem. Biophys. 1993; 306: 200-208Crossref PubMed Scopus (47) Google Scholar), which results in increased affinity for a member of the Janus family of cytoplasmic tyrosine kinases, JAK2 (9Watowich S.S. Hilton D.J. Lodish H.F. Mol. Cell. Biol. 1994; 14: 3535-3549Crossref PubMed Scopus (187) Google Scholar, 10Miura O. Ihle J.N. Arch. Biochem. Biophys. 1993; 306: 200-208Crossref PubMed Scopus (47) Google Scholar, 11Witthuhn B. Quelle F.W. Silvennoinen O. Yi T. Tang B. Miura O. Ihle J.N. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1005) Google Scholar, 12Miura O. Nakamura N. Quelle F.W. Witthuhn B.A. Ihle J.N. Aoki N. Blood. 1994; 84: 1501-1507Crossref PubMed Google Scholar). JAK2 is activated by transphosphorylation of its active site and phosphorylates some or all of the eight tyrosine residues in the intracellular domain of the Epo 1The abbreviations EpoerythropoietinEpo-REpo receptormEpoRmurine Epo receptorCHOChinese hamster ovaryCMVcytomegalovirusGFPgreen fluorescent proteinwtwild-typeSH2Src homology 2PLCphospholipase C 1The abbreviations EpoerythropoietinEpo-REpo receptormEpoRmurine Epo receptorCHOChinese hamster ovaryCMVcytomegalovirusGFPgreen fluorescent proteinwtwild-typeSH2Src homology 2PLCphospholipase Creceptor. JAK2 also is activated by other cytokine receptors, including thrombopoietin, prolactin, and growth hormone (2Ihle G.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (549) Google Scholar, 3Ihle J.N. Nature. 1995; 377: 591-594Crossref PubMed Scopus (1144) Google Scholar, 13Drachman J.G. Griffin J.D. Kaushansky K. J. Biol. Chem. 1995; 270: 4979-4982Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Phosphorylation of the intracellular tyrosines attracts other intracellular proteins that bind to Epo-R via their Src homology 2 (SH2) domains; many are in turn tyrosinephosphorylated (14Damen J. Krystal G. Exp. Hematol. 1996; 24: 1455-1459PubMed Google Scholar). erythropoietin Epo receptor murine Epo receptor Chinese hamster ovary cytomegalovirus green fluorescent protein wild-type Src homology 2 phospholipase C erythropoietin Epo receptor murine Epo receptor Chinese hamster ovary cytomegalovirus green fluorescent protein wild-type Src homology 2 phospholipase C Identification of the phosphorylated tyrosines of the erythropoietin receptor to which specific SH2-containing proteins bind is important in defining the pathways involved in erythropoietin stimulated proliferation and differentiation. Tyrosine at position 343 or 401 of the murine Epo-R mediates maximal STAT5 activation (15Chin H. Nakamura N. Kamiyama R. Miyasaka N. Ihle J.N. Miura O. Blood. 1996; 88: 4415-4425Crossref PubMed Google Scholar, 16Fujitani Y. Hibi M. Fukada Y. Takahashi-Tezuka M. Yoshida H. Yamaguchi T. Sugiyama K. Yamanaka Y. Nakajima K. Hirano T. Oncogene. 1997; 14: 751-761Crossref PubMed Scopus (141) Google Scholar, 17Wakao H. Harada N. Kitamura T. Mui A.L.-F. Miyajima A. EMBO J. 1995; 14: 2527-2535Crossref PubMed Scopus (214) Google Scholar, 18Klingmuller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 19Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 20Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). The proliferation stimulating SH2-containing tyrosine phosphatase SHP2 is phosphorylated by JAK2 after binding at position 401 (21Tauchi T. Feng G.-S. Shen R. Hoatlin M. Bagby Jr., G.C. Kabat D. Lu L. Broxmeyer H.E. J. Biol. Chem. 1995; 270: 5631-5635Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22Tauchi T. Damen J.E. Toyama K. Feng G.-S. Broxmeyer H.E. Krystal G. Blood. 1996; 87: 4495-4501Crossref PubMed Google Scholar). The proliferation inhibiting SH2-containing tyrosine phosphatase SHP1 is recruited to the Epo receptor following the phosphorylation of tyrosine 429, resulting in inactivation of JAK2 and termination of proliferative signals (23Klingmuller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (841) Google Scholar, 24Yi T. Zhang J. Miura O. Ihle J.N. Blood. 1995; 85: 87-95Crossref PubMed Google Scholar). Phosphatidylinositol 3-kinase directly associates with the murine erythropoietin receptor at amino acids 479 and 343 (25Damen J.E. Cutler R.L. Jiao H. Yi T. Krystal G. J. Biol. Chem. 1995; 270: 23402-23408Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 26He T.-C. Zhuang H. Jiang N. Waterfield M.D. Wojchowski D.M. Blood. 1993; 82: 3530-3538Crossref PubMed Google Scholar, 27Damen J.E. Mui A.L.-F. Puil L. Pawson T. Krystal G. Blood. 1993; 81: 3204-3210Crossref PubMed Google Scholar). Other kinases/signal transducers that are tyrosine-phosphorylated in response to erythropoietin include GAP (28Torti M. Marti K.B. Altschuler D. Yamamoto K. Lapetina E.G. J. Biol. Chem. 1992; 267: 8293-8298Abstract Full Text PDF PubMed Google Scholar), Shc (14Damen J. Krystal G. Exp. Hematol. 1996; 24: 1455-1459PubMed Google Scholar), Vav (29Miura O. Miura Y. Nakamura N. Quelle F.W. Witthuhn B.A. Ihle J.N. Aoki N. Blood. 1994; 84: 4135-4141Crossref PubMed Google Scholar), c-fps/fes (30Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar), Lyn (31Chin H. Arai A. Wakao H. Kamiyama R. Miyasaka N. Miura O. Blood. 1998; 91: 3734-3745Crossref PubMed Google Scholar), phospholipase C-γ1 (32Ren H.-Y. Komatsu N. Shimizu R. Okada K. Miura Y. J. Biol. Chem. 1994; 269: 19633-19638Abstract Full Text PDF PubMed Google Scholar), and c-Cbl (33Odai H. Sasaki K. Iwamatsu A. Hanazono Y. Tanaka T. Mitani K. Yazaki Y. Hirai H. J. Biol. Chem. 1995; 270: 10800-10805Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 34Barber D.L. Mason J.M. Fukazawa T. Reedquist K.A. Druker B.I. Band H. D'Andrea A.D. Blood. 1997; 89: 3166-3174Crossref PubMed Google Scholar). It is not known whether these transducers are recruited directly to the Epo-R or bind indirectly via other adaptor proteins. Ligand binding of the Epo-R causes an increase in intracellular Ca2+, and it is believed that this is part of the signaling mechanisms controlling proliferation/differentiation of human (35Miller B.A. Scaduto Jr., R.C. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 37Mladenovic J. Kay N.E. J. Lab. Clin. Med. 1988; 112: 23-27PubMed Google Scholar) and murine (38Misiti J. Spivak J.L. J. Clin. Invest. 1979; 64: 1573-1579Crossref PubMed Scopus (51) Google Scholar, 39Gillo B. Ma Y.-S. Marks A.R. Blood. 1993; 81: 783-792Crossref PubMed Google Scholar, 40Hensold J.O. Dubyak G. Housman D.E. Blood. 1991; 77: 1362-1370Crossref PubMed Google Scholar, 41Levenson R. Houseman D. Cantley L. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5948-5952Crossref PubMed Scopus (84) Google Scholar, 42Sawyer S.T. Krantz S.B. J. Biol. Chem. 1984; 259: 2769-2774Abstract Full Text PDF PubMed Google Scholar) erythroid progenitors and precursors. The mechanism of regulation of calcium by erythropoietin has been examined at the single cell level using normal human burst-forming units-erythroid-derived cells at defined stages of differentiation and fluorescence microscopy coupled to digital video imaging (35Miller B.A. Scaduto Jr., R.C. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 43Yelamarty R.V. Miller B.A. Scaduto Jr., R.C. Yu F.T.S. Tillotson D.L. Cheung J.Y. J. Clin. Invest. 1990; 85: 1799-1809Crossref PubMed Scopus (38) Google Scholar,44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar), patch clamp (44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 45Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. 1997; 89: 92-100Crossref PubMed Google Scholar), and microinjection (46Miller B.A. Bell L. Hansen C.A. Robishaw J.D. Linder M.E. Cheung J.Y. J. Clin. Invest. 1996; 98: 1728-1736Crossref PubMed Scopus (25) Google Scholar). Our laboratory has shown that erythropoietin induces a dose-dependent increase in [Ca]i in single normal human BFU-E-derived erythroblasts modulated through a voltage-independent ion channel permeable to calcium (44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 45Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. 1997; 89: 92-100Crossref PubMed Google Scholar) and dependent on tyrosine phosphorylation (47Miller B.A. Bell L.L. Lynch C.J. Cheung J.Y. Cell Calcium. 1994; 16: 481-490Crossref PubMed Scopus (21) Google Scholar). Here, we identified the domain of the Epo receptor required for Epo-induced calcium influx using a system in which single cells that expressed transfected wild-type or mutant erythropoietin receptors were identified by detection of green fluorescent protein (GFP) with digital video imaging and cytosolic Ca2+ changes simultaneously measured in transfected cells. CHO cells were cultured in DMEM with 10% fetal calf serum at 37 °C in 5% CO2. All constructs were based on the pTracer-CMV vector (Invitrogen). This mammalian expression vector contains an SV40 early promoter that drives expression of the GFP gene fused to a zeocin resistance gene and a CMV immediate-early promoter driving expression of a second cDNA. A panel of Epo-R deletion mutants (see Fig. 2) was generated as described previously (48D'Andrea A.D. Yoshimura A. Youssoufian H. Zon L.I. Koo J.W. Lodish H.F. Mol. Cell. Biol. 1991; 11: 1980-1987Crossref PubMed Scopus (224) Google Scholar). Briefly, a region from nucleotide 1083 to the specific 3′-end of each construct was amplified via polymerase chain reaction. After subcloning the polymerase chain reaction fragment into pCR-Script, the fragment was digested with SphI andEcoRI and subcloned intoSphI-EcoRI-digested pCDNA3-EPO-R. The Epo-R −221 construct was subcloned from pXM into pcDNA3 (48D'Andrea A.D. Yoshimura A. Youssoufian H. Zon L.I. Koo J.W. Lodish H.F. Mol. Cell. Biol. 1991; 11: 1980-1987Crossref PubMed Scopus (224) Google Scholar). Epo-R tyrosine mutants were generated via overlap extension polymerase chain reaction. These included a series of single tyrosine mutants in which phenylalanine was substituted for different tyrosine residues in the Epo receptor (see Fig. 3) and a series of add back mutants to an Epo receptor devoid of tyrosine residues. Oligonucleotide primers were selected that produced either a phenylalanine at amino acid position 443, 460, or 464, or a tyrosine at amino acid position 443, 460, or 464. Polymerase chain reaction was performed using either pBSK-Epo-R or pBSK-Epo-R F8 (all eight cytoplasmic tyrosines converted to phenylalanine) to generate a SphI-EcoRI fragment in pCR-Script. The fidelity of all constructs was confirmed by sequencing both strands of the 440-base pair fragment. EachSphI-EcoRI fragment was subcloned intoSphI-EcoRI-digested pBSK-Epo-R. The Epo-R cDNA was subcloned into pCDNA3 or pTracer-CMV usingKpnI and EcoRI sites in the pTracer vectors.Figure 3Schematic representation of wt mEpo-R receptor and tyrosine substitution mutants. F8 has all eight cytoplasmic tyrosines converted to phenylalanine. The Y7 receptor has phenylalanine substituted for single tyrosines at position 443, 460, or 464. Single-letter amino acid codes used.View Large Image Figure ViewerDownload (PPT) CHO cells were transfected with transfection solution containing pTracer-CMV vectors (6 μg/ml) and LipofectAMINE™ (Life Technologies, Inc.; 16 μg/ml) for 5 h at 37 °C. Successful transfection of CHO cells was verified by detection of expression of green fluorescent protein by digital video imaging (44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 45Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. 1997; 89: 92-100Crossref PubMed Google Scholar, 46Miller B.A. Bell L. Hansen C.A. Robishaw J.D. Linder M.E. Cheung J.Y. J. Clin. Invest. 1996; 98: 1728-1736Crossref PubMed Scopus (25) Google Scholar). The excitation peaks of GFP are 395 and 478 nm, and the emission peak is 507 nm. Cells were excited at 380 nm, and emission was detected at 510 nm. The optimal time for expression of transfected pTracer CMV Epo-R was found to be 48–72 h after transfection by Western blotting, and this time interval posttransfection was selected to examine the response of transfected CHO cells to Epo. At this time interval, 40–50% of CHO cells expressed GFP. A fluorescence microscopy coupled digital video imaging system was used to measure [Ca]i (35Miller B.A. Scaduto Jr., R.C. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 43Yelamarty R.V. Miller B.A. Scaduto Jr., R.C. Yu F.T.S. Tillotson D.L. Cheung J.Y. J. Clin. Invest. 1990; 85: 1799-1809Crossref PubMed Scopus (38) Google Scholar, 44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 45Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. 1997; 89: 92-100Crossref PubMed Google Scholar, 46Miller B.A. Bell L. Hansen C.A. Robishaw J.D. Linder M.E. Cheung J.Y. J. Clin. Invest. 1996; 98: 1728-1736Crossref PubMed Scopus (25) Google Scholar). To study changes in intracellular calcium in transfected cells, we were not able to use Fura-2 as the detection fluorophore because its excitation and emission wavelengths overlap with those of GFP. Instead, we used the fluorescence indicator rhod-2 (Molecular Probes, Eugene, OR) (49Mitani A. Takeyasu S. Yanase H. Nakamura Y. Kataoka K. J. Neurochem. 1994; 62: 626-634Crossref PubMed Scopus (93) Google Scholar, 50Yoshino M. Kamiya H. Brain Res. 1995; 695: 179-185Crossref PubMed Scopus (28) Google Scholar). Fluorescence of GFP does not interfere with rhod-2 fluorescence. Rhod-2 is a single wavelength excitation Ca2+ fluorophore (excitation, 540 nm; emission, 590 nm). We cannot obtain absolute [Ca]i values in single cells, because fluorescence is proportional to [Ca]i, fluorophore concentration, optical path, and excitation light intensity. Fo (fluorescence at baseline) and Ft (fluorescence at time t) were measured and used to quantitate changes in [Ca]i in rhod-2 loaded CHO cells. Using the peak Ft measurement 20 min after Epo stimulation, Ft/Fo × 100% was calculated and used for standardization to compare changes between groups. CHO cells were loaded by incubation with rhod-2 (2 μm) for 30 min at 37 °C and stimulated with recombinant erythropoietin (>100,000 units/mg; R & D Systems, Inc., Minneapolis, MN). Baseline measurements of Fo were taken before stimulation, and measurements of Ft were taken at 1, 5, 10, 15, and 20 min after Epo stimulation. Experiments were performed with or without physiological calcium (0.7 mm) (36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar). Pertussis toxin was obtained from List Biological Laboratory (Campbell, CA) and was heat-inactivated by incubating at 95 °C for 20 min (51Miller B.A. Foster K. Robishaw J.D. Whitfield C.F. Bell L. Cheung J.Y. Blood. 1991; 77: 486-492Crossref PubMed Google Scholar). Whole cell lysates were prepared, and ECL was performed as described previously (52Zhang M.-Y. Harhaj E.W. Bell L. Sun S.-C. Miller B.A. Blood. 1998; 92: 1225-1234Crossref PubMed Google Scholar). Membranes were incubated with anti-mEpo-R antibody (sc-697, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:100). Donkey anti-rabbit antibody (1:1500) was used as the secondary antibody. To analyze Epo-dependent JAK2 tyrosine phosphorylation in stably transfected Ba/F3 cells, we used a JAK2 polyclonal antibody for immunoprecipitations (53Barber D.L. D'Andrea A.D. Mol. Cell. Biol. 1994; 14: 6506-6514Crossref PubMed Scopus (51) Google Scholar) and a different JAK2 polyclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY) for Western analysis. The monoclonal anti-phosphotyrosine 4G10 antibody was generously provided by Dr. Brian Druker (Portland, OR). To confirm our results in a factor-dependent hematopoietic cell line, we studied Ba/F3 cells stably transfected with wild-type or mutant mEpo-R (34Barber D.L. Mason J.M. Fukazawa T. Reedquist K.A. Druker B.I. Band H. D'Andrea A.D. Blood. 1997; 89: 3166-3174Crossref PubMed Google Scholar). Ba/F3 cells transfected with wt or mutant mEpo-R were cultured with 1 mg/ml G418 (Life Technologies, Inc., Gaithersburg, MD) and 500 pg/ml IL-3. Ba/F3 and Ba/F3 mEpo-R cells were adhered to fibronectin coated coverslips, growth factor deprived for 5 h, and loaded with 2 μm Fura-2 acetoxymethyl ester (Molecular Probes, Inc.) for digital video imaging studies (35Miller B.A. Scaduto Jr., R.C. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar, 46Miller B.A. Bell L. Hansen C.A. Robishaw J.D. Linder M.E. Cheung J.Y. J. Clin. Invest. 1996; 98: 1728-1736Crossref PubMed Scopus (25) Google Scholar, 47Miller B.A. Bell L.L. Lynch C.J. Cheung J.Y. Cell Calcium. 1994; 16: 481-490Crossref PubMed Scopus (21) Google Scholar). To determine the domains of the Epo-R required for erythropoietin regulation of calcium channels, we established a system in which single cells transfected with wild-type or mutant Epo-R could be identified by GFP fluorescence, and [Ca]i was measured simultaneously in the identical cells with digital video imaging. CHO cells were used for these transfections, because they lack endogenous Epo-R and have been shown to contain all the necessary transducers required for growth hormone-induced [Ca]i increase (54Billestrup N. Bouchelouche P. Allevato G. Ilondo M. Nielsen H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2725-2729Crossref PubMed Scopus (85) Google Scholar). In Fig.1, five CHO cells are shown 24 h after transfection with Epo-R subcloned into the pTracer-CMV vector. Successfully transfected CHO cells were detected by fluorescence from GFP (Fig. 1 B). [Ca]i detected by intracellular rhod-2 is shown for the same five cells in Fig. 1 C. A significant increase in [Ca]i was observed in CHO cells stimulated with Epo following transfection with pTracer-CMV mEpo-R (Table I, p < 0.02). The rate of rise in intracellular calcium was similar to that previously observed in BFU-E-derived cells (35Miller B.A. Scaduto Jr., R.C. Tillotson D.L. Botti J.J. Cheung J.Y. J. Clin. Invest. 1988; 82: 309-315Crossref PubMed Scopus (57) Google Scholar, 36Miller B.A. Cheung J.Y. Tillotson D.L. Hope S.M. Scaduto Jr., R.C. Blood. 1989; 73: 1188-1194Crossref PubMed Google Scholar). The magnitude of [Ca]i changes cannot be directly compared because we were unable to calibrate rhod-2 signals into [Ca]i, and rhod-2 was observed to have a smaller dynamic range than Fura-2. No change in Ft measured at 10-s intervals compared with Fo was observed over the first minute (data not shown). No increase in [Ca]i was observed in Epo treated CHO cells transfected with vector alone. Nontransfected CHO cells (e.g. the other four cells in Fig. 1 C) did not demonstrate a significant change in percentage of Ft/Fo over the experimental period (20 min), indicating that problems due to leakage and photobleaching of rhod-2 are minimal (no decrease in Ft/Fo), and that the increase in [Ca]i in successfully transfected CHO cells was specific for Epo.Table IPertussis toxin inhibits the response of transfected CHO cells to EpoPreincubated withpTracer vectorFoFtFt/Fon%CMV mEpo-R42 ± 462 ± 5151 ± 9aSignificant increase in percentage of Ft/Fo above cells not transfected with vector (p < 0.05), which had no detectable GFP fluorescence.65 μg/ml PTCMV mEpo-R50 ± 552 ± 5105 ± 3115 μg/ml HI PTCMV mEpo-R42 ± 464 ± 6153 ± 8aSignificant increase in percentage of Ft/Fo above cells not transfected with vector (p < 0.05), which had no detectable GFP fluorescence.13None50 ± 753 ± 6110 ± 46CHO cells transfected with or without pTracer-CMV mEpo-R were preincubated at 37 °C for 80 minutes with 0 or 5 μg/ml pertussis toxin (PT) or 5 μg/ml heat-inactivated pertussis toxin (HI/PT) and then stimulated with 2 units/ml Epo. Fo, the baseline rhod-2 measurement, and Ft, the peak measurement of rhod-2 20 min after Epo stimulation, were measured in cells that express GFP. Mean ± S.E. of Ft/Fo × 100% were calculated. n = number of cells studied.a Significant increase in percentage of Ft/Fo above cells not transfected with vector (p < 0.05), which had no detectable GFP fluorescence. Open table in a new tab CHO cells transfected with or without pTracer-CMV mEpo-R were preincubated at 37 °C for 80 minutes with 0 or 5 μg/ml pertussis toxin (PT) or 5 μg/ml heat-inactivated pertussis toxin (HI/PT) and then stimulated with 2 units/ml Epo. Fo, the baseline rhod-2 measurement, and Ft, the peak measurement of rhod-2 20 min after Epo stimulation, were measured in cells that express GFP. Mean ± S.E. of Ft/Fo × 100% were calculated. n = number of cells studied. The Ca2+ response to erythropoietin was then further characterized in CHO cells. The rise in intracellular Ca2+was dependent on extracellular calcium, because no response to erythropoietin was seen in medium without calcium (data not shown). Pretreatment with pertussis toxin, but not heat-inactivated pertussis toxin, significantly inhibited the increase in percentage of Ft/Fo observed in Epo-stimulated pTracer-CMV mEpo-R transfected CHO cells (Table I, p < 0.002). These results demonstrated that the influx of calcium in CHO cells is regulated by a pertussis toxin-sensitive G protein. CHO cells transfected with pTracer-CMV mEpo-R were then pretreated with the l-type Ca2+ channel blocker nifedipine. Nifedipine blocked the increase in [Ca]i seen in response to erythropoietin, but only at doses higher than typically required to block voltage-sensitivel-type calcium channels (TableII). These data confirm that the Epo-R in transfected CHO cells behaved similarly to the endogenous Epo-R on BFU-E-derived cells (44Cheung J.Y. Elensky M.B. Brauneis U. Scaduto Jr., R.C. Bell L.L. Tillotson D.L. Miller B.A. J. Clin. Invest. 1992; 90: 1850-1856Crossref PubMed Scopus (35) Google Scholar, 45Cheung J.Y. Zhang X.-Q. Bokvist K. Tillotson D.L. Miller B.A. Blood. 1997; 89: 92-100Crossref PubMed Google Scholar, 46Miller B.A. Bell L. Hansen C.A. Robishaw J.D. Linder M.E. Cheung J.Y. J. Clin. Invest. 1996; 98: 1728-1736Crossref PubMed Scopus (25) Google Scholar, 51Miller B.A. Foster K. Robishaw J.D. Whitfield C.F. Bell L. Cheung J.Y. Blood. 1991; 77: 486-492Crossref PubMe
'%3e%3ccircle r='20' fill='%23008'/%3e%3ccircle r='17.5' fill='%23fff'/%3e%3ccircle r='3.5' fill='%23008'/%3e%3cg id='d'%3e%3cg id='c'%3e%3cg id='b'%3e%3cg id='a' fill='%23008'%3e%3ccircle r='.875' transform='rotate(7.5 -8.75 133.5)'/%3e%3cpath d='M0 17.5.6 7 0 2l-.6 5L0 17.5z'/%3e%3c/g%3e%3cuse xlink:href='%23a' transform='rotate(15)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(30)'/%3e%3c/g%3e%3cuse xlink:href='%23c' transform='rotate(60)'/%3e%3c/g%3e%3cuse xlink:href='%23d' transform='rotate(120)'/%3e%3cuse xlink:href='%23d' transform='rotate(-120)'/%3e%3c/g%3e%3c/svg%3e)